WO1993023547A1 - ENZYMATIC PROCESS FOR PRODUCTION OF S-6-METHOXY-α-METHYL-2-NAPHTHALENEACETIC ACID - Google Patents

Abstract

The enantioselective hydrolysis of racemic naproxen esters by ester hydrolases using hydrolases derived from a panel of microorganisms is described, the most suitable of which was the microorganism Zopfiella latipes. The ester hydrolases are of use in a low cost, high yield hydrolysis of racemic naproxen esters.

Description

ENZYMATIC PROCESS FOR PRODUCTION OF

(S)-6-METHOXY-α-METHYL-2-NAPHTHALENEACETIC ACID

Field of the Invention

This invention relates to the preparation of (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid by the enantioselective hydrolysis of racemic esters using microorganisms and enzymes derived therefrom.

Background of the Invention

6-Methoxy-α-methyl-2-napthaleneacetic acid, which has the following structural formula:

is a nonsteroidal, anti-inflammatory drug described, for example, in U.S. Patent No. 3,904,682. It has a center of asymmetry at the α-carbon

(indicated by the asterisk), and thus possesses two enantiomers.

It is known that the S-enantiomer, (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid, has 28-fold greater anti-inflammatory activity than the corresponding R-enantiomer (I.T. Harrison, et al . , J. Med. Chem. , 13 : 203 (1970)); and the S-enantiomer alone is marketed as the

anti-inflammatory drug Naprosyn^® (Barnhart, et al . , Physician 's Desk Reference, 44:2200 (1990)).

"Naproxen" is the USAN and INN nonproprietary name for (S)-6-methoxy-α-methyl-2-napththaleneacetic acid. For convenience, however, in this application the terms "naproxen" and "R,S-naproxen" mean a mixture of the R- and S-enantiomers of 6-methoxy-α-methyl-2-napththaleneacetic acid, especially a racemic mixture; and "R-naproxen" and "S-naproxen" mean the two enantiomers individually. Thus, the term "S-naproxen" used in this application corresponds to the USAN/INN name "naproxen". Further, for convenience in this application, although naproxen is an acid, the terms "naproxen", "R,S-naproxen", "R-naproxen", and "S-naproxen" include not only the acid form of the compound, but also the anion form and pharmaceutically acceptable salts of the acid form, unless the context requires otherwise.

The chemical synthesis of 6-methoxy-α-methyl-2-naphthaleneacetic acid typically leads to a racemic mixture of the R- and S-enantiomers (R,S-naproxen), which must be resolved to obtain the desired S-naproxen from the racemic mixture. A number of microorganisms, as well as a few selected commercial lipases and esterases, belonging to the genera

Bacillus, Pseudomonas, Arthrobacter, Mucor and Streptomyces have been reported to hydrolyze racemic mixtures of R,S-naproxen esters with some chiral preference. The actual application of these microorganisms or enzymes in S-naproxen manufacturing, however, has been made impractical by their lack of sufficient chiral specificity and relatively low rates of conversion of R,S-naproxen ester into S-naproxen.

Iriuchijima and Keiyu, Agr. Biol. Chem. 45:1389 (1981) describe the hydrolysis of racemic mixtures of A,S-naproxen esters by Mycobacterium smegmatis to R-naproxen having an 89% enantiomeric excess ("ee"). The extent of conversion of R,S-naproxen ester to R-naproxen was 20%.

Nakagawa, et al . , J. Biochem 95:1047 (1984) describe an

intracellular esterase isolated from Pseudomonas fluorescens, which catalyzes the hydrolysis of methyl esters of short chain length. The intracellular esterase differs from known extracellular lipases in its sensitivity to inhibitors, molecular weight and substrate specificity.

Gu, et al. , Tetrahedron Lett. 27:1763 (1986), describe the

preparation of S-naproxen from A,S-naproxen esters using a lipase derived from Candida cylindracea. Although the resulting S-naproxen had >98% ee, it took 216 hours to achieve 39% conversion of the R,S-naproxen ester at 22°C. This low rate of conversion is unacceptable for a high yield, low cost industrial process.

EP 0 153 474 describes the process of preparing S-naproxen from R,S-naproxen ester using microbial enzymes, but requires a two step hydrolysis process. The R,S-naproxen ester is first enantioselectively hydrolyzed to S-naproxen ester and R-naproxen with a microbial esterase, preferably from Aspergillus, and the R-naproxen separated. The S-naproxen ester is then nonselectively hydrolyzed by esterase from hog liver or Pleurotus ostreatus to form the desired S-naproxen.

U.S. Patent No. 4,762,793 describes an enzymatic process in which enantioselective hydrolysis of R,S-α-arylalkanoic esters is carried out using a lipase enzyme isolated from Candida cylindracea. When used in the production of S-naproxen, this process took over two days at 32°C to convert 40% of R,S-naproxen ester to S-naproxen. Moreover, the enzyme loses about 80% of its activity over a 96 hour reaction period. (See also, EP 0 195 717). EP 0205 215 describes the process of preparing

S-α-methylareneacetic acids by the microbial asymmetric oxidation of α-methylareneethanes. In one embodiment, 6-methoxy-α-methyl-2-naphthaleneethane is oxidized to (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid using Cordyceps militaris. EP 0 227 078 describes the process of preparing

S-α-methylareneacetic acids from R,S-naproxen esters using extracellular lipases of microbial origin, preferably Candida cylindracea. At 22°C, Candida cylindracea lipase required several days to convert 41% of methyl R,S-naproxen ester into S-naproxen. This rate of conversion is too slow to be suitable for a high yield, low cost industrial process.

EP 0 328 125 describes a process for the enzymatically catalyzed enantioselective transesterification of racemic alcohols, such as (R,S)-6-methoxy-α-methyl-2-naphthaleneethanol, with an ester such as ethyl acetate, methyl acetate or methyl propionate, to afford the ester of the S-alcohol. The resulting esters are said to be useful in the preparation of anti-inflammatory agents such as S-naproxen. Preferred enzymes are steapsin and the lipase from Pseudomonas fluorescens. EP 0 330 217 describes a continuous enzymatic process for the preparation of S-naproxen from an alkoxyethyl R,S-naproxen ester using a lipase isolated from Candida cylindracea. The enzymatic reaction gave a 37% conversion of R,S-naproxen ester at 35°C after 500 hours. This rate of conversion is too low for a high yield, low cost process.

U.S. Patents Nos. 4,886,750 and 5,037,751 describe a process using microorganisms having the esterase ability for enantioselective hydrolysis of R,S-naproxen esters into S-naproxen having at least 60% ee. In particular, the patents describe an esterase that has the ability to enantioselectively hydrolyze R,S-naproxen ester into S-naproxen having at least 98.8% ee. However, the conversion of R,S-naprcxen ester to S-naproxen is limited to low substrate concentrations. The esterases do not act in a biphasic aqueous/organic system or on insoluble R,S-naproxen ester. Moreover, the disclosed esterases require a surfactant, such as Tween^®, to be active; thereby restricting their use to a process requiring additional equipment and time to remove the surfactant.

PCT/NL90/00058 describes the stabilization of esterases used to enantioselectively hydrolyze R,S-naproxen ester to S-naproxen. The enzymes being stabilized are disclosed in U.S. Patents Nos. 4,886,750 and 5,037,751. At the high substrate concentrations of R,S-naproxen ester required for high yield, low cost production, the described esterase is almost completely inactivated by the S-naproxen formed by the hydrolysis. By treating the esterase with an aldehyde or anhydride, the stability of the esterase is enhanced in the presence of S-naproxen. However, these stabilizing agents (including the preferred agent, formaldehyde) are known carcinogens that must be removed by extensive processing for the product to be used in humans. Hence, the ability to run such a hydrolysis reaction without the need for carcinogenic stabilizing agents is a highly desirable characteristic.

Commercially available enzymes are limited in the extent to which they can be used at elevated temperatures. Use of an enzyme that exhibits high thermal stability is especially desirable in a process for

hydrolyzing R,S-naproxen ester. The economics of an enzyme-catalyzed chemical reaction, whether a continuous or batch process, depend greatly on the lifespan of the catalytically active enzyme. Because thermal inactivation is the most common cause of enzyme inactivation, an increase in thermal stability acts to prolong the life span of the enzyme, which in turn improves the economics of the overall process.

In addition, the rate of an enzymatic reaction depends on the reaction temperature. An enzyme exhibiting thermal stability permits running the reaction at a higher temperature which accelerates the rate, which in turn increases the production throughput. In the hydrolysis of n-propyl R,S-naproxen ester, high temperature also drives the solid ester substrate towards its molten form, rendering the control of solid particle size less critical. It is, therefore, desirable to conduct the reaction at the highest temperature that can be tolerated by the enzyme. To this end, it is desirable to develop an enzyme that exhibits high thermal stability.

Many enzymes, particularly those that are traditionally referred to as esterases, require the addition of surfactants for optimal activity as described, for example, in connection with U.S. Patent No. 4,886,750.

Employment of surfactants in a manufacturing process can be quite costly and their removal requires additional process technology, equipment and labor. Also, many surfactants can be hydrolyzed by such enzymes; for example, soybean oil or Tween^® 80 are hydrolyzed by some esterases.

Hydrolysis of surfactant results in the introduction of undesirable contaminants. Hence, the ability to perform an enantioselective

hydrolysis without the need for surfactants is a highly desirable characteristic. To this end, it is desirable to have an enzyme that exhibits high stability without the addition of surfactants. Therefore there is still a great need for a commercial scale process giving rise to economically attractive yields with high enantioselectivity and high rates of conversion of R,S-naproxen ester into S-naproxen. In particular, there has remained a need for a process that can be run without surfactants or carcinogenic stabilizing agents at high levels of R,S-naproxen ester and S-naproxen and at relatively high temperatures, while maintaining a high rate of conversion. SUMMARY OF THE INVENTION

In one embodiment of the invention, a process for the production of S-naproxen comprising the enantioselective hydrolysis of A,S-naproxen ester by an ester hydrolase selected from ester hydrolases produced by a microorganism of the group Absidia griseola, Aspergillus sydowii,

Doratomyces stemonitis, Eupenicillium baarnenses, Graphium sp . ,

Heterocephalum aurantiacum, Pencillium roguefortii and Zopfiella latipes is described.

In another embodiment of the invention, a coding region of a gene encoding for an ester hydrolase capable of enantioselective hydrolysis of an A,S-naproxen ester, which region comprise the nucleotide sequence as set forth in Sequence I.D. No. 2, Sequence I.D. No. 5, Sequence I.D.

No. 8, Sequence I.D. No. 11 or Sequence I.D. No. 14, or a sequence that hybridizes thereto is described.

In yet another embodiment of the invention, an ester hydrolase capable of the enantioselective hydrolysis of an A,S-naproxen ester to S-naproxen wherein said ester hydrolase hydrolyzes the reaction of R,S-naproxen ester at a temperature range from about 35°C to about 65°C is described.

In another embodiment of the invention, there is described an ester hydrolase capable of the enantioselective hydrolysis of ethyl R,S-naproxen ester to S-naproxen, which ester hydrolase comprises an amino acid sequence as set forth in Sequence I.D. No. 3, Sequence I.D. No. 6,

Sequence I.D. No. 9, Sequence I.D. No. 12 or Sequence I.D. No. 15. In still another embodiment of the invention, there is described an ester hydrolase capable of the enantioselective hydrolysis of n-propyl R,S-naproxen ester to S-naproxen, which ester hydrolase comprises an amino acid sequence as set forth in Sequence I.D. No. 3, Sequence I.D. No. 6, Sequence I.D. No. 9, Sequence I.D. No. 12 or Sequence I.D. No. 15.

FIGURES

Figure 1(a) is a diagram illustrating cDNA synthesis for the ester hydrolase gene in E. coli.

Figure 1(b) shows the construction of the yeast expression plasmid for the ester hydrolase gene.

Figure 2 shows the degenerate oligonucleotide primers based on the partial amino acid sequences determined for the first 20 amino acids at the N-terminus as well as the four internal cyanogen bromide cleaved fragments of the Zopfiella ester hydrolase.

Figure 3 shows the nucleotide junction sequences and the inferred amino acid sequences between the Zopfiella cDNA and the plasmid vector.

Figure 4 shows the enhanced thermal tolerance of rec 780-m10 over rec 780.

Figure 5 is a schematic flowsheet for an immobilized Zopfiella bioreactor system.

SEQUENCE I.D. DESIGNATION

Seq. I.D. No. 1: fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 511 gene.

Seq. I.D. No. 2: nucleotide sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 511 gene,

Seq. I.D. No. 3 : inferred amino acid sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 511 gene. Seq. I.D. No. 4 fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 780 gene.

Seq. I.D. No. 5 : nucleotide sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780 gene,

Seq. I.D. No. 6 inferred amino acid sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780 gene.

Seq. I.D. No. 7: fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 780-m10 gene.

Seq. I.D. No. 8 nucleotide sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m10 gene.

Seq. I.D. No. 9: inferred amino acid sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m10 gene.

Seq. I.D. No. 10: fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 780-165 gene.

Seq. I.D. No. 11: nucleotide sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m165 gene.

Seq. I.D. No. 12: inferred amino acid sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m165 gene.

Seq. I.D. No. 13: fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 780-m210 gene.

Seq. I.D. No. 14: nucleotide sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m165r210 gene. Seq. I.D. No. 15: inferred amino acid sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m165r210 gene. Figure 4 :

Thermal Stability at 54 C was assessed. Equal amounts of enzyme activity were added to tubes containing 1mg/ml of (S) Naproxen-p-nitrophenol solubilized by DMSO in 0.1M MOPS buffer. Enzyme added per tube at temperature and aliquots removed at indicated timepoints. Emzyme activity stopped by acetonitrile on ice. Unhydrolyzed substrate pelleted by centrifugation at 3000 X G for 15 minutes. Enzyme activity measured by accumulation of p-nitrophenyl measured by absorbance at 410nm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for producing S-naproxen by presenting R,S-naproxen ester to the action of an ester hydrolase isolated from a microorganism selected from the group Absidia griseola, Aspergillus sydowii, Doratomyces stemonitis, Eupenicillium baarnenses, Graphium sp. , Heterocephalum aurantiacum, Pencillium roguefortii and Zopfiella latipes to enantioselectively catalyze the hydrolysis of

R,S-naproxen ester to S-naproxen.

More particularly, this invention relates to the screening of a panel of microorganisms in order to identify a microorganism that produces an ester hydrolase of use in the high yield, low cost production of S-naproxen. To improve the efficiency of production of the enzyme, once identified, the gene for the native enzyme is cloned and expressed in a suitable host. The recombinant enzyme is then used in the high yield, low cost production of S-naproxen. The Zopfiella latipes (hereinafter

Zopfiella) family of microorganisms was found to produce an ester hydrolase enzyme that met the stringent criteria for commercial

production, including yielding S-naproxen having an enantiomeric excess greater than 98%.

Most particularly, this invention relates to a high yield, low cost process for the production of S-naproxen.

Before proceeding further with the description of the specific embodiments of the present invention, a number of terms will be defined.

As stated before, the term "S-naproxen" includes the

pharmaceutically acceptable salts of S-naproxen, in particular the sodium salt. The invention thus includes those processes wherein the S-naproxen formed by enantioselective hydrolysis is converted to a pharmaceutically acceptable salt and those processes in which it is not. The terms "R,S-naproxen ester" or "racemic naproxen ester" mean a mixture of the R- and S-enantiomers of varying or equal ratios of an ester of 6-methoxy-α-methyl-2-naphthaleneacetic acid. R,S-naproxen ester is defined by the following formula:

where R is alkyl, cycloalkyl, aralkyl or aryl. Preferably, R is lower alkyl, and more preferably R is ethyl or n-propyl.

The term "alkyl" refers to both straight and branched chain alkyl groups having total of 1 to 12 carbon atoms, thus including primary, secondary and tertiary alkyl groups. Typical alkyls include, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-amyl, n-hexyl and the like. "Lower alkyl" refers to alkyl groups having 1 to 4 carbon atoms. Typical lower alkyls include, for example, methyl, ethyl, n-propyl and the like.

"Cycloalkyl" refers to cyclic hydrocarbon groups having from 3 to 12 carbon atoms such as, for example, cyclopropyl, cyclopentyl, cyclohexyl, and the like. "Lower cycloalkyl" refers to cycloalkyl groups having 3 to 6 carbon atoms.

"Aryl" refers to a monovalent unsaturated aromatic carbocyclic radical having a single ring (e.g., phenyl) or two condensed rings (e.g., naphthyl).

"Aralkyl" refers to an aryl substituted alkyl group, such as, for example, benzyl or phenethyl.

An alkyl, cycloalkyl, aryl or aralkyl group can be optionally substituted with one or more non-interfering electron-withdrawing substituents, for example, halo, nitro, cyano, phenyl, hydroxy, alkoxy, alkylthio, or -C(O)R¹ wherein R¹ is lower alkyl, lower cycloalkyl, hydroxy, alkoxy, cycloalkoxy, phenoxy, benzyloxy, NR²R³ (in which R² and R³ are independently H, lower alkyl, lower cycloalkyl, or jointly form a 5- or 6-membered ring together with the nitrogen, the ring optionally including a hetero group selected from O, NH, or N-(lower alkyl)), or -OM wherein M is an alkali metal. The term "non-interfering" characterizes the substituents as not adversely affecting any reactions to be performed in accordance with the process of this invention.

"Halo" refers to iodo, bromo, chloro and fluoro.

"Alkoxy" refers to the group having the formula -OR⁴, wherein R⁴ is lower alkyl, as defined above. Typical alkoxy groups include, for example, methoxy, ethoxy, t-butoxy and the like.

"Alkylthio" refers to the group having the formula -SR⁵, wherein R⁵ is lower alkyl, as defined above. Typical alkylthio groups include, for example, thiomethyl, thioethyl and the like. "Cycloalkoxy" refers to the group having the formula -OR⁶, wherein R⁶ is lower cycloalkyl, as defined above. Typical cycloalkoxy groups include, for example, cyclopropyloxy, cyclopentyloxy, cyclohexyloxy, and the like. "Alkali metal" refers to sodium, potassium, lithium and cesium.

The electron-withdrawing substituents, if present, are preferably at the α- or β- position of the R group, to the extent consistent with the stability of the group. Esters in which the R groups contain

electron-withdrawing substituents are referred to as activated esters, since they generally hydrolyze more rapidly than those where the R group is not so substituted.

Specific examples of alkyl groups, R, are methyl, ethyl, n-propyl, t-butyl, n-hexyl, i-octyl, n-dodecyl, benzyl, 2-chloroethyl,

2,2,2-trichloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, 2-bromoethyl, cyanomethyl, 2-nitropropyl, carboethoxymethyl, methoxymethyl,

2-hydroxy-1,2-dimethoxycarbonylethyl, 2-hydroxy-1,2-dicarboxyethyl, 2-hydroxy-1,2-diethoxycarbonylethyl, and the like.

"Organic solvents" includes solvents such as methanol, ethanol, acetic acid, methylene chloride, chloroform, tetrahydrofuran,

dimethoxyethane, dimethylformamide, dimethylsulfoxide, benzene, toluene, carbon tetrachloride and the like.

"Base" refers to bases such as alkali metal hydroxides, alkali metal alkoxides, alkali metal hydrides, alkali metal di(lower alkyl)amines, alkali metal acetates, alkali metal bicarbonates, alkali metal, tri(lower alkyl)amines, and the like, for example, potassium hydroxide, sodium hydroxide, potassium methoxide, sodium carbonate, sodium salt of diethyl amine, sodium acetate, potassium bicarbonate, and the like. A "resolving agent" is an optical isomer of a chiral amine base such as α-methylbenzylamine, cinchonidine, cinchonine, quinine, quinidine, strychnine, brucine, morphine, α-phenylethylamine, arginine,

dehydroabietylamine, 2-amino-1-propanol, amphetamine, glucosamine, conessine, anabasine, ephedrine and the like.

"Methyl naproxen ester" or "MeNPR" refers to the compound of Formula I when R is methyl. "Ethyl naproxen ester" or "EtNPR" refers to the compound of Formula

I when R is ethyl.

"n-Propyl naproxen ester" or "n-PrNPR" refers to the compound of Formula I when R is n-propyl.

"S-p-nitrophenyl ester" or "S-PEN" refers to the compound of Formula I when R is p-nitrophenyl.

Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography, thick-layer (preparative) chromatography, distillation, or a combination of these procedures. Specific illustration of suitable separation and isolation procedures can be had by references to the examples herein.

However, other equivalent separation or isolation procedures can, of course, also be used.

"Recombinant enzyme" refers to an ester hydrolase obtained by the cloning of a Zopfiella ester hydrolase gene into a suitable expression system. Whether used in the singular or plural form, "recombinant enzyme" refers to these particularly defined ester hydrolase enzymes, either as a group or individually. When referring to particular recombinant enzymes of the invention, the identifier "rec" will be used with the name of the clone, for example, rec 511 refers to the recombinant enzyme from

Zopfiella strain 511, and so on.

"Fusion protein" means a fusion protein of the recombinant ester hydrolase having a sequence expressed as a fusion between all or a portion of the sequence for the Zopfiella ester hydrolase and a heterologous protein.

"Regulatory region" means the expression control sequence, for example, a promoter and ribosome binding site, necessary for transcription and translation. "Stability" means the retention of enzymatic activity under defined reaction conditions.

"High Thermal Stability" refers to a temperature of about 10ºC above T½, where T½ is defined as the temperature at which one-half of the enzymatic activity of a reference ester hydrolase is lost within one hour.

"Enantiomeric excess" or "ee" means the excess of one enantiomer over the other in a mixture of two enantiomers, such as in the product of an enantioselective reaction; and is typically expressed as a percentage. Thus, in the enantioselective hydrolysis of R,S-naproxen ester to

S-naproxen, the %ee of the S-naproxen reaction product refers to the percentage of S-naproxen present minus the percentage of R-naproxen present.

"Conversion", in the enantioselective hydrolysis of R,S-naproxen ester to S-naproxen, means the ratio of S-naproxen produced to the initial R,S-naproxen ester present in a reaction mixture in a given time, and is usually expressed as a percentage.

"KNPR" means the potassium salt of S-naproxen. I. Screening of Microorganisms

The microorganisms that produce the enzymes of this invention were discovered after selecting over 600 Class 1, i.e. non-pathogenic, microorganisms for screening. The panel of microorganisms screened included 284 fungi, 180 true bacteria, 69 yeasts, 51 filamentous bacteria, 8 algae and 16 unclassified strains. The microorganisms were obtained from the American Type Culture Collection ( "ATCC" ) . Of the 600 carefully selected microorganisms, only eleven demonstrated the level of enzyme activity suitable for the enantioselective conversion of R,S-naproxen esters to S-naproxen with at least a 95% ee for use in the high yield, low cost production of S-naproxen. The eleven microorganisms identified appear in Table 1. TABLE 1

Microorganism ATCC Number

Absidia griseola 22618

Aspergillus sydowii 1017

Aspergillus sydowii 52077

Doratomyces stemonitis 12249

Eupenicillium baarnenses 34719

Graphium sp. 58400

Heterocephalum aurantiacum 36778

Pencillium roguefortii 46766

Zopfiella latipes 22015

Zopfiella latipes 26183

Zopfiella latipes 44575

To grow the microorganisms for the enantioselective hydrolysis of A,S-naproxen esters, the dehydrated microorganism is rehydrated and plated out to assess growth and purity of the transported culture. If the microorganism passes a visual inspection for purity, the microorganism is transferred onto slants of a culture medium for initial growth. The microorganisms can be kept on agar slants, in 50% glycerol at -20ºC or lyophilized.

The culture media used contain an assimilable carbon source, for example glucose, lactate, sucrose and the like; a nitrogen source, for example ammonium sulphate, ammonium nitrate, ammonium chloride and the like; with an agent for an organic nutrient source, for example yeast extract, malt extract, peptone, meat extract and the like; and an inorganic nutrient source, for example phosphate, magnesium, potassium, zinc, iron and other metals in trace amounts.

The preferred medium for a particular microorganism is defined by the slant with the best growth and is used for the liquid culture and assay. Following identification of the preferred medium for liquid culture and assay, a 5% (v/v) inoculum was grown in the defined medium for 24-48 hours, depending on the growth rate of the organism. A preferred growth medium for Absidia griseola is 28, Aspergillus sydowii is 325, Doratomyces stemonitis is 323, Eupenicillium baarnenses is 28,

Graphium sp. is 323, Heterocephalum aurantiacum is 28, Penicillium roguefortii is 325, Zopfiella latipes(ATCC #22015 and #44575) is 200 and Zopfiella latipes(ATCC #26183) is 325. The descriptions of these media can be found in R. Cote, ATCC Media Handbook, First Edition, 1984, which is incorporated by reference. A temperature between about 10°C and about 40°C and a pH between 4 and 10 is maintained during the growth of the microorganism. Preferably the microorganisms are grown at a temperature between about 23°C and about 36°C and at a pH between 5 and 9.

The aerobic conditions required during the growth of the

microorganisms can be provided according to any of the well-established procedures, provided that the supply of oxygen is sufficient to meet the metabolic requirement of the microorganisms. This is most conveniently achieved by exposing the slant to air. During the hydrolysis of R,S-naproxen ester, the microorganisms might be in a growing stage using a culture medium, as described above, or might be preserved in any system (buffer or medium) preventing degradation of enzymes. During the hydrolysis of R,S-naproxen ester, an ordinary culture medium, as described above, can be used. The preferred medium for the enantioselective hydrolysis of R,S-naproxen ester with a particular microorganism is the preferred medium used for growth of that

microorganism.

The microorganisms can be kept in the non-growing stage, for example, by exclusion of the assimilable carbon source or by exclusion of the nitrogen source. A preferred storage medium for Absidia griseola is 336, for Aspergillus sydowii(ATCC #1017) is 312, for Aspergillus

sydowii(ATCC #52077) is 325, for Doratomyces stemonitis is 323, for Eupenicillium baarnenses is 325, for Graphium sp. is 336, for

Heterocephalum aurantiacum is 325, for Penicillium roguefortii is 336, for Zopfiella latipes(ATCC #22015 and #44575) is 200 and for Zopfiella latipes(ATCC #26183) is 340. The descriptions of these media can be found in ATCC Media Handbook, supra. A temperature between about 10°C and about 40°C and a pH between about 4 and 9 is maintained during the storage.

A temperature between about 10°C and about 40°C and a pH between about 4 and 10 is maintained during the assay of the enantioselective hydrolysis of A,S-naproxen ester. Preferably the microorganisms are kept at a temperature between about 23°C and about 36°C and at a pH between 5 and 9. The aerobic conditions required during the assay can be provided according to any of the well-established procedures, provided that the supply of oxygen is sufficient to meet the metabolic requirement of the microorganisms. This is most conveniently achieved by supplying oxygen, suitably in the form of air by agitating the reaction liquid.

Racemic naproxen ester, preferably lower alkyl naproxen ester, is dissolved in a sterile organic solvent, preferably sterile soybean oil, to a concentration of 200-300 mg/ml, preferably 250 mg/ml. To aqueous culture medium containing the microorganism is added the R,S-naproxen ester solution to obtain a concentration of 0.20-0.30 mg/ml, most preferably 0.25 mg/ml. Aliquots are removed from the mixture at defined intervals from duplicate cultures. The processing is preferably done by robotic sample preparation. Processing of the samples includes extraction into an organic solvent, preferably ethyl acetate, centrifugation, sampling of the organic layer and evaporation. The sample is then derivatized with a resolving agent, preferably (S)-α-methylbenzylamine, to form

diastereomeric amides. The amides are then dissolved in the desired solvent, preferably a mixture of acetonitrile/water, for HPLC analysis to assess S-naproxen concentration and enantioselectivity in the hydrolysis of racemic naproxen esters.

Isolated standards are run of R,S-naproxen, S-naproxen and the media in which the assay is run. Each sample can be run in duplicate. Standard organisms can also be run to check on the reproducibility of the analysis.

Eleven strains of microorganisms were identified containing an enzyme suitable for the enantioselective hydrolysis of R,S-naproxen esters to S-naproxen with an ee of great than 95%. Of the microorganisms listed in Table 1, Zopfiella latipes yields an ee of greater than 98%.

Emphasis was placed on the isolation and purification of enzymes from microorganisms in the Zopfiella strains, since the enzymes showed activity capable of producing S-naproxen in greater than 98% ee from racemic mixtures of naproxen esters. Such a high chiral specificity at high conversion levels is unusual and was not seen in similar studies using commercially available enzymes. In terms of high activity, ease of purification, and enantioselectivity, enzymes from Zopfiella are are most preferred to others similarly listed, despite the high ee values for all the listed microorganisms in Table 1.

II. Cloning of the Ester Hydrolase

In a method of the invention a recombinant enzyme is obtained by isolating the ester hydrolase enzyme from a suitable microorganism, e.g., Absidia griseola, Aspergillus sydowii, Doratomyces stemonitis,

Eupenicillium baarnenses, Graphium sp . , Heterocephalum aurantiacum,

Pencillium roguefortii or Zopfiella latipes, preferably from a Zopfiella strain, more preferably Zopfiella Strain 780: ATCC #44575, determining the amino acid sequence of the isolated enzyme and thereafter cloning and expressing the recombinant enzyme using an E. coli, yeast, such as

Saccharomyces cerevisiae, or other expression system. Other expression systems that can be used include Bacillus subtilis, Aspergillus niger, and Pichia pastoris. Preferably, the E. coli bacterium is used for the production of the recombinant enzyme. Cloning and expression can be obtained rapidly in E. coli and high levels of gene expression are common. In addition, production in E. coli results in a system easily scaled up for large scale fermentation and protein purification.

The ester hydrolase from the microorganism can be isolated and purified by standard techniques. Preferably the enzyme is purified by a straight forward series of purification steps, i.e. cell disruption, ammonium sulfate precipitation, gel filtration, anion exchange

chromatography and hydrophobic interaction chromatography. The purified enzyme is then used for determination of the internal amino acid sequence. The internal amino acid sequence of the enzyme is determined by CNBr cleavage and the isolation of the resulting polypeptide fragments, preferably by reverse phase-HPLC. The results provide a partial amino acid sequence (first 10 amino acids at the N-terminal), which aids in the cloning of the ester hydrolase gene.

The cloning and expression of a recombinant ester hydrolase enzyme from the microorganism is carried out using the lac promoter of E. coli. E. coli promoters in addition to the lac promoter which may be used in the practice of the invention include, for example, trp, tac, lambda P_L, lambda P_R and T7 phage promoter.

The molecular cloning of the ester hydrolase gene from the

microorganism can be carried out using standard molecular cloning techniques as described in Maniatis, et.al. Molecular Cloning: A

Laboratory Manual, 2nd. Edition, Vol.1-3, Cold Spring Harbor Laboratory

Press (1989), which is incorporated by reference. The preferred molecular constructs used in the practice of the invention are set forth in diagram form in Figure 1(a). Double stranded cDNA may be prepared from mRNAs isolated from the microorganism in the manner more completely described in Examples 4 and 6. The cDNA is ligated with λgt-11 phage DNA suitable for use as a cloning plasmid. After in vitro packaging, the recombinant phage DNA is used to infect an E. coli strain devoid of detectable basal enzyme activity.

Plasmid DNA is prepared as more fully described in Examples 4 and 6.

A gene encoding the recombinant enzyme is identified from a cDNA library prepared from microorganism by DNA hybridization with polymerase chain reaction (PCR) generated probes using oligonucleotide primers that were based on the partial amino acid sequence previously determined for that microorganism's native enzyme. A subset of the clones, which hybridized positively with the PCR generated probes, should also test positive in the agar-overlay esterase/lipase activity assay as described in Higerd and Spizizen, J.Bacteriol.. 114:1184(1973), which is

incorporated by reference. A complete description of the cloning procedures and confirmation assays used can be found in the examples. From these positive clones, the gene is subcloned into a vector,

preferably pGEM-13Zf(+) (Promega Corp., Madison, WI). The resulting fusion protein (Seq. I.D. No. 1 and 4) is expressed in high levels in an overnight fermentation of E. coli.

The correct identity of a recombinant ester hydrolase gene can be confirmed by determining the DNA sequence of the insert and comparing its inferred amino acid sequence with that previously partially determined for the purified enzyme. In addition, the cloned enzyme, upon comparison with the natural enzyme isolated from the microorganism, should show identical substrate preference for the S- versus the R-naproxen ester.

When using a lac promoter, the expression of the ester hydrolase gene in E. coli is driven by the lac P-O promoter of the lac operon.

Addition of isopropylthio-galactoside (IPTG), which is known to induce the lac promoter, increases the enzyme expression, whereas addition of high levels of glucose, which is known to shut off lac expression via catabolic repression, shut off the production of the cloned enzyme. Moreover, insertion of four nucleotides at the junction between the lac Z and the ester hydrolase genes, which moves the ester hydrolase gene out of frame with respect to lac Z in the pGEM vector, effectively abolished the expression of a functional enzyme.

To further characterize the ester hydrolases of the invention, the complete DNA sequence of the gene encoding Zopfiella ATCC #26183: Strain 511 enzyme was determined (Seq. I.D. No. 2 ) , as was the DNA sequence of the gene encoding a more preferred embodiment, Zopfiella ATCC #44575:

Strain 780 enzyme (Seq. I.D. No. 5). Activity staining of a recombinant enzyme of this invention, preferably a Zopfiella recombinant enzyme, and other commercially available enzymes in a non-denaturing system verifies the enzyme activity of the recombinant enzyme while indicating that the enzymes of the invention are distinctly different from known commercially available enzymes.

Using A,S-naproxen esters, a subset of the positive recombinant clones from a microorganism selected from the group Absidia griseola, Aspergillus sydowii, Doratomyces stemonitis, Eupenicillium baarnenses, Graphium sp. , Heterocephalum aurantiacum, Pencillium roguefortii and Zopfiella latipes, preferably Zopfiella latipes, shows activity and evidences enantioselectivity. In particular, when the S- or R-enantiomer of naproxen ester is reacted with the recombinant enzymes, the recombinant enzymes show a strong preference for the S-naproxen ester. A detailed discussion of the experimental methods used to characterize the enzymes can be found in Examples 5 and 7. III. Enantioselectivity and Stability of the Zopfiella Enzymes

The enantioselectivity of a recombinant enzyme is confirmed by analyzing hydrolysis products of racemic mixtures of R,S-naproxen esters. In a preferred embodiment, the enantioselectivity of the recombinant enzyme from Zopfiella Strain 511 (the "rec 511") was confirmed by analyzing hydrolysis products of racemic mixtures of methyl, ethyl, and n-propyl naproxen esters. In a more preferred embodiment, the

enantioselectivity of the recombinant enzyme from Zopfiella Strain 780 (the "rec 780") was confirmed by analyzing hydrolysis products of racemic mixtures of methyl, ethyl, and n-propyl naproxen esters. Example 9, Table 2 shows that the enantioselectivity of the ester hydrolase enzyme isolated from the Zopfiella Strain 780 (the "780 enzyme"), the ester hydrolase enzyme isolated from the Zopfiella Strain 511 (the "511 enzyme"), the rec 511 enzyme, rec 780 enzyme and rec 780-m165r210 all yield an average ee of greater than 99%.

As shown in Example 9, Table 2, the enantiomeric excess ("ee") was an average of 99.3% for all three naproxen esters (not corrected for background levels of racemic acid), and was highest for ethyl ester, which had the lowest level of background naproxen acid.

The rec 511, rec 780 and rec 780-m165r210 enzymes compare favorably with the native enzymes on the basis of their ability to hydrolyze a broad range of naproxen esters with a high enantioselectivity.

The stability of the enzymes towards inactivation by S-naproxen, preferably KNPR, was also studied. The Zopfiella enzymes (both native and recombinant) have been found to be less sensitive towards S-naproxen inactivation than other untreated commercial enzymes. Strain 780 enzyme was shown to be more stable than Strain 511 enzyme towards KNPR

inactivation when incubated at about 45°C.

A non-ionic stabilizer, preferably bovine serum albumin ("BSA") or polyethylene glycol ("PEG"), more preferably PEG 8000, optionally can be added to the reaction mixture containing Zopfiella enzymes (both native and recombinant). Unlike the esterases, it was found that surfactants, such as Tween or soybean oil, did not stabilize the Zopfiella enzymes from inactivation by naproxen and formaldehyde treatment did not stabilize the Zopfiella enzymes from inactivation by naproxen.

Addition of a non-ionic stabilizer, preferably PEG or BSA, to a concentration of about 0.05% to about 2.0%, preferably to a concentration of about 0.2%, has a stabilizing effect, nearly doubling the half-life of enzymes denatured by S-naproxen. However, as can be seen from the data in Example 9, Table 3, the addition of a non-ionic stabilizer is not required for the Zopfiella ester hydrolase to enantioselectively hydrolyze

A,S-naproxen ester.

Mutagenesis experiments, as described herein, have been carried out with rec 780 enzyme in order to minimize the extent of inactivation by S-naproxen. Inactivation by S-naproxen is also a highly temperature-dependent process. Mutagenesis experiments, as described herein, have also been carried out for rec 780 enzyme in order to minimize the extent of thermal denaturation.

IV. Enhancement of recombinant enzyme characteristics

In order to improve the performance of the hydrolysis reaction for R,S-naproxen ester, it was desirable to develop Zopfiella enzyme mutants having improved performance characteristics in terms of thermal stability and S-naproxen stability while maintaining a high rate of hydrolysis and chiral specificity. In particular, it was highly desirable to develop a thermally stable recombinant hydrolase that could perform the hydrolysis reaction at about 30° to about 65°C.

Chemical mutagenesis, for example, nitrous acid mutagenesis and hydroxylaraine mutagenesis, and other forms of mutagenesis can be employed, including site-directed mutagenesis (Smith, Ann. Rev. Genet, 19:423

(1985)) to enhance thermal stability (Matthews, Biochemistry, 26:6885 (1987)) and S-naproxen stability, the methods of which are known to those skilled in the art. These references are incorporated by reference.

Mutagenesis experiments were carried out on the ester hydrolase gene of this invention in an attempt to develop a more thermally stable, tolerant ester hydrolase. These experiments are described in Example 10.

In one practice this invention, ester hydrolase recovered from a mutant with high thermal stability ("rec 780-m10") was purified and sequenced to determine the genotype changes. At position 443 of rec 780 the threonine was changed to an isoleucine to give the rec 780-m10 enzyme (Seq. I.D. No. 8 and 9). This specific change gave high thermal tolerance of the rec 780-m10 enzyme as shown in Figure 4.

In another practice of this invention, ester hydrolase recovered from a mutant with improved stability to S-naproxen, ("rec 780-m165") was purified and sequenced to determine the genotype changes. At position 53 the threonine was changed to alanine, at position 72 the alanine was changed to threonine, at position 133 the lysine was changed to arginine, at position 330 the valine was changed to leucine and at position 400 the threonine was changed to isoleucine in the rec 780 enzyme (Seq. I.D. No. 11 and 12). Among these specific changes were those which gave the rec 780-m165 enzyme high tolerance to KNPR inactivation as shown in Table 4, Example 10.

Ester hydrolase recovered from a mutant (identified as rec 780-m165r210) with improved stability to KNPR was purified and sequenced to determine the genotype. At position 53 the threonine was changed to alanine, at position 72 the alanine was changed to threonine, at position 133 the lysine was changed to arginine, at position 210 the serine was changed to arginine, at position 330 the valine was changed to leucine and at position 400 the threonine was changed to isoleucine in the rec 780 enzyme (Seq. I.D. No. 14 and 15). Among these specific changes were those which gave the rec 780-m165r210 enzyme high resistance to KNPR

inactivation as shown in Table 4, Example 10.

V. Use of Recombinant Enzymes in the Production of S-Naproxen

A. Isolation of Enzyme

The recombinant enzymes of the invention are especially suitable for use in the high yield, low cost production of S-naproxen. The recombinant enzymes can be purified from an E. coli or a yeast culture using various standard protein purification techniques, for example, affinity, ion exchange, size exclusion or hydrophobic interaction chromatography.

Active enzyme recovered from such purification techniques can be

concentrated using ammonium sulphate, or alternatively, by lyophilization neat or in the presence of sucrose.

An exemplary preparation and purification scheme comprises 1) growing the transformed E. coli cells in LB broth and inducing with IPTG; 2) harvesting the culture by centrifugation; 3) resuspending the cell pellet in buffer followed by cell disruption; 4) centrifuging the cell lysate; and 5) purifying the soluble enzyme by passage of the cell lysate over a two-step chromatographic column.

In terms of enzyme stabilization, in addition to genetic

modifications (protein engineering), one can chemically modify the enzyme as well. These include modification of surface amino-groups by alkylation or acylation (Torchillin, Biochem. Biophys. Acta., 557:1,(1979)), intramolecular cross-linking (Torchillin, Biochem. Biophys. Acta, 522:277 (1977)), enzyme immobilization which involves a multitude of different approaches (Chibata, J. Mol. Catal . , 63 (Review Issue) (1986); Trevan:

Immobilized Enzymes: Introduction and Application in Biotechnology, John Wiley, Chichester, UK, (1980)). In addition to those techniques which immobilize the isolated enzyme, it is also possible to immobilize cells which contain the enzyme, thereby indirectly immobilizing the enzyme. Such techniques are well known in the art and are described, e.g. Wood, L.L. and Calton, G.J.,"A Novel Method of Immobilization and Its Use in Aspartic Acid Production," Biotechnology, 12:1081 (1984). These

references are incorporated by reference.

B. Production of S-Naproxen

Employment of an ester hydrolase (as purified enzyme from the microorganism or as a recombinant enzyme), preferably the Zopfiella enzyme, in the production of S-naproxen can be carried out in many formats. For example, in one practice of the invention the enzyme can be added into a continuous stirred tank reactor. Likewise, it can be immobilized onto matrixes either as immobilized enzyme or as host cells containing the enzyme.

In a preferred practice of the invention, the enzyme is immobilized on a solid support. Preferably the immobilization is carried out by glutaraldehyde binding of the recombinant enzyme to an inert substance, such as silica or the like. Preferably the inert substance is Manville Celite^® R-648, R-649 or R-685. Figure 5 shows a schematic of the immobilization process and a description of the immobilization procedure for the isolated enzyme is set forth in Examples 11 and 12. The items identified in Figure 5 are as follows:

1. Immobilized Enzyme Packed Bed Reactor

2. Circulating water bath for reactor heating media

3. Flowmeter to measure circulation through reactor

4. Back pressure regulator to control reactor inlet pressure

5. Feed reservoir for water, A,S naproxen ester and organic

solvent

6. Base feed to control pH

7. A,S-naproxen ester to reactor

8. Excess substrate returned to feed reservoir from back pressure regulator

9. Heating media into reactor jacket

10. Heating media return from reactor jacket

11. Substrate/solvent/water feed to reactor

12. Substrate/solvent/water effluent from reactor

In another practice of the invention, host cells, such as E. coli, preferably E. coli Strain JM109 or Strain BL₂₁DE₃, that express the recombinant ester hydrolase gene are immobilized without isolating the enzyme. The use of whole cells is less expensive and time-consuming than the use of isolated enzyme. The rate of hydrolysis may be stimulated by a biphasic system having organic solvents at about 5% - about 40%(v/v), preferably about 20% to about 25% (v/v). Preferably hexane or toluene is used. DMSO may also be used in a monophase system. Example 13 sets forth a description of the intact cell immobilization procedure. Example 13, Table 9 shows the activity of intact E. coli carrying rec 780-m165r210 immobilized with Polymer 1195 and Polyazetidine.

A reactor configuration for using immobilized enzyme to hydrolyze R,S-naproxen esters to S-naproxen in an organic/aqueous solution is in theory, relatively easy to operate. In practice, however, the

hydrodynamics of the packed bed reactor require careful control. Focus of attention is on the reactor itself with regards to R,S-naproxen ester concentrations and relative amounts of organic and aqueous phases.

Preferably the R,S-naproxen ester concentration is approximately 100-500 g/1 in the organic phase and the relative amounts of organic and aqueous phase are approximately 3:1.

In a high yield, low cost production, A,S-naproxen ester, preferably a lower alkyl ester, more preferably ethyl or n-propyl naproxen ester, is introduced continuously into the reactor as a slurry, preferably 50-250 gm per liter. Optionally, a non-ionic surfactant, preferably PEG, may be added at a concentration of about 0.05% to about 2.0%. The actual residence time of the enzyme in the enzyme reactor will depend on the substrate infusion rate, the removal rate of the final product and the reaction volume. Preferably the residence time is 12-36 hours. The enzymatic hydrolysis can be conducted in a continuous or batch mode. The reaction is generally carried out at the temperature range between about 30°C and about 65°C, preferably between about 40°C and about 55°C. When using recombinant enzymes, preferably rec 780, more preferably rec 780-m165r210, in the reaction protocol, the incubation temperature should be between about 40°C and about 55°C.

A feed reservoir contains water as the aqueous phase and R,S-naproxen ester dissolved in an organic solvent, preferably in an aliphatic solvent. Preferably the solvent should have a normal boiling point equal to or greater than water, such as heptane, octane, decane, and dodecane. The preferred solvent is heptane. This biphasic mixture is agitated to keep the phases well mixed.

The biphasic mixture is fed to the hydrolysis reactor where the S-naproxen ester in the organic phase is hydrolyzed to s-naproxen. The S-naproxen then transfers to aqueous phase and both phases return to the feed reservoir. Because naproxen is acidic, a base, preferably an alkali metal salt, more preferably potassium hydroxide, is added to the feed reservoir to maintain a constant pH of 6-10, preferably 8.0-9.5.

In a continuous process, water. R,S-naproxen ester, and organic solvent can be continuously added to the feed reservoir while a portion of the reactor effluent is withdrawn from the system and sent to product recovery, wherein a phase separation technique is used to separate the S- naproxen (KNPR) from any R-naproxen ester and unhydrolyzed S-naproxen ester. The use of a lower alkyl naproxen ester is preferable.

The use of ethyl or n-propyl naproxen ester is more preferable in the hydrolysis reaction of the invention. The use of the ethyl ester results in the highest enantioselectively as shown in Example 9, Table 2 and the n-propyl ester is an oil at low temperatures, allowing greater freedom in the design of a hydrolysis bioreactor. Ethylene glycol based esters, such as the ethoxyethyl ester, can also be used, as can other esters previously described in the specification. S-naproxen, the product of the ester hydrolysis, is preferably removed from the process stream by passing through a series of filtration membranes that have different and specific molecular weight cut-offs.

This avoids the entry of either the unreacted naproxen ester substrate or the recombinant enzyme into the final product. The final product can then be further purified by crystallization. Potential impurities (such as, naproxen esters, ester hydrolase, proteins, DNA associated with production of the ester hydrolase, etc.) from the process can be monitored using analytical methods that have been modified and validated to demonstrate selectivity for such impurities. Stringent standards for acceptable levels of impurity are established and maintained.

The unreacted R-naproxen ester, as well as any residual S-naproxen ester, can be recycled through a separate reactor in which both are racemized chemically. The resultant 50-50 racemic mixture of naproxen ester, as well as fresh R,S-naproxen ester, can again be introduced into the bioreactors and the processing cycle repeated.

EXAMPLES

The examples which follow are illustrative and not limiting of the invention. Enzymes used in cloning experiments were obtained from commercial sources and were used substantially in accordance with the manufacturer's instructions. Except where otherwise indicated,

procedures such as DNA preparation, cleavage with restriction enzymes, ligation and transformation, were carried out essentially as described by Maniatis, et al . , supra, which is incorporated by reference. EIAMPLE 1(A)

Screening of Zopfiella

The dehydrated Zopfiella(ATCC# 26183) microorganism, purchased from ATCC, was rehydrated and plated out on medium 325 to assess growth and purity of the culture by visual inspection. The medium is described in R. Cote, ATCC Media Handbook, First Edition, 1984, which is incorporated by reference. After passing the visual purity test, Zopfiella was then transferred to slants of medium 340 for initial growth. Several media were also screened at this point in an attempt to define the optimum liquid medium in which to conduct the enzymatic assay. The preferred medium was determined by the slant with the beet growth and was used for the liquid culture and assay. Following identification of the preferred medium 200 for liquid culture and assay, a 5% (v/v) inoculum was grown in medium 200 for 24 hours. A qualitative assessment of biomass by turbidity was used to keep the same biomass concentration in a culture volume of 25 ml for all microorganisms grown.

A 5% (v/v) inoculum was then added to 25 ml of medium 200 for the assay. 25 μl of a suspension of 2.5g racemic naproxen ethyl ester in 10 ml sterile soybean oil was added to the medium to a final concentration of 0.25 mg/ml. This mixture was then agitated at 150 r.p.m. at approximately 25ºC for 48 hours.

Aliquots of 2 ml were removed at 24 hour intervale from duplicate cultures of Zopfiella. The aliquot of the culture was retained in a sterile 15 ml centrifuge tube, acidified to pH 1 with 0.1 M hydrochloric acid and frozen.

The contents were later thawed and the tubes processed. Processing consisted of extraction into ethyl acetate, centrifugation, sampling of the ethyl acetate layer, evaporation, derivatization with (S)-α-methylbenzylamine to form the diastereomeric amides and dissolution in a mixture of 80% acetonitrile and 20% water for liquid chromatography. The sample containing 10 μg/ml was then assessed for KNPR concentration and enantioselectivity by HPLC analysis (Hypersil, 3 micron, 4.6 × 100 mm, C-18 or equivalent, UV at 235 nm, 0.2 aufsd). The S-naproxen amide (5-amide) peak was 35.2 unite and the R-amide was O units with an ee of >99%. Isolated standards were also run of R,S-naproxen, S-naproxen and medium 200. EXAMPLE 1(B)

Screening of Absidia griseola

The dehydrated Absidia griseola microorganism, purchased from ATCC, was rehydrated and plated out on medium 325 to assess growth and purity of the transported culture by visual inspection and for determination of the optimum culture medium using the procedure essentially as described in Example 1(a). For Absidia griseola the S-amide peak was 28.7 unite and the R-amide was 0.4 unite with an ee of 97%. Isolated standards were also run of R,S- naproxen, S-naproxen and medium 28. EXAMPLE 2(A)

Isolation and Purification of Zopfiella 511 enzyme

Enzyme from Zopfiella (ATCC #26183: Strain 511) was prepared from 3-day cultures by cell lysis in a bead beater, removal of cell wall debris by centrifugation, and concentration by ammonium sulfate precipitation (40-60% saturation). The pellet was redissolved in 10 ml of 20 mM

Trie HCl/1 mM EDTA pH 8 buffer, loaded on and eluted from a Sephacryl HR300 gel filtration column (Pharmacia, Piscataway, NJ) with 50 mM Tris HCl/1 mM EDTA pH 8.

Enzyme was then adsorbed to a 2.5 × 20 cm column of anion exchange Q-Sepharose-Fast Flow (Pharmacia, Piscataway, NJ) equilibrated in 20 mM Trie HCl/1 μM EDTA pH 8, and was eluted in a 0.35 - 0.5 M NaCl gradient. Following elution, hydrophobic interaction chromatography was used as the next purification step. Enzyme was adsorbed to a 1.6 × 20 cm column of Phenyl Sepharose (Pharmacia, Piscataway, NJ) equilibrated in 0.2 M NaCl/20 mM Tris HCl/1 mM EDTA pH 8, and was eluted with a 0-50% ethylene glycol gradient in 10 mM Tris HCl/1 mM EDTA pH 7.5 buffer. The activity peak was pooled and was concentrated 40-fold in a Centriprep 30 (Amicon ultrafiltration devices, 30 kD cut off).

SDS-PAGE analysis was conducted on the eluted fractions. 1-50 ng of the enzyme was applied to a 11-23% gradient gel. The eluted fractions from the purification steps were also analyzed on 10% acrylamide native gels. 10-50 ng of protein were applied to the gel. The gels were stained with Coomassie Blue and analyzed for enzyme activity with Fast Blue using β-naphthyl acetate as the substrate, as described in Higerd and Spizizen, J.Bacteriol., 114:1184(1973). which is incorporated by reference. The activity gels showed a single, active protein band that corresponded to the protein on the SDS-PAGE gels at a molecular weight of approximately 46.5 kD. The SDS-PAGE gels also showed that the enzyme fraction recovered after hydrophobic interaction chromatography with Phenyl Sepharose contained no other detectable proteins. Purified enzyme was used for determination of the internal amino acid sequence as described in Example 3.

EXAMPLE 2(B)

Isolation and Purification of Zopfiella 780 enzyme Enzyme from Zopfiella (ATCC #44575: Strain 780) was prepared from 2-day cultures using the procedure essentially as described in Example 2(A).

Activity gels showed a single, active protein band that corresponded to the protein on the SDS-PAGE gels at a molecular weight of approximately

46.5 kD. The SDS-PAGE gels also showed that the enzyme fraction recovered after hydrophobic interaction chromatography with Phenyl Sepharose contained no other detectable proteins.

EXAMPLE 3

Internal amino acid sequencing of purified Strain 511 enzyme

Following the isolation and purification of the ester hydrolase from Zopfiella Strain 511, amino acid sequence analysis was performed.

Identification of the N-terminal sequence and the internal sequences from four peptide fragments provided sufficient information for the design of oligonucleotide probes for screening a cDNA library prepared from the Strain 511 gene. Construction of the cDNA library was as described in Example 4. The internal amino acid sequence of the Strain 511 enzyme was determined by CNBr cleavage and the isolation of the nine resulting polypeptide fragments by reverse phase-HPLC. In particular, a purified preparation of the enzyme was electrophoresed on a SDS-polyacrylamide gel. The resolved protein band(s) were then electro-blotted onto an Immobulon filter (Millipore Corporation, Medford, MA) and the protein band of interest was cut out and subjected to the standard micro-sequencing technique as described in Matsudira, J. Biol . Chem. 262 :10035 (1987), which is incorporated by reference. The products were then analyzed on an automated gas-phase microsequentor (Applied Biosystem Inc., Foster City, CA) using the methods as described by Hunkapellier et al. , Meth. Enz. ,

91:399 (1983), which is incorporated by reference. The results provided a partial amino acid sequence (first 10 amino acids at the N-terminal sequence) as follows: D X P S G A G S I T T E I Q S A I X K X, which allowed for the cloning of the rec 511 ester hydrolase gene as described in Example 4. EXAMPLE 4

Cloning of Zopfiella rec 511 ester hydrolase enzyme

The cloning of the Zopfiella ester hydrolase enzyme was carried out as follows:

1. Mycelial growth and harvest

Zopfiella (ATCC #26183: Strain 511) was propagated in YM broth using sheared glass broken mycelia as seed to provide uniform growth. The mycelia were harvested by filtering through sterile gauze after 2-3 days of growth and prior to asci formation according to the methods of Davis and DeSerres, Methods of Enzymology, Vol.17a (1970) and Weigel et al . , J. of Bacteriol . , 170 (9 ):3187 (1988), which are incorporated by reference. 2. mRNA preparation

Zopfiella mycelia were pulverized under nitrogen by mortar and pestle. mRNA was prepared according to Chirgwin, Biochem. , 18: 5294

(1979), which is incorporated by reference. Frozen Zopfiella cells were resuspended in buffer containing 4 M guanidinium thiocyanate, 0.5% sodium N-laurylsarcosine, 25 mM sodium citrate and 0.1 mM β-mercaptoethanol. The suspension was Polytron (Brinkmann Instruments Inc., Westbury, N.Y.) treated twice at 30 seconds each. The lysate was repeatedly drawn into a hypodermic syringe fitted with a 18 gauge needle and then expelled into polypropylene tubes. This was repeated 10 times to shear the cellular DNA.

After 15 min at room temperature, the lysate was clarified by centrifugation (10 min at 7,000 rpm in a HS4 rotor). LiCl was added to the supernatant to give a final concentration of 3.6 M. After overnight incubation at 4°C, the mixture was centrifuged at 7,000 rpm/HS4 rotor for 1 hour. Freshly prepared 3 M LiCl/4 M urea was added to the RNA pellet and the suspension was re-centrifuged. The RNA pellet was resuspended in 10 mM Trie HCl/0.1 mM EDTA pH 7.4 buffer containing 0.1% SDS and

immediately extracted with hot phenol (65°C). RNA recovered from the aqueous phase was extracted with phenol and chloroform. The RNA was then precipitated with ethanol. PolyA RNA was selected from the bulk of cellular RNA by affinity chromatography on oligo(dT)-cellulose as described in Edmonds, Proc. Natl . Acad. Sci . USA, 68: 1336 (1971) and Aviv, Proc. Natl . Acad. Sci. USA, 69:1408 (1972), which are incorporated by reference.

3. cDNA synthesis (Figure 1)

A cDNA library was constructed using a Promega Riboclone Kit

(Madison, WI) and an Invitrogen Kit (San Diego, CA) . Ten μg of polyA(+) mRNA was mixed with 3 μg of Not I/oligo(dT)-primer. The mixture (50 μl) was heated to 70°C for 5 min before cooling slowly to room temperature (via water baths of intermediate temperatures). After 15 min at room temperature, the mixture was placed in ice. AMV reverse transcriptase (final cone. 22 U/μl) was added to the reaction mixture which contains 50 mM Trie HCl, pH 8.3, 75 mM KCl, 16 mM MgCl₂, 0.5 mM Spermidine, 10 mM DTT, 1 mM dNTP mix, 1 U/μl RNasin Ribonuclease inhibitor and 4 mM sodium pyrophosphate. After incubation at 42°C for 1 hour, the reaction was phenol/CHCl₃ extracted and the first strand synthesis product was ethanol precipitated. The second strand synthesis was carried out using an Invitrogen Kit

(San Diego, CA). Ethanol precipitated first strand synthetic product was resuspended in buffer containing 20 mM Tris HCl, pH 7.5, 5 mM MgCl₂, 10 mM NH₄SO₄, 100 mM KCl, 0.5 mM BSA, 0.2 mM NAD, 0.2 mM dNTP's. 18 Unite of RNaseH/JE. coli ligase (1:1) and 30 Units of E. coli DNA polymerase were added to the reaction and the mixture was first incubated at 15°C for 90 min, and subsequently at room temperature for an additional 30 min. The reaction was then heat denatured at 70°C for 10 min and set at room temperature for 2 min. After the addition of T₄ DNA polymerase (27

Units), the reaction was further incubated for 10 min at 37°C. The reaction was then extracted with phenol/CHCl₃.

4. Addition of Eco RI Adaptor

cDNA products were size-selected on agarose gels. DNA molecules greater than 0.8 Kb were electro-eluted and concentrated by ethanol precipitation. The cDNA molecules (2.5 μg) were resuspended in buffer containing 30 mM Tris HCl, pH 7.8, 10 mM MgCl₂, 10 mM DTT, 0.5 mM ATP and 100 μg/ml BSA. 10 pmoles of synthetic Eco RI adaptors and 7.5 Weiss unite of T₄ DNA ligase were added to the mixture and the reaction was incubated at 15°C overnight. Following heat inactivation, the reaction was adjusted to IX Not I buffer (NEB, Beverly, MA), 100 μg/ml BSA, and 5 μM ATP. After the addition of T₄ polynucleotide kinase (10 Unite) and Not I (30 Unite), the reaction was incubated at 37°C for 60 min followed by a second incubation with Not I (30 Units) for 60 min. DNA samples following phenol/CHCl₃ extractions were purified using Promega CE802 Spin columns (Madison, WI) and ethanol-precipitated.

5. Ligation to Lambda-gt11 arms

5, 15, 45 and 200 ng of the Eco RI/Not I adaptor-flanked double stranded cDNAs were mixed with 0.5 μg of lambda gtll Sfi/Not I arms (Promega lot SN402, Madison, WI) previously digested by Eco RI and Not I according to vendor specification. T₄ ligase (0.5 Weiss unit) was added to the reaction which contains 50 mM Tris HCl, pH 8.0, 7 mM MgCl₂, 1 mM DTT and 1 mM ATP. The ligation reaction was incubated overnight at 14°C. 6. In vitro phage packaging

In vitro phage packaging was carried out using a "Gigapack Gold Extract" according to the vendor's protocol (Stratagene, La Jolla, CA). E. coli LE392 infected with the in vitro packaged phages were plated onto NZY plates in NZY soft agar. Alternatively, E. coli Y1090 cells were used and plated onto LB plates in LB soft agar containing 1.2 mM IPTG and 0.07% X-gal. The packaging efficiency of recombinant phages was about 1-2 × 10⁶ pfu per μg of lambda arms. 7. Screening of recombinant library

A. Hybridization with PCR generated probes

Partial amino acid sequences were previously determined for the first 20 amino acids at the N-terminus and four internal cyanogen bromide cleaved fragments (order unknown) of the Zopfiella ester hydrolase (Example 3). Based on these sequences, degenerate oligonucleotide primers were synthesized. In Figure 2, Oligonucleotides 1 to 3 correspond to the sense strand of the mRNA that encodes the amino-terminal sequence. In Figure 2, Oligonucleotides 4 to 7 correspond to the antisense strand of the mRNA that encode the internal tryptic fragments. Primers 1, 2 and 3 were independently paired with 4, 5, 6 and 7 anti-sense primers to generate polymerase chain reaction (PCR) products using the Zopfiella 511 genomic DNA as template.

The PCR products were generated using an USB GeneAmp Kit (Perkin Elmer Cetus, through United States Biochemical, Cleveland, OH).

Oligonucleotide primers (10 pmoles each) were mixed with 250 ng of genomic DNA in buffer containing 10 mM Trie HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin and 250 μM dNTPs. 1.25 units of AmpliTaq DNA polymerase was added to the reaction. Temperatures for annealing were increased step-wise: 4 cycles at 37°C, 3 cycles at 43°C and 26 cycles at 50°C. All extensions were performed at 72°C for 3 min except for the last 50°C annealing cycle which was for 10 min. Between cycles, reactions were denatured at 94°C for 2.5 min in the very first cycle and 1 min in all subsequent cycles. The products were analyzed on 1.8% agarose gels.

Depending on the sets of primers used, discrete DNA bands with varied complexity were detected. The exception was with primer 5, which gave no PCR product when combined with primers 1, 2 or 3. When PCR products generated by one set of primers were probed with radioactively labelled PCR products generated by another set of primers, certain common fragments showed positive hybridization. Based on the intensity and simplicity of hybridization pattern, PCR products generated by primers 1 + 6 and 2 + 7 were regarded as most likely to be specific for the Zopfiella ester hydrolase gene. Four phage plaque lifts were made per plate using

Hybond-N membranes (Amersham Corp., Arlington Hts., Il.) Filters in duplicates were hybridized to the primer 1 + 6 and 2 + 7 generated probes. The generation of radioactively labelled PCR probes, conditions for hybridization (at 42ºC, in 50% formamide, 750 mM NaCl, 250 mM Tris HCl, pH 8, 5 mM EDTA and 0.01% NaPi, and 100 μg salmon sperm DNA), and washing (at 60°C in 0.5 X SSC, 0.1% SDS) were according to Strub and Walter, Proc. Natl. Acad. Sci. USA, 86:9747-9751 (1989), which is incorporated by reference. 179 plaques from a pool of 1.2 million showed positive hybridization to the two PCR generated probes.

B. In situ enzymatic assay

Concurrently, the phage plaques were induced with IPTG and assayed in situ for ester hydrolase activity. If the open reading frame of the ester hydrolase gene was in the same translational reading frame as the lac gene and without interruption by stop codons within the 5' untranslated region, a functional ester hydrolase would be produced, as evidenced by the development of purple color when the phage plaques were overlaid with soft agar containing β-naphthyl-acetate and fast blue BB salt (Sigma, St. Louis, MO) (Higerd and Spizizen, supra, (1973)).

Three ester hydrolase positive phages were identified among 5,000 plaques screened. It was subsequently shown that these three phages also hybridized positively to the radioactive PCR generated probes (1 + 6 and 2 + 7). The three phages, designated Zl-2, Z2-5 and Z3-2, were extensively purified and phage DNAs prepared.

8. Transfer of lambda DNA insert into plasmid vector pGEM-13Zf(+)

To facilitate DNA sequencing and expression work, the DNA inserts from the three ester hydrolase positive phages were excised using restriction enzyme Sfi I and Not I. The DNA inserts, which were about 1.55 kb in length, were transferred onto pGEM-13Zf(+) plasmid DNA

(Promega, Madison, WI) via the same restriction sites. The ligated mixture was transformed into E. coli JM109 (Messing, Gene, 19:269 (1982)). The resultant plasmids were subjected to DNA sequencing. Figure 3 shows the junction sequences between the Zopfiella cDNA and the plasmid vector. The cDNA inserts are in the same translational reading frame as the lac sequence. In addition, the 5' portion of the cDNA molecule encodes amino acids which correspond to those previously determined for the N terminus of the purified Zopfiella ester hydrolase. The complete DNA sequence for the Zopfiella ester hydrolase gene (clone 1-2) was subsequently determined using an Applied Biosystem DNA sequenator (Applied Biosystems, Inc., Foster City, CA). The complete DNA sequence as identified is set forth as Seq. I.D. No. 12.

The amino acid sequence inferred from the DNA sequence indicated that the rec 511 enzyme was 430 amino acids in length with predicted pi of 7.4. Except for peptide 5, amino acid sequences corresponding to the cyanogen bromide fragments could be located within the recombinant enzyme. EXAMPLE 5

Expression of the Zopfiella rec 511 enzyme in E. coli

E. coli cells harboring the pGEM-13Zf(+) /enzyme plasmids were propagated overnight in LB broth in the presence of 1 mM IPTG. The cells were harvested by centrifugation and disrupted by sonication. After centrifugation at 10,000 rpm for 30 min (JA20 rotor), the supernatants were assayed for enzyme activity. Enzyme activity was measured using the S-enantiomer of p-nitrophenyl naproxen ester (5-PEN). Hydrolysis of S-PEN was carried out at 37°C in a 1 ml reaction consisting of 50 mM NaMOPS pH 7.5, 50 μg BSA, 1-5 μg extract and 20 μl of 100 mM S-PEN in DMSO. The reaction was terminated when visible yellow color appeared (approximately 20 min) by placing the reaction in a dry ice bath. The reaction was thawed and centrifuged at

15k x g for 5 min (4°C). Next the supernatant was passed through a 0.2 μ filter and the absorbance at 410 nm was determined. Among the three clones, clone (1-2) had the highest enzyme activity. When the protein extracts were analyzed by SDS-PAGE, clone (1-2) also showed a prominent protein band at about 46 kD. When compared with untransformed E. coli host, the rec 511 enzyme has a major protein band of approximately 46.5 kD which is slightly larger than the naturally occurring Strain 511 enzyme. Also, there is a major protein band in the native (non-reduced) gels of rec 511 enzyme that corresponds to the migration pattern of the activity stain. This indicates that major protein being expressed is not only the correct size for a rec 511 enzyme, but also that the protein being expressed has enzymatic activity. The migration rate of the recombinant protein is slightly slower in native gels than the authentic fungal derived 511 enzyme.

Upon further purification as described in Example 2(a), the 46.5 kD protein was subsequently shown to have good enzyme activity. Moreover, it preferentially hydrolyzed S-naproxen esters as described in Example 9.

EXAMPLE 6

Cloning of the Zopfiella rec 780 ester hydrolase enzyme A cDNA library was constructed using mRNA isolated from Zopfiella

780 using methodologies identical to that described in Example 4. The cDNA library was screened using a radioactively labelled rec 511 DNA as hybridization probe and approximately 30 (out of half a million plaques screened) positive plaques were identified. 10% of these plaques were also positive for ester hydrolase activity as evidenced by their

exhibiting purple color in the soft agar-overlay assay. The DNA inserts (Sfi I to Not I) from the positive phages were transferred onto the pGEM plasmid vector as described in Example 4. EXAMPLE 7

Expression of the Zopfiella 780 enzyme in E. coli

Enzyme activity was assayed using methodologies identical to that described in Example 5. When analyzed by SDS-PAGE, the purified Strain 780 enzyme, like the Strain 511 enzyme had a major protein band that migrated at a rate consistent with a 46.5 kD size protein. The two activity bands associated with the purest preparation migrated with an R_f number of 0.67 and 0.56. Given the extreme differences in specific activity between the two protein bands, the slower migrating protein (R_f = 0.56) is believed to be either a breakdown or deaminated product of the 46.5 kD protein. Alternatively, it can be another unrelated nonspecific enzyme. EXAMPLE 8

Expression of Zopfiella ester hydrolase in yeast

1. Construction of Expression Plasmid

Yeast shuttle plasmid pSRF137 was constructed to allow galactoseinducible expression of the Zopfiella 511 enzyme in Saccharomyces cerevisiae. Figure 1(b) sets forth a diagram of the expression plasmid construction. cDNA from clone 1-2 (Figure 3) was first subcloned into the Sma I site of pUC18, creating pSRF115, by digesting with Eco RI and Not I, and treating with the Klenow fragment of DNA polymerase. The cDNA was excised from pSRF114 as a Bam HI-Asp718I fragment and inserted between the BAM HI and Asp718I sites of pSEY303 to create pSRF16, as described by Emr, Douglas, J. Cell Biol. , 102:523 (1986), which in incorporated by

reference. pYRF102 is a 2μ-based shuttle plasmid that contains LEU2 and URA3 selectable markers, the GAL4 gene, and the GAL1 regulatory region promoter with a unique Bam HI site about 65bp distal to the transcription initiation site as described in U.S. Patent No. 4,661,454, which is incorporated by reference. The Bam HI-SnaB I CDNA-SUC2 fragment from pSRF16 was inserted at the Bam HI site of pYRF102, to create pSRF137. 2. Growth, Induction of Activity and Preparation of Extracts

Yeast cells (DA2102) Barnes, D.A. and J. Thorner, Mol. Cell. Biol . 6:2828 (1986) were grown and plasmid pSRF137 selection was maintained in media lacking uracil (0.67% Yeast Nitrogen Base without amino acids (Difco), 0.5% vitamin-assay Casamino acids (Difco), 50 μg/ml adenine sulfate, 40 μg/ml histidine hydrochloride, and 25 μg/ml tryptophan).

Non-inducing media was supplemented with 2% glucose whereas inducing media was supplemented with 2% galactose plus 0.1% glucose. Cells were grown to mid-logarithmic phase (klett=25-125, a klett reading of 100 represents approximately 4 × 10⁷ cells/ml) by shaking at 30°C in non-inducing media. Cells were pelleted at 22°C-30°C and resuspended in inducing media

(klett=20-100) and shaken at 30° for 24 to 48 hours. Cells were pelleted at 4°C, resuspended in ice cold 50 mM NaMOPS pH 7.5 (1000-6000 klett unite/ml, klett unit - klett reading × culture vol. in ml) and repelleted. Extracts were either prepared immediately or the cell pellets were stored at -80°C.

Extracts were prepared by disrupting cells with glass beads, as modified from a previously described procedure in Asubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, ed., Current Protocols in Molecular Biology. , New York: Greene publishing and Wiley-Interscience, 1991, which is incorporated by reference. Cell pellets were resuspended in ice cold MOPS buffer (1 ml for each 10,000-12,000 klett unite) and 0.25 to 0.6 ml aliquots were placed in 1.5 ml microfuge tubes and 1 μl antifoam A. (Sigma, St. Louis, MO) was added.

Next a line was drawn on these tubes to indicate the volume occupied by the cell suspension and glass beads were added until they reached this line. The tubes were then vortexed for 12 min alternating 20 sec of vortexing with 20 sec on ice. Tubes were then centrifuged for 1 min at 10 k × g (4°C). The supernatant was removed and assayed for protein and enzyme activity.

3. Assays of Enzyme Expression

The protein concentration of the extracts was determined either by a Bradford Bio-Rad Protein Assay (BioRad Laboratories, Richmond, CA) or Pierce BCA Protein Assay Kit (Pierce Chemical Co., Rockford, IL) assay using BSA as a standard. Enzyme activity was measured using either the S-enantiomer of p-nitrophenyl naproxen ester (S-PEN) or racemic naproxen ethyl ester.

Non-denaturing gels contained 12.5% acrylamide (acrylamide:bis is 30:0.8) and 370 mM Trie HCl pH 8.8 in the running buffer and 4%

acrylamide, 125 mM Tris HCl pH 6.8 in the stacking buffer. Running buffere contained 37.7 mM Trie HCl, 40 mM glycine pH 8.9 in the top reservoir; and 62.5 mM Trie HCl pH 7.5 in the bottom reservoir. Gels were stained for lipase activity using a β-naphthyl acetatefast blue assay. The gels were incubated for 15 min at room temperature in 100 ml of NaPi pH 7.4, 5 ml isopropanol, 0.4 mg/ml fast blue (Sigma F-0500), 0.03% β-naphthyl acetate (Sigma N-6875, 1.5 ml of a prepared/2% solution in acetone). Gels were subsequently destained in 7% acetic acid.

Proteine were eluted from non-denaturing gels using an in situ gel assay, "Elutrap" (Schleicher and Scheull, Woburn, MA, ) with 25 mM Tris HCl/192 mM glycine pH 8.3 (Jacobs and Clad, Anal. Biochem. 154:583

(1986)). Hydrolysis of S-PEN was carried out at 37°C in a 1 ml reaction consisting of 50 mM NaMOPS pH 7.5, 50 μg BSA (Bovine Fraction V, defatted and protease-free, Sigma #A-3294), 1-5 μg extract and 20 μl of 100 mM S-PEN in DMSO. The reaction was terminated when visible yellow color appeared (approximately 20 min) by placing the reaction in a dry ice bath. The reaction was thawed and centrifuged at 1500 × g for 5 min (4°C). Next the supernatant was passed through a 0.2 μ filter and the absorbance at 410 nm was determined.

Hydrolysis of ethyl ester was carried out at 37°C in a 1 ml reaction consisting of 50 mM NaPi pH 8.5, 50 μg BSA, 1-5 μg extract, and 20 μl of

100 mM ester in DMSO. The reaction was terminated at various times by the addition of 1 ml of CH₂Cl₂. The aqueous phase was then passed through a 0.2 μ filter. The filtrate was analyzed on a chiral HPLC column as described in Kern, J.R., J. Chromatography, 543 : 355 (1991), which is incorporated by reference.

4. Preparation of Antibodies and Immunoblot

Polyclonal antisera to the Zopfiella ester hydrolase were prepared by injecting rabbits with recombinant or native enzyme (initial injection, 0.1 mg subcutaneous in complete Freund's adjuvant; subsequent boosts, 0.5 mg IM in incomplete Freund's adjuvant).

5. Amino-Terminal Analysis

Enzyme expression was induced in strain DA2102 carrying the pSRF137 by growth in galactose for 26 hrs. Extracts were prepared from these cells as well as a control strain carrying pYRF102. These extracts were subjected to native gel electrophoresis and stained with an ester hydrolase specific stain. A single band was stained in the pSRF137 lane but not in the control lane. This band was eluted and subjected to amino-terminal sequencing. Sequence data indicated that this band contained the yeast-derived Zopfiella enzyme.

The eluted band was subjected to SDS-polyacrylamide gel

electrophoresis along with the crude extracts from the pSRF137 and control strains. The eluted band contained a single 43 kD species, the molecular weight predicted from the DNA sequence of the Zopfiella enzyme. Together with the amino-terminal sequencing, these data suggest that the yeast-derived enzyme is unmodified and has the expected carboxy terminus. Following SDS gel electrophoresis these samples were immunoblotted with anti-ester hydrolase antibodies. A protein of approximately 43 KD, present in both the eluted band and extracts from the pSRFl37-containing strain, but absent in the control strain, reacted with the antibodies. This is a further confirmation that the eluted band contained the yeast-derived enzyme. Also, the yeast-derived enzyme was shown to be slightly smaller than the recombinant enzyme produced in E. coli which is expressed with a 3 kilodalton fusion partner, as expression in yeast results in a full length authentic enzyme. EXAMPLE 9

Enantioselectivity of Zopfiella enzymes

to R,S-naproxen esters

Each enzyme was incubated with 5 mg/ml of solid R,S-EtNPR or MeNPR and 20% (v/v) of the liquid R,S-PrNPR at 42°C in 0.1 M Tris

HCl/0.2% PEG 8000 pH 8.0 overnight.

The hydrolysate was diluted 1:3 in 50 mM KH₂PO₄ and ultrafiltered through a 3000 MWCO membrane (Centricon microconcentrator, Amicon,

Beverly, MA). The filtrate was analyzed on a chiral HPLC column (Chiral AGP Column, ChromTech, Stockholm, Sweden) using procedures recommended by the manufacturer.

Strain 511 enzyme was harvested from a 3-day culture and prepared by 30%-60% ammonium sulfate fractionation, followed by DEAE and hydrophobic interaction chromatography as described in Example 2(A). Strain 780 enzyme was harvested from a 2-day culture and prepared by 30%-60% ammonium sulfate fractionation, followed by DEAE and hydrophobic interaction chromatography as described in Example 2(B).

Rec 511, rec 780 and rec 780-ml65r210 enzymes were obtained from an overnight E. coli fermentation in LB broth and were concentrated with a 30%-60% ammonium sulfate precipitation, followed by purification with DEAE and size exclusion HPLC.

The ee values given in Table 2 are not corrected for background levels of racemic acid. The levels of background acid are methyl ester»n-propyl ester>ethyl ester and may well account for the differences in ee's between these naproxen alkyl esters. As the results indicate, the Zopfiella enzymes studied showed an enantioselectivity of greater than 98%.

A comparison of the ability of Zopfiella Strain 511 ester hydrolase to hydrolyze ethyl and n-propyl naproxen ester was carried out. A 2.5 ml solution of heptane containing 2% ethyl A,S-naproxen ester was mixed with 2.5 ml of 0.1 M Tris HCl, pH 8.0. The reaction was started by adding 200 μl of Zopfiella Strain 511 unpurified ester hydrolase solution (16 mg dry weight/ml) to the appropriate reaction mixture.

In the case of n-propyl naproxen ester, 1 ml of ester was mixed with 4 ml of 0.1 M Tris-Hcl, pH 8.0 buffer, and the reactions were the same as described above. At the times indicated in Table 3, 125 μl samples were taken and mixed with 875 μl acetonitrile to quench the reaction. The results from this study are given in Table 3(a) and 3(b) below.

EXAMPLE 10

Mutagenesis of the 780 ester hydrolase gene

The 780 ester hydrolase gene was cloned into the vector pSELECT-1 (Promega) . The pSELECT-1 vector containing the 780 gene will be

hereinafter referred to as "pS780". pSELECT-1 DNA contains lac operon sequences and transformants show β-galactosidase activity. pS780 was transformed into E. coli JM109 cells. The cells were then infected with helper phage R408 (Promega) to generate single stranded DNA copies of pS780. The single stranded pS780 was packaged into phage, harvested, and isolated.

The isolated DNA was treated with 0.2 M nitrous acid for 15 minutes at room temperature. Following nitrous acid mutagenesis, the single stranded DNA was primer extended using a T7 primer and AMV reverse transcriptase in the presence of deoxynucleotides. The mutated 780 gene was excised by Hind III/Bam HI digestion and gel purified and then ligated into gel purified Hind III/Bam HI digested p-SELECT-1. The mutagenized single stranded DNA was then transformed into E. coli JM109.

Transformants generated were replica plated into LB + tetracycline (15 μg/ml) + IPTG (1 mM) medium. The replica platea were allowed to grow overnight at 37ºC.

The pSELECT-1 plasmid offered an easy way to determine the

effectiveness of the mutagenesis. Cells grown on solid medium in the presence of isopropylthiogalactoside (IPTG) and 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) formed easily recognizable blue colonies. If β-galactosidase was inactivated through mutation, the colonies were white. 1. Screening for thermostable mutants

Replica plates containing 200-700 colonies were heated at 55°C for several hours. The plates were then cooled to room temperature and overlaid with 0.5% agarose containing β-naphthyl acetate and the indicator fast blue. Ester hydrolase activity was indicated by the colonies turning a red-purple color. Colonies showing the most rapid color changes were restreaked onto the same medium. After outgrowth, the plates were replicated and the replates examined again for enzyme activity after heating at 55°C for up to 7 hours.

Enzyme inactivation kinetics of cells transformed with mutant DNA were compared with those of non-mutant pS780 transformed cells. Cell extracts (in 0.2% PEG 8000 and 0.1 M Trie HCl, pH 8.0) were incubated at 50°C and at various times, samples were taken and assayed for activity using the S-PEN assay. After 3 hours at 50°C, the mutant enzyme retained approximately 45% of its original activity, while the non-mutant enzyme had lost almost all activity. The DNA from the mutant enzyme rec 780-m10 was then purified and sequenced to determine genotype changes. The thermal stability of rec 780-m10 at 54°C was assessed. Equal amounts of enzyme activity of rec 780 and rec 780-m10 were added to tubes containing 1 mg/ml of S-PEN solubilized in DMSO in 0.1 M MOPS buffer.

Aliquots were removed at the time points indicated in the graph of

Figure 4. Enzyme activity was then stopped by acetonitrile on ice. The unhydrolyzed S-PEN was then pelleted by centrifugation at 3000 × g for 15 minutes. Enzyme activity was measured by accumulation of S-PEN, measured at an absorbance at 410 nm (Figure 4).

2. KNPR resistance of thermostable mutants

Resistance to KNPR inactivation of thermostable mutants was compared with that of non-mutants by incubating cell extracts at 45ºC in the presence of KNPR at 20 g/l and 33 g/l. At various times, samples were taken and assayed for enzyme activity by the S-PEN method. Ester hydrolase activity was more stable with the mutant extracts than with the non-mutant extracts when incubated with 20 g/l KNPR. At 33 g/l KNPR, the mutant extract was rapidly inactivated.

Thermostable mutants were subjected further to nitrous acid mutagenesis as described above. The mutated DNA was made double stranded, excised, and ligated into the appropriate vector. E. coli JM109 was transformed and about 200,000 transformants were obtained. Replicas were made onto LB plates supplemented with 15 μg/ml tetracycline and 0.5 mM IPTG. After grow out, the plates were incubated at 60°-65°C for various lengths of time and subsequently screened for enzyme activity using the β-naphthyl acetate overlay method. Mutants that exhibited strong

temperature stability were isolated and screened for stability in the presence of KNPR. A mutant was isolated that exhibited stability in the presence of 33 g/l KNPR at 45°C. Therefore, the second generation of mutants were much more KNPR resistant than the original pS780.

A third generation of KNPR resistant thermostable mutants were prepared using the above procedure. Third-generation mutants exhibited enzyme stability in the presence of 40 g/l KNPR at 40°C. Fourth- generation mutants, similarly prepared, exhibited enzyme stability in the presence of 60 g/l KNPR at 40°C.

E. coli JM109 containing rec 780, rec 780-ml65 and rec 780-ml65r210 were grown overnight in LB broth supplemented with IPTG (1 μm) and tetracycline (15 μg/ml). The cells were harvested, suspended in 1 ml of 0.1 M Trie HCl, pH 8.0, supplemented with 0.2% PEG 8000 and disrupted by vigorous agitation in the presence of glass beads. Cellular debris and glass beads were removed by centrifugation (10,000 × g for 10 min). Three tenths ml of extract was added to a KNPR stock solution of 50 g/l so that the final incubation mixture (1 ml) contained 50 μm Tris HCl, pH 8.0, 0.1% PEG 8000 and KNPR (50 g/l). The mixture was incubated at 40°C. At the time points indicated in Table 4, 10 ml aliquots were taken and assayed for activity using the PEN assay at 37°C. Enzyme activity was measured by accumulation of S-PEN measured at an absorbance at 405 nm as shown in Table 4. Activity at the start of the experiment was taken at 100%.

EXAMPLE 11

Immobilization of Enzyme

1. Production of ester hydrolase

Inoculum was started from frozen seed stocks of Zopfiella stored at -70°C in 20% glycerol. One vial was thawed and inoculated into the basal media containing 0.6% glucose (w/v), 5 g/l (NH₄)₂PO₂, 6 g/l Na₂HPO₄, 3 g/l KH₂PO₄, 1.1 g/l Na₂SO₄, 5 mg/1 thiamine, 500 mg/1 MgSO₄7H₂O, 100 mg/l ampicillin and 0.5 ml/l trace metal solution. The culture was incubated in a baffled flask on a rotary shaker at 37°C for 7-8 hours. The cells were then passaged into fresh medium containing 1% glucose and incubated 14-16 hours. The fermentor was inoculated with these cells at a

concentration of 1 part to 20 parts of minimal medium. Specifically, eight liters of basal medium are inoculated with 400 ml of the seed.

Dissolved oxygen is maintained at 20-40% through control of agitation speed and addition of supplemental oxygen. The pH is regulated at 6.9-7.0 by addition of 5N NH₄OH. Feed solution #1 of 400 g/l glucose, 10 g/1 MgSO₄.7H₂O and 100 mg/l thiamine is added at a rate to maintain the glucose concentration at 1-3 g/l. Feed solution #2 of 100 g/l (NH₄)₂HPO₄ is added when dissolved oxygen starts to increase and feed rate is adjusted to maintain a steady growth rate based on dissolved oxygen status. The E. coli culture was then induced (lac promoter of the plasmid is induced) with 1 mM IPTG when the cell density reached an absorbence of .20 at 550 nm. The feed streams were discontinued at this time and the culture was harvested five to six hours post induction. The cells were then concentrated by centrifugation. The cells can also be concentrated by cross filtration.

2. Purification

After cell disruption, the cell lysate, including insoluble cellular debris, was extracted in 17% (w/v) PEG 1550, 8% (w/v) sodium phosphate and 20% (weight wet cells prior to disruption/v) biomass. After mixing for 20 minutes, the mixture was centrifuged at 2000 rpm. Eighty percent of the enzyme partitions to the upper PEG rich phase. The PEG was removed from the enzyme utilizing ultrafiltration (30,000 molecular weight cutoff, Amicon spiral cartridge).

3. Preparation of Support

Preparation of the silica support (Manville Celite^® R-648) for binding of the Zopfiella enzyme was carried out by using the following procedures: a. Nitric Acid Treatment

To 100 g of Manville Celite^® R-648 was added 250 ml of 10% HNO₃. The flask was evacuated several times to ensure that the pores were liquid filled. The flask was then heated to 70°C and held at that temperature for one hour. The flask was then cooled to room temperature and washed extensively with deionized (DI) water. The acid washed Celite was then dried in a vacuum oven at 70°C. b. Treatment with Silane

To a flask was added 100 g of acid-washed Manville Celite^® R- 648. A solution of 3-aminopropyltriethoxysilane at a concentration of 10% v/v in DI water was prepared with the pH between 3 and 4. The pH was adjusted with 1.0 N HCl or 1.0 N KOH. To the flask was added 300 ml of 10% silane solution per gram of Celite. The flask was evacuated several times to ensure that the pores were liquid filled. The flask was heated to 70°C and held at that temperature for three hours. The acid washed-silanized Celite was cooled to room temperature, washed extensively with DI water and dried in a vacuum oven at 70°C. c. Grafting of glutaraldehyde to silanized Celite^®

A 10% solution of glutaraldehyde in DI water was prepared. To a flask containing 20 g of silanized Celite^® was added 100 ml

glutaraldehyde solution. The flask was evacuated several times to ensure that the pores were liquid filled. The flask then stood at room

temperature for six hours. The glutaraldehyde-grafted Celite was then washed extensively with DI water and dried in a vacuum oven at 70°C.

Alternatively the enzyme can be added to wet support after washing with DI water. d. Attachment of enzyme to glutaraldehyde-grafted Celite^®

To a flask was added 8.0 g of glutaraldehyde-grafted Celite^®

R-648 and 25.0 ml of enzyme solution. The enzyme solution contained 1.0 mg/ml protein in 50 mM Bicine buffer at a pH of 8.5. The flask was evacuated several times to ensure that the pores were liquid filled. The mixture stood overnight for 12-15 hours at room temperature.

The surface moisture was removed from the support by vacuum filtration. The mixture was then washed three times with 50 ml of 50 mM Bicine buffer. The wet support was then transferred to the hydrolysis bioreactor. 2. Evaluation of the Immobilized Enzyme

The first immobilization technique used was glutaraldehyde linking of the Zopfiella enzyme to a silica support. The support used was

Manville Celite^® R-648 comprised of spherical particles of -30+50 mesh with a surface area of 46 m²/g. The procedure for support preparation and enzyme attachment was as described above.

Two immobilization variables were investigated. The first was the glutaraldehyde concentration used for grafting to the silanized support. The second variable was the amount of protein that could be attached to the glutaraldehyde-grafted support. Based on the surface area of the support and an estimate of the size of an enzyme molecule, it was estimated that the maximum protein loading on the substrate, assuming monolayer formation, was in the range of 0.2 to 0.4 mg/m². The initial immobilization was performed with protein loading below and above this range. Table 5 summarizes the results of the immobilization experiments. A control was used in which the support was silanized, but not treated with glutaraldehyde prior to incubation with the enzyme. The enzyme used was the 40-60% ammonium sulfate fraction (Fraction II) recovered from the crude lysate.

It was concluded from these results that the maximum protein loading for the Manville Celite R-648 support is on the order of 0.6 mg/m².

Each of the immobilized enzyme samples prepared as described above was used in a hydrolysis experiment with n-propyl naproxen ester. A schematic flowsheet of the immobilized bioreactor system is shown in Figure 5.

The experiments were conducted by adding 8.0 g of the immobilized enzyme to 50 ml of 0.05 M KH₂PO₄ containing 500 ppm of PEG 8000 (Sigma, St. Louis, MO). After the aqueous phase was heated to 40°C and the pH adjusted to 8.5, 10.0 ml of heptane containing 1.63 g PrNPR was added. The reaction was allowed to proceed for 24 hours. The aqueous phase was sampled for S-naproxen analysis, including concentration and ee. Table 6 summarizes the results of the hydrolysis experiments with the immobilized enzyme preparations.

During the hydrolysis, the amount of 0.5 M KOH added to control pH was recorded. For comparison, two hydrolysis experiments were conducted using soluble enzyme at different concentrations (Example 12). The data generated in these experiments verified that the enzyme was immobilized with high retention of activity and selectivity. Experiments were also conducted to test the activity of immobilized enzyme recovered from batch hydrolysis experiments. Results showed that there was essentially no loss in activity through two hydrolysis cycles.

EXAMPLE 12

Use of the Zopfiella rec 511 enzyme in a bioreactor for production of S-naproxen As an alternative to using the immobilized system as described in Example 11, the reaction can be carried out using soluble enzyme.

Approximately 900 Unite of the recombinant enzyme, rec 511, in 0.1 M Trie HCl buffer, pH 8.0, was added in the initial reaction mixture containing 100 ml of water, 2.0 g PEG 8000, 25 ml of hexane and 6.0 g n-propyl ester of A,S-naproxen. The reaction mixture was maintained at room temperature and the pH maintained at 8.0 by the addition of 1.0 M KOH. After 28 hours of hydrolysis, the organic and aqueous phases of the reaction were separated in a separatory funnel. The KNPR content in the aqueous phase was measured by HPLC using a Hypersil C8 column (Alltech, Deerfield, Il). The optical purity of the KNPR was measured by chiral HPLC using a chiral AGP column(ChromTech, Stockholm, Sweden) . The unreacted ester was recovered from the hexane by evaporation and analyzed for optical purity using the same chiral HPLC column. The aqueous phase contained 13.2 g/l naproxen as the potassium salt with an enantiomeric excess of 99.0%. This represented an R,S-ester conversion of 35.0%. The unreacted n-propyl ester of naproxen contained 68.2% of the R-enantiomer and 31.8% of the S- enantiomer. EXAMPLE 13

Immobilization of intact cells

A. Immobilization Using Polyazetidine

E. coli cells carrying the rec 511 gene, and cells carrying the rec 780 gene were grown overnight in LB broth supplemented with 100 μg/ml ampicillin, harvested, and suspended in distilled water. The cells were permeabilized with 1% v/v toluene. The permeabilized cells were then mixed with an equal volume of polyazetidine. The pH was maintained around pH 8.0 by adding a email volume of 1.0 M NaOH. The mixture was then poured into a plastic container and a vacuum was pulled. After a short period of vigorous bubbling, the suspension solidified into a wafer. The wafer was ground into a powder using a coffee mill.

After immobilization and grinding, the cells were assayed for n-propyl naproxen ester hydrolysis at 35°C in the presence of 25% v/v hexane. This was done by adding varying amounts of immobilized cells to flasks containing 15 ml of 0.1 M Tris HCl buffer, pH 8.0 containing 0.2% PEG 8000 and 25% v/v hexane containing 100 mg of PrNPR. Samples of the aqueous phase were taken at various times and analyzed for naproxen concentration by HPLC using a Hypersil C8 column. The results of these hydrolysis experiments are shown in Table 7. The immobilized cells hydrolyzed n-propyl naproxen ester at rates dependent upon catalyst concentration.

Stability of the immobilized cells in KNPR was determined by adding 200 mg of immobilized cells to 13 ml of 1 mM Trie HCl buffer, pH 8.0 containing 0.2% PEG 8000 and 48.75 g/l KNPR, and 4.0 ml DMSO. After the enzyme was allowed to stand in this solution at room temperature for ten minutes, 2 ml of PrNPR was added to the flask and the pH monitored with time. The decrease in pH shown in Table 8 below indicates the formation of S-naproxen.

B. Immobilization Using Polymeric Flocculating Agents

E. coli JM109 carrying rec 780-ml65r210 was suspended to an optical density of 20 at 620 nm in 120 ml of 0.1 M Tris HCl containing 0.2% PEG 8000. 1.5 ml of polyazetidine (Hercules, Inc., Wilmington, DE) was added to the cell suspension while stirring. Then 1.5 ml of polymer 1195 (Betz, Trevose, PA) was added, while continuing to stir. The flocculated cells were then pelleted by low speed centrifugation and the resulting pellets were combined, pressed and dried overnight at 37°C. This material was then cut into thin strips, dried for an additional 24 hours at 37°C and cut into email pellets (approx. 1 mm).

The reaction mixture contained 100 mM of 3 mm Trie HCl, 0.2% PEG 8000, 15 ml of PrNPR and 1 g of the pellets. The reaction was carried out at 37°C and a pH of 7.9 was maintained by adding 20% KOH to the reaction mixture. The results of this study are presented in Table 9.

EXAMPLE 14

Use of the Soluble Zopfiella rec 780-m165r210 Enzyme in a Bioreactor for Production of S-Naproxen As an alternative to using the immobilized system as described in

Example 11, the reaction can be carried out using soluble enzyme. A reaction flask containing 300 ml of 50 mM potassium phosphate buffer and 30.0 g of the ethyl ester of A,S-naproxen was heated to 50°C. To this flask was added 7,800 Unite of the recombinant enzyme, rec 780-ml65r210, in 22.4 ml of 30 mM Trie HCl buffer. The reaction mixture was maintained at 50°C and the pH was maintained at 8.5 by the addition of 1.0 M KOH. After 24 hours of hydrolysis, the reaction slurry was separated by filtration. The KNPR content in the aqueous phase was measured by HPLC using a Hypersil C8 column (Alltech, Deerfield, IL) and the optical purity of the KNPR was measured by chiral HPLC using a chiral AGP column

(ChromTech, Stockholm, Sweden). The aqueous phase contained 30.1 g/l of S-naproxen as the potassium salt with an ee of 99.3%. This represented a conversion of 39.0%. The above description and examples serve to fully disclose the invention including preferred embodiments thereof. Modifications obvious to those of ordinary skill in molecular biology, protein chemistry, biochemical engineering and related sciences are intended to be within the scope of the following claims. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) INVENTORS: Beck, Steven R.

Cain, Robert O.

Chan, Hardy W.

Freedman, Richard

Heefner, Donald L.

Phalphs, Trish

Roberts, Christopher R.

Salazar, Falix H.

Snyder, Roger C.

(ii) TITLE OP INVENTION: Enzymatic Process for Production of

(S)-6-Msthoxy-α-Msthyl-2-Naphthalsnsacstic Acid

(iii) PRIORITY APPLICATION DATA:

(A) PRIORITY APPLICATION NUMBER: United States Serial No. 07/883,658

(B) FILING DATE: May 15, 1992

(iv) NUMBER OF SEQUENCES: 15

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatantIn Release #1.0, Version #1.25

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1552 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 511

(C) INDIVIDUAL ISOLATE: ATCC # 26183

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-gt11

(B) CLONE: 21511 1-2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATGACCATGA TTACGCCAAG CTATTTAGGT GACACTATAG AATACTCAAG CTTGGGCCAA 60

GTCGGCCAGA ATTCCGTTGC TGTCGCCACC ATCAAAATGC CTCCACCATC CGGCGCCGGC 120

TCCATCACCA CCGAGATCCA ATCCGCCATC TCAAAAGGCA TCCTCAATGG CGCCATCCTC 180

CTCGCAACTG ACTCAACTTC CTCCTTCACT TTCTCCTCTG CCGTAGGCAC TCGCACTCTT 240

CTCTCAGGGG AAACCGTCCC CCAGGCCCTC GAAGACGTCC TGTACCTCGC CTCGGCGACC 300

AAGCTACTCG CCTCCATCGC AGCCCTGCAG GTCGTCGAAG ATGGGCTCCT AACCCTCACC 360

TCCGACCTGT CCTCCATCGT ACCGGAATTG ACCTCCAAGA AAGTCTTCAA AGGCTGGTCC 420

GACGCCACCT CCGATCCCCC GATCGCCATC CTGGAAGATC AAATCCCCGA AAACCAACCC 480

ATCACTCTCA AGTCCCTCCT TACCCATTCT TCCGGAATCA TCTACGATTT CTTCGACCCC 540

GCCGGCCTCG CCAAATGGAA CGCCAAGTTC AATCCCGTCG AGACTCTCCC CGACGGAAAA 600

TCGAAACCCC GCCCCGTCGA AAAAGCCTTT GCTTATCCTC TCGCCTTTCA GCCCAACACA 660

AGCTGGATGT ATGGTCCCTC GATCGACTGG GCGGGCTTGA TCGTGGAACG TCTCACGGGA 720

CGCACTCTAG GCGATCATAT CCGCGAGCGA ATCATCAAGG CCGTGGGCGG GAACCCTGTC 780

GACGCGGAGT TTTACCCGCC CAAGAATGAA GACGTCCGGA AGAGACTGAT TGACTTGCAC 840

CCTGACGACC CTCTCGCTAC AGGGAAGCAA GTTCTCGCTG GGGGCGGGAA TATGAACCTT 900

GTTGCTGATG GTGATTTCGG TGGACACGGG ATGTTCACCA CCGGCGAGAA TTACCTCAAG 960

GTGTTGAAGA GTTTGCTGGC TAATGATGGG AAACTCCTCA GCCCCGAGAT GGTCAACCTC 1020

ATGTTTGAAG ATCATCTCAC GGGGGGAGCT AAAAAGGGTC ACGAGGACGC GCTGAATGGG 1080

CCGGTGGGAT CATTCTTTGC CGTGGGGACT GATGAGTTTG GCATGAAGGT GGGTCATGGA 1140

CTGGGTGGGC TGGTCACGTT GGAGAGTGTC GAAGGGTGGT ATGGCAAGGG GACTATGAGT 1200

TGGGGCGGCG GGCATACATT GGTTTGGTTT ATCGATCGGG AGAATGACCT GTGTGGAATC 1260

TGTGCGTTGC AGGCGAAGTT GCCGGTTACG GAGACACAAA AGATTGCGGA TGTGAAGCAG 1320

TGCTTTAGGA GGGATATTTA TCGGGTTAGA GAGGCTTGGA AGGCTAGTGG GGGTGGGAAG 1380

GAGGAGTAAG TACGAGGATT TGGGGCTAAG GATGTTAGTA TATGGTTCTT TTTGTTATGG 1440

TGGATGATAA TAGAGATTTG AGAAAGGCGG GAAATAGGCG ATTCATTAGG CATTATTCAG 1500 ATACATTCCC CAAATCGAAC CAAGACGTTT TCCTTTAAAA AAAAAAAAAA AA 1552

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1290 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 511

(C) INDIVIDUAL ISOLATE: ATCC #26183

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA into lambda-gt11

(B) CLONE: Z1511 1-2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

ATGCCTCCAC CATCCGGCGC CGGCTCCATC ACCACCGAGA TCCAATCCGC CATCTCAAAA 60

GGCATCCTCA ATGGCGCCAT CCTCCTCGCA ACTGACTCAA CTTCCTCCTT CACTTTCTCC 120

TCTGCCGTAG GCACTCGCAC TCTTCTCTCA GGGGAAACCG TCCCCCAGGC CCTCGAAGAC 180

GTCCTGTACC TCGCCTCGGC GACCAAGCTA CTCGCCTCCA TCGCAGCCCT GCAGGTCGTC 240

GAAGATGGGC TCCTAACCCT CACCTCCGAC CTGTCCTCCA TCGTACCGGA ATTGACCTCC 300

AAGAAAGTCT TCAAAGGCTG GTCCGACGCC ACCTCCGATC CCCCGATCGC CATCCTGGAA 360

GATCAAATCC CCGAAAACCA ACCCATCACT CTCAAGTCCC TCCTTACCCA TTCTTCCGGA 420

ATCATCTACG ATTTCTTCGA CCCCGCCGGC CTCGCCAAAT GGAACGCCAA GTTCAATCCC 480

GTCGAGACTC TCCCCGACCG AAAATCGAAA CCCCGCCCCG TCGAAAAAGC CTTTGCTTAT 540

CCTCTCGCCT TTCAGCCCAA CACAAGCTGG ATGTATGGTC CCTCGATCGA CTGGGCGGGC 600

TTGATCGTGG AACGTCTCAC GGGACGCACT CTAGGCGATC ATATCCGCGA GCGAATCATC 660

AAGGCCGTGG GCGGGAACCC TGTCGACGCG GAGTTTTACC CGCCCAAGAA TGAAGACGTC 720

CGGAAGAGAC TGATTGACTT GCACCCTGAC GACCCTCTCG CTACAGGGAA GCAAGTTCTC 780

GCTGGGGGCG GGAATATGAA CCTTGTTGCT GATGGTGATT TCGGTGGACA CGGGATGTTC 840 ACCACCGGCG AGAATTACCT CAAGGTGTTG AAGAGTTTGC TGGCTAATGA TGGGAAACTC 900

CTCAGCCCCG AGATGGTCAA CCTCATGTTT GAAGATCATC TCACGGGGGG AGCTAAAAAG 960

GGTCACGAGG ACGCGCTGAA TGGGCCGGTG GGATCATTCT TTGCCGTGGG GACTGATGAG 1020

TTTGGCATGA AGGTGGGTCA TGGACTGGGT GGGCTGGTCA CGTTGGAGAG TGTCGAAGGG 1080

TGGTATGGCA AGGGGACTAT GAGTTGGGGC GGCGGGCATA CATTGGTTTG GTTTATCGAT 1140

CGGGAGAATG ACCTGTGTGG AATCTGTGCG TTGCAGGCGA AGTTGCCGGT TACGGAGACA 1200

CAAAAGATTG CGGATGTGAA GCAGTGCTTT AGGAGGGATA TTTATCGGGT TAGAGAGGCT 1260

TGGAAGGCTA GTGGGGGTGG GAAGGAGGAG 1290

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 430 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipea

(B) STRAIN: 511

(C) INDIVIDUAL ISOLATE: ATCC #26183

(vii) IMMEDIATE SOURCE:

(B) CLONE: ZL511 1-2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met Pro Pro Pro Ser Gly Ala Gly Ser Ile Thr Thr Glu Ile Gln Ser 1 5 10 15 Ala Ile Ser Lye Gly Ile Leu Asn Gly Ala Ile Leu Leu Ala Thr Asp

20 25 30

Ser Thr Ser Ser Phe Thr Phe Ser Ser Ala Val Gly Thr Arg Thr Leu

35 40 45

Leu Ser Gly Glu Thr Val Pro Gln Ala Leu Glu Asp val Leu Tyr Leu 50 55 60

Ala Ser Ala Thr Lys Leu Leu Ala Ser Ile Ala Ala Leu Gln Val Val

65 70 75 80

Glu Asp Gly Leu Leu Thr Leu Thr Ser Asp Leu Ser Ser Ile Val Pro

85 90 95

Glu Leu Thr Ser Lye Lye Val Phe Lye Gly Trp Ser Asp Ala Thr Ser

100 105 110

Asp Pro Pro Ile Ala Ila Leu Glu Asp Gln Ile Pro Glu Asn Gln Pro

115 120 125

Ile Thr Leu Lys Ser Leu Leu Thr His Ser Ser Gly Ile Ile Tyr Asp 130 135 140

Phe Phe Asp Pro Ala Gly Leu Ala Lys Trp Asn Ala Lys Phe Asn Pro 145 150 155 160

Val Glu Thr Leu Pro Asp Gly Lys Ser Lys Pro Arg Pro Val Glu Lys

165 170 175 Ala Phe Ala Tyr Pro Leu Ala Phe Gln Pro Asn Thr Ser Trp Met Tyr

180 185 190

Gly Pro Ser Ile Asp Trp Ala Gly Leu Ile Val Glu Arg Leu Thr Gly

195 200 205

Arg Thr Leu Gly Asp His Ile Arg Glu Arg Ile Ile Lys Ala Val Gly

210 215 220

Gly Asn Pro Val Asp Ala Glu Phe Tyr Pro Pro Lys Asn Glu Asp Val 225 230 235 240 Arg Lya Arg Leu Ile Asp Leu His Pro Asp Asp Pro Leu Ala Thr Gly

245 250 255

Lys Gln Val Leu Ala Gly Gly Gly Asn Met Asn Leu Val Ala Asp Gly

260 265 270

Asp Phe Gly Gly His Gly Met Phe Thr Thr Gly Glu Asn Tyr Leu Lys

275 280 285

Val Leu Lye Ser Leu Leu Ala Asn Asp Gly Lys Leu Leu Ser Pro Glu

290 295 300

Met Val Asn Leu Met Phe Glu Asp His Leu Thr Gly Gly Ala Lys Lys 305 310 315 320

Gly His Glu Asp Ala Leu Aen Gly Pro Val Gly Ser Phe Phe Ala Val

325 330 335

Gly Thr Asp Glu Phe Gly Met Lys Val Gly His Gly Leu Gly Gly Leu

340 345 350

Val Thr Leu Glu Ser Val Glu Gly Trp Tyr Gly Lys Gly Thr Met Ser

355 360 365

Trp Gly Gly Gly His Thr Leu Val Trp Phe Ile Asp Arg Glu Asn Asp 370 375 380

Leu Cys Gly Ile Cys Ala Leu Gln Ala Lya Leu Pro Val Thr Glu Thr 385 390 395 400 Gln Lys Ile Ala Asp Val Lys Gln Cys Phe Arg Arg Asp Ile Tyr Arg 405 410 415

Val Arg Glu Ala Trp Lys Ala Ser Gly Gly Gly Lys Glu Glu

420 425 430

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1584 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipea

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: CDNA in lambda-gt11

(B) CLONE: Zl780-3a

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

ATGACCATGA TTACGCCAAG CTATTTAGGT GACACTATAG AATACTCAAG CTTGGGCCAA 60

GTCGGCCAGA ATTCCGTTGC TGTCGAATCA TCACAGCATT CTTCAGTCAA ACCCCAAACC 120

ATCACTTCCA TCACCACCAT CAAGATGCCT CCACCGTCCG GCGCCGGCTC CATCACCATC 180

GAGATCCAAT CCGCTATCTC AAAAGGCGTC CTCAATGGTG CCATCCTCCT CGCCACTGAC 240

TCAACCTCCT CCTTCACTTT CTCCTCCGCC GCGGGCACTC GAACTCTTCT CTCAGGAGAA 300 ACCGTCCCTC AGGCCCTCGA CGACGTCCTC TACCTCGCCT CCGCCACCAA ACTCCTGGCC 360

TCCATCGCAG CCCTGCAAGT CGTCGAAGAT GGTCTTCTAA CCCTCACCTC CGACCTATCC 420 ATCATCGTCC CGGAATTGAC CTCCAAGAAA GTCTTCAAAG GCTGGTCCGA CGCCACCTCC 480

GATCCCCCGG TCGCCATCCT CGAAGACCAA TTCCCCGACA ACCAACCCAT CACTCTCAAG 540

TCCCTCCTGA CTCACTCCTC GGGAATGATC TACGATTTCT TCGACCCCGG CGGGCTCGTC 600

AAATGGAACG GCAAGTTCAA TCCTATCGAG ACTCTCCCCG ACGGGAAACC CAAGCCCCGC 660

CCCGTCGAAA AAGCCTTTGC TTATCCACTG GCTTTTCAGC CCAACACAAG CTGGATGTAT 720

GGTCCCTCAA TCGACTGGGC GGGCCTGATC GTGGAACGTC TCACGGGGCG CAGTCTAGGC 780

GATCATATCC GCGAGAGAAT CATCAAGGCC GTTGGCGGGA ACCCTGCCGA TGCGGAGTTT 840

TACCCGCCCA AGAATGAAGA CGTCCGGAAG AGACTGATTG ACTTGCACCC TGACGACCCT 900

CTCGCTACAG GGAAACAGGT ACTCGCGGGT GGCGGGAATA TGAACCTTGT TGCGGATGGT 960

GATTTCGGTG GACACGGGAT GTTCACCACC GGCGAGAATT ACCTCAAGGT GTTGAAGAGT 1020

TTGCTGGCTA ATGATGGGAA ACTCCTCAGC CCCGAGATGG TCAACCTCAT GTTTGAAGAT 1080

CATCTCACGG GGGGAGCTAA AAAGGGTCAC GAAGACGCGC TGAATGGGCC GGTGGGATCA 1140

TTCTTTCCCG TGGGGACTGA TGAGTTTGGC ATGAAGGTGG GTCATGGACT GGGTGGCCTG 1200

GTCACGTTGG AGAGTGTCGA AGGGTGGTAT GGCAAGGGGA CTATGAGTTG GGGCGGCGGG 1260

CATACATTGG TTTGGTTTAT CGATCGGGAG AATGACCTGT GTGGAATCTG TCCGTTGCAG 1320

GCGAAGTTGC CGGTTACGGA GACACAAAAG ATTGCGGATG TGAAGCAGTG CTTTAGGAGG 1380

GATATTTATC GGGTTAGAGA GGCTTGGAAG GCTAGTGGGG GTGGGAAGGA GGAGTAAGTA 1440

CGAGGATTTG GGGCTAGGGA TGTTATTATA TGGTTCTTTT TGATGTGATG AATAATAATG 1500

GAGATTGTAG AAGGCGGGAA GCAGGCGAGT TATTAGAATA GTTATTATTC AGATACATTC 1560

CCCACATTGA AAAAAAAAAA AAAA 1584

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1290 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN* 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-gt11

(B) CLONE: Z1780-3A

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

ATGCCTCCAC CGTCCGGCGC CGGCTCCATC ACCATCGAGA TCCAATCCGC TATCTCAAAA 60

GGCGTCCTCA ATGGTGCCAT CCTCCTCGCC ACTGACTCAA CCTCCTCCTT CACTTTCTCC 120

TCCGCCGCGG GCACTCGAAC TCTTCTCTCA GGAGAAACCG TCCCTCAGGC CCTCGACGAC 180

GTCCTCTACC TCGCCTCCGC CACCAAACTC CTGGCCTCCA TCGCAGCCCT GCAAGTCGTC 240

GAAGATGGTC TTCTAACCCT CACCTCCGAC CTATCCATCA TCGTCCCGGA ATTGACCTCC 300

AAGAAAGTCT TCAAAGGCTG GTCCGACGCC ACCTCCGATC CCCCGGTCGC CATCCTCGAA 360

GACCAATTCC CCGACAACCA ACCCATCACT CTCAAGTCCC TCCTGACTCA CTCCTCGGGA 420

ATGATCTACG ATTTCTTCGA CCCCGGCGGG CTCGTCAAAT GGAACGGCAA GTTCAATCCT 480

ATCGAGACTC TCCCCGACGG GAAACCCAAG CCCCGCCCCG TCGAAAAAGC CTTTGCTTAT 540

CCACTGGCTT TTCAGCCCAA CACAAGCTGG ATGTATGGTC CCTCAATCGA CTGGGCGGGC 600

CTGATCGTGG AACGTCTCAC GGGGCGCAGT CTAGGCGATC ATATCCGCGA GAGAATCATC 660

AAGGCCGTTG GCGGGAACCC TGCCGATGCG GAGTTTTACC CGCCCAAGAA TGAAGACGTC 720 CGGAAGAGAC TGATTGACTT GCACCCTGAC GACCCTCTCG CTACAGGGAA ACAGGTACTC 780

GCGGGTGGCG GGAATATGAA CCTTGTTGCG GATGGTGATT TCGGTGGACA CGGGATGTTC 840 ACCACCGGCG AGAATTACCT CAAGGTGTTG AAGAGTTTGC TGGCTAATGA TGGGAAACTC 900

CTCAGCCCCG AGATGGTCAA CCTCATGTTT GAAGATCATC TCACGGGGGG AGCTAAAAAG 960

GGTCACGAAG ACGCGCTGAA TGGGCCGGTG GGATCATTCT TTCCCGTGGG GACTGATGAG 1020

TTTGGCATGA AGGTGGGTCA TGGACTGGGT GGCCTGGTCA CGTTGGAGAG TGTCGAAGGG 1080

TGGTATGGCA AGGGGACTAT GAGTTGGGGC GGCGGGCATA CATTGGTTTG GTTTATCGAT 1140

CGGGAGAATG ACCTGTGTGG AATCTGTCCG TTGCAGGCGA AGTTGCCGGT TACGGAGACA 1200

CAAAAGATTG CGGATGTGAA GCAGTGCTTT AGGAGGGATA TTTATCGGGT TAGAGAGGCT 1260

TGGAAGGCTA GTGGGGGTGG GAAGGAGGAG 1290

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 430 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(B) CLONE: Z1780-3A

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Pro Pro Pro Ser Gly Ala Gly Ser Ile Thr Ile Glu Ile Gln Ser 1 5 10 15 Ala Ile Ser Lya Gly Val Leu Asn Gly Ala Ile Leu Leu Ala Thr Asp

20 25 30

Ser Thr Ser Ser Phe Thr Phe Ser Ser Ala Ala Gly Thr Arg Thr Leu

35 40 45

Leu Ser Gly Glu Thr Val Pro Gln Ala Leu Asp Asp Val Leu Tyr Leu 50 55 60

Ala Ser Ala Thr Lya Leu Leu Ala Ser Ile Ala Ala Leu Gln Val val 65 70 75 80

Glu Asp Gly Leu Leu Thr Leu Thr Ser Asp Leu Ser Ile Ile Val Pro

85 90 95

Glu Leu Thr Ser Lys Lys Val Phe Lys Gly Trp Ser Asp Ala Thr Ser

100 105 no

Asp Pro Pro Val Ala Ila Leu Glu Asp Gln Phe Pro Asp Asn Gln Pro

115 120 125

Ile Thr Leu Lys Ser Leu Leu Thr His Ser Ser Gly Met Ha Tyr Asp 130 135 140

Phe Phe Asp Pro Gly Gly Leu Val Lys Trp Asn Gly Lys Phe Asn Pro 145 150 155 160 Ile Glu Thr Leu Pro Asp Gly Lys Pro Lys Pro Arg Pro Val Glu Lys

165 170 175 Ala Phe Ala Tyr Pro Leu Ala Phe Gln Pro Asn Thr Ser Trp Met Tyr

180 185 190

Gly Pro Ser Ile Asp Trp Ala Gly Leu Ile Val Glu Arg Leu Thr Gly

195 200 205

Arg Ser Leu Gly Asp His Ile Arg Glu Arg Ile Ila Lya Ala Val Gly 210 215 220

Gly Asn Pro Ala Asp Ala Glu Phe Tyr Pro Pro Lya λan Glu Asp Val 225 230 235 240 Arg Lys Arg Leu Ile Asp Leu His Pro Asp Asp Pro Leu Ala Thr Gly

245 250 255

Lya Gln Val Leu Ala Gly Gly Gly Asn Met Asn Leu Val Ala Asp Gly

260 265 270

Asp Phe Gly Gly His Gly Met Phe Thr Thr Gly Glu Asn Tyr Leu Lys

275 280 285

Val Leu Lye Ser Leu Leu Ala Asn Asp Gly Lys Leu Leu Ser Pro Glu 290 295 300

Met Val Asn Leu Met Phe Glu Asp His Leu Thr Gly Gly Ala Lya Lys 305 310 315 320

Gly His Glu Asp Ala Leu Asn Gly Pro Val Gly Ser Phe Phe Pro Val

325 330 335

Gly Thr Asp Glu Phe Gly Met Lya Val Gly His Gly Leu Gly Gly Leu

340 345 350

Val Thr Leu Glu Ser Val Glu Gly Trp Tyr Gly Lys Gly Thr Mat Ser

355 360 365

Trp Gly Gly Gly His Thr Leu Val Trp Phe Ile Asp Arg Glu Asn Asp 370 375 380

Leu Cys Gly Ile Cys Pro Leu Gln Ala Lys Leu Pro Val Thr Glu Thr

385 390 395 400 Gln Lys Ile Ala Asp Val Lys Gln Cys Phe Arg Arg Asp Ile Tyr Arg 405 410 415

Val Arg Glu Ala Trp Lys Ala Ser Gly Gly Gly Lys Glu Glu

420 425 430

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1584 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-gt11

(B) CLONE: Z1780-B10

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ATGACCATGA TTACGCCAAG CTATTTAGGT GACACTATAG AATACTCAAG CTTGGGCCAA 60

GTCGGCCAGA ATTCCGTTGC TGTCGAATCA TCACAGCATT CTTCAGTCAA ACCCCAAACC 120 ATCACTTCCA TCACCACCAT CAAGATGCCT CCACCGTCCG GCGCCGGCTC CATCACCATC 180

GAGATCCAAT CCGCTATCTC AAAAGGCGTC CTCAATGGTG CCATCCTCCT CGCCACTGAC 240

TCAACCTCCT CCTTCACTTT CTCCTCCGCC GCGGGCACTC GAACTCTTCT CTCAGGAGAA 300 ACCGTCCCTC AGGCCCTCGA CGACGTCCTC TACCTCGCCT CCGCCACCAA ACTCCTGGCC 360

TCCATCGCAG CCCTGCAAGT CGTCGAAGAT GGTCTTCTAA CCCTCACCTC CGACCTATCC 420

ATCATCGTCC CGGAATTGAC CTCCAAGAAA GTCTTCAAAG GCTGGTCCGA CGCCACCTCC 480

GATCCCCCGG TCGCCATCCT CGAAGACCAA TTCCCCGACA ACCAACCCAT CACTCTCAAG 540

TCCCTCCTGA CTCACTCCTC GGGAATGATC TACGATTTCT TCGACCCCGG CGGGCTCGTC 600

AAATGGAACG GCAAGTTCAA TCCTATCGAG ACTCTCCCCG ACGGGAAACC CAAGCCCCGC 660

CCCGTCGAAA AAGCCTTTGC TTATCCACTG GCTTTTCAGC CCAACACAAG CTGGATGTAT 720 GGTCCCTCAA TCGACTGGGC GGGCCTGATC GTGGAACGTC TCACGGGGCG CAGTCTAGGC 780

GATCATATCC GCGAGAGAAT CATCAAGGCC GTTGGCGGGA ACCCTGCCGA TGCGGAGTTT 840

TACCCGCCCA AGAATGAAGA CGTCCGGAAG AGACTGATTG ACTTGCACCC TGACGACCCT 900

CTCGCTACAG GGAAACAGGT ACTCGCGGGT GGCGGGAATA TGAACCTTGT TGCGGATGGT 960

GATTTCGGTG GACACGGGAT GTTCACCACC GGCGAGAATT ACCTCAAGGT GTTGAAGAGT 1020

TTGCTGGCTA ATGATGGGAA ACTCCTCAGC CCCGAGATGG TCAACCTCAT GTTTGAAGAT 1080

CATCTCACGG GGGGAGCTAA AAAGGGTCAC GAAGACGCGC TGAATGGGCC GGTGGGATCA 1140

TTCTTTCCCG TGGGGACTGA TGAGTTTGGC ATGAAGGTGG GTCATGGACT GGGTGGCCTG 1200

GTCACGTTGG AGAGTGTCGA AGGGTGGTAT GGCAAGGGGA CTATGAGTTG GGGCGGCGGG 1260

CATACATTGG TTTGGTTTAT CGATCGGGAG AATGACCTGT GTGGAATCTG TCCGTTGCAG 1320

GCGAAGTTGC CGGTTACGGA GATACAAAAG ATTGCGGATG TGAAGCAGTG CTTTAGGAGG 1380

GATATTTATC GGGTTAGAGA GGCTTGGAAG GCTAGTGGGG GTGGGAAGGA GGAGTAAGTA 1440

CGAGGATTTG GGGCTAGGGA TGTTATTATA TGGTTCTTTT TGATGTGATG AATAATAATG 1500

GAGATTGTAG AAGGCGGGAA GCAGGCGAGT TATTAGAATA GTTATTATTC AGATACATTC 1560

CCCACATTGA AAAAAAAAAA AAAA 1584

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1290 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipea

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-grt11

(B) CLONE: Z1780-910

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

ATGCCTCCAC CGTCCGGCGC CGGCTCCATC ACCATCGAGA TCCAATCCGC TATCTCAAAA 60

GGCGTCCTCA ATGGTGCCAT CCTCCTCGCC ACTGACTCAA CCTCCTCCTT CACTTTCTCC 120

TCCGCCGCGG GCACTCGAAC TCTTCTCTCA GGAGAAACCG TCCCTCAGGC CCTCGACGAC 180

GTCCTCTACC TCGCCTCCGC CACCAAACTC CTGGCCTCCA TCGCAGCCCT GCAAGTCGTC 240

GAAGATGGTC TTCTAACCCT CACCTCCGAC CTATCCATCA TCGTCCCGGA ATTGACCTCC 300

AAGAAAGTCT TCAAAGGCTG GTCCGACGCC ACCTCCGATC CCCCGGTCGC CATCCTCGAA 360

GACCAATTCC CCGACAACCA ACCCATCACT CTCAAGTCCC TCCTGACTCA CTCCTCGGGA 420

ATGATCTACG ATTTCTTCGA CCCCGGCGGG CTCGTCAAAT GGAACGGCAA GTTCAATCCT 480

ATCGAGACTC TCCCCGACGG GAAACCCAAG CCCCGCCCCG TCGAAAAAGC CTTTGCTTAT 540

CCACTGGCTT TTCAGCCCAA CACAAGCTGG ATGTATGGTC CCTCAATCGA CTGGGCGGGC 600

CTGATCGTGG AACGTCTCAC GGGGCGCAGT CTAGGCGATC ATATCCGCGA GAGAATCATC 660

AAGGCCGTTG GCGGGAACCC TGCCGATGCG GAGTTTTACC CGCCCAAGAA TGAAGACGTC 720 CGGAAGAGAC TGATTGACTT GCACCCTGAC GACCCTCTCG CTACAGGGAA ACAGGTACTC 780 GCGGGTGGCG GGAATATGAA CCTTGTTGCG GATGGTGATT TCGGTGGACA CGGGATGTTC 840 ACCACCGGCG AGAATTACCT CAAGGTGTTG AAGAGTTTGC TGGCTAATGA TGGGAAACTC 900 CTCAGCCCCG AGATGGTCAA CCTCATGTTT GAAGATCATC TCACGGGGGG AGCTAAAAAG 960

GGTCACGAAG ACGCGCTGAA TGGGCCGGTG GGATCATTCT TTCCCGTGGG GACTGATGAG 1020

TTTGGCATGA AGGTGGGTCA TGGACTGGGT GGCCTGGTCA CGTTGGAGAG TGTCGAAGGG 1080

TGGTATGGCA AGGGGACTAT GAGTTGGGGC GGCGGGCATA CATTGGTTTG GTTTATCGAT 1140

CGGGAGAATG ACCTGTGTGG AATCTGTCCG TTGCAGGCGA AGTTGCCGGT TACGGAGATA 1200

CAAAAGATTG CGGATGTGAA GCAGTGCTTT AGGAGGGATA TTTATCGGGT TAGAGAGGCT 1260

TGGAAGGCTA GTGGGGGTGG GAAGGAGGAG 1290

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 430 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-gt11

(B) CLONE: zl780-m10

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Met Pro Pro Pro Ser Gly Ala Gly Ser Ile Thr Ile Glu Ile Gln Ser 1 5 10 15 Ala Ile Ser Lye Gly Val Leu Asn Gly Ala Ile Leu Leu Ala Thr Asp

20 25 30

Ser Thr Ser Ser Phe Thr Phe Ser Ser Ala Ala Gly Thr Arg Thr Leu

35 40 45

Leu Ser Gly Glu Thr Val Pro Gln Ala Leu Asp Asp Val Leu Tyr Leu 50 55 60

Ala Ser Ala Thr Lys Leu Leu Ala Ser Ile Ala Ala Leu Gln Val Val 65 70 75 80

Glu Asp Gly Leu Leu Thr Leu Thr Ser Asp Leu Ser Ile Ile Val Pro

85 90 95

Glu Leu Thr Ser Lya Lya Val Phe Lys Gly Trp Ser Asp Ala Thr ser

100 105 110

Asp Pro Pro Val Ala Ile Leu Glu Asp Gln Phe Pro Asp Asn Gln Pro

115 120 125 Ile Thr Leu Lys Ser Leu Leu Thr His Ser Ser Gly Met Ile Tyr Asp 130 135 140

Phe Phe Asp Pro Gly Gly Leu Val Lys Trp Asn Gly Lys Phe Asn Pro 145 150 155 160 Ile Glu Thr Leu Pro Asp Gly Lys Pro Lys Pro Arg Pro Val Glu Lya

165 170 175

Ala Phe Ala Tyr Pro Leu Ala Phe Gln Pro Asn Thr Ser Trp Met Tyr

180 185 190

Gly Pro Ser Ile Asp Trp Ala Gly Leu Ile Val Glu Arg Leu Thr Gly

195 200 205

Arg Ser Leu Gly Asp His Ile Arg Glu Arg Ile Ile Lys Ala Val Gly 210 215 220

Gly Asn Pro Ala Asp Ala Glu Phe Tyr Pro Pro Lys Asn Glu Asp Val 225 230 235 240

Arg Lya Arg Leu Ile Asp Leu His Pro Asp Asp Pro Leu Ala Thr Gly

245 250 255

Lys Gln Val Leu Ala Gly Gly Gly Asn Met Asn Leu Val Ala Asp Gly

260 265 270

Asp Pha Gly Gly His Gly Mat Phe Thr Thr Gly Glu Asn Tyr Leu Lys

275 280 285

Val Leu Lya Ser Leu Leu Ala Asn Asp Gly Lys Leu Leu Ser Pro Glu 290 295 300

Met Val Asn Leu Met Phe Glu Asp His Leu Thr Gly Gly Ala Lys Lys 305 310 315 320

Gly His Glu Asp Ala Leu Asn Gly Pro Val Gly Ser Phe Phe Pro Val

325 330 335

Gly Thr Asp Glu Phe Gly Met Lya Val Gly His Gly Leu Gly Gly Leu

340 345 350

Val Thr Leu Glu Ser Val Glu Gly Trp Tyr Gly Lys Gly Thr Mat Ser

355 360 365

Trp Gly Gly Gly His Thr Leu Val Trp Phe Ile Asp Arg Glu Asn Asp 370 375 380

Leu Cys Gly Ile Cys Pro Leu Gln Ala Lys Leu Pro Val Thr Glu Ile 385 390 395 400 Gln Lys Ile Ala Asp Val Lys Gln Cys Phe Arg Arg Asp Ile Tyr Arg 405 410 415

Val Arg Glu Ala Trp Lys Ala Ser Gly Gly Gly Lys Glu Glu

420 425 430

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1584 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-gt11

(B) CLONE: Zl780-m165

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

ATGACCATGA TTACGCCAAG CTATTTAGGT GACACTATAG AATACTCAAG CTTGGGCCAA 60

GTCGGCCAGA ATTCCGTTGC TGTCGAATCA TCACAGCATT CTTCAGTCAA ACCCCAAACC 120

ATCACTTCCA TCACCACCAT CAAGATGCCT CCACCGTCCG GCGCCGGCTC CATCACCATC 180

GAGATCCAAT CCGCTATCTC AAAAGGCGTC CTCAATGGTG CCATCCTCCT CGCCACTGAC 240

TCAACCTCCT CCTTCACTTT CTCCTCCGCC GCGGGCACTC GAACTCTTCT CTCAGGAGAA 300

GCCGTCCCTC AGGCCCTCGA CGACGTCCTC TACCTCGCCT CCGCCACCAA ACTCCTGACC 360

TCCATCGCAG CCCTGCAAGT CGTCGAAGAT GGTCTTCTAA CCCTCACCTC CGACCTATCC 420

ATCATCGTCC CGGAATTGAC CTCCAAGAAA GTCTTCAAAG GCTGGTCCGA CGCCACCTCC 480

GATCCCCCGG TCGCCATCCT TGAAGACCAA TTCCCCGACA ACCAACCCAT CACTCTCAGG 540

TCCCTCCTGA CTCACTCCTC GGGAATGATC TACGATTTCT TCGACCCCGG CGGGCTCGTC 600

AAATGGAACG GCAAGTTCAA TCCTATCGAG ACTCTCCCCG ACGGGAAACC CAAGCCCCGC 660

CCCGTCGAAA AAGCCTTTGC TTATCCACTA GCTTTTCAGC CCAACACAAG CTGGATGTAT 720 GGTCCCTCAA TCGACTGGGC GGGCCTGATC GTGGAACGTC TCACGGGGCG CAGTCTAGGC 780

GATCATATCC GCGAGAGAAT CATCAAGGCC GTTGGCGGGA ACCCTGCCGA TGCGGAGTTT 840

TACCCGCCCA AGAATGAAGA CGTCCGGAAG AGACTGATTG ACTTGCACCC TGACGACCCT 900

CTCGCTACAG GGAAACAGGT ACTCGCGGGT GGCGGGAATA TGAACCTTGT TGCGGATGGT 960

GATTTCGGTG GACACGGGAT GTTCACCACC GGCGAGAATT ACCTCAAGGT GTTGAAGAGT 1020

TTGCTGGCTA ATGATGGGAA ACTCCTCAGC CCCGAGATGG TCAACCTCAT GTTTGAAGAT 1080

CATCTCACGG GGGGAGCTAA AAAGGGTCAC GAAGACGCGC TGAATGGGCC GTTGGGATCA 1140

TTCTTTCCCG TGGGGACTGA TGAGTTTGGC ATGAAGGTGG GTCATGGACT GGGTGGCCTG 1200

GTCACGTTGG AGAGTGTCGA AGGGTGGTAT GGCAAGGGGA CTATGAGTTG GGGCGGCGGG 1260

CATACATTGG TTTGGTTTAT CGATCGGGAG AATGACCTGT GTGGAATCTG TCCGTTGCAG 1320

GCGAAGTTGC CGGTTACGGA GATACAAAAG ATTGCGGATG TGAAGCAGTG CTTTAGGAGG 1380

GATATTTATC GGGTTAGAGA GGCTTGGAAG GCTAGTGGGG GTGGGAAGGA GGAGTAAGTA 1440

CGAGGATTTG GGGCTAGGGA TGTTATTATA TGGTTCTTTT TGATGTGATG AATAATAATG 1500

GAGATTGTAG AAGGCGGGAA GCAGGCGAGT TATTAGAATA GTTATTATTC AGATACATTC 1560

CCCACATTGA AAAAAAAAAA AAAA 1584

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1290 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-gt11

(B) CLONE: Z1780-ml65

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

ATGCCTCCAC CGTCCGGCGC CGGCTCCATC ACCATCGAGA TCCAATCCGC TATCTCAAAA 60

GGCGTCCTCA ATGGTGCCAT CCTCCTCGCC ACTGACTCAA CCTCCTCCTT CACTTTCTCC 120

TCCGCCGCGG GCACTCGAAC TCTTCTCTCA GGAGAAGCCG TCCCTCAGGC CCTCGACGAC 180

GTCCTCTACC TCGCCTCCGC CACCAAACTC CTGACCTCCA TCGCAGCCCT GCAAGTCGTC 240

GAAGATGGTC TTCTAACCCT CACCTCCGAC CTATCCATCA TCGTCCCGGA ATTGACCTCC 300

AAGAAAGTCT TCAAAGGCTG GTCCGACGCC ACCTCCGATC CCCCGGTCGC CATCCTTGAA 360

GACCAATTCC CCGACAACCA ACCCATCACT CTCAGGTCCC TCCTGACTCA CTCCTCGGGA 420 ATGATCTACG ATTTCTTCGA CCCCGGCGGG CTCGTCAAAT GGAACGGCAA GTTCAATCCT 480

ATCGAGACTC TCCCCGACGG GAAACCCAAG CCCCGCCCCG TCGAAAAAGC CTTTGCTTAT 540

CCACTAGCTT TTCAGCCCAA CACAAGCTGG ATGTATGGTC CCTCAATCGA CTGGGCGGGC 600

CTGATCGTGG AACGTCTCAC GGGGCGCAGT CTAGGCGATC ATATCCGCGA GAGAATCATC 660

AAGGCCGTTG GCGGGAACCC TGCCGATGCG GAGTTTTACC CGCCCAAGAA TGAAGACGTC 720 CGGAAGAGAC TGATTGACTT GCACCCTGAC GACCCTCTCG CTACAGGGAA ACAGGTACTC 780

GCGGGTGGCG GGAATATGAA CCTTGTTGCG GATGGTGATT TCGGTGGACA CGGGATGTTC 840

ACCACCGGCG AGAATTACCT CAAGGTGTTG AAGAGTTTGC TGGCTAATGA TGGGAAACTC 900

CTCAGCCCCG AGATGGTCAA CCTCATGTTT GAAGATCATC TCACGGGGGG AGCTAAAAAG 960

GGTCACGAAG ACGCGCTGAA TGGGCCGTTG GGATCATTCT TTCCCGTGGG GACTGATGAG 1020

TTTGGCATGA AGGTGGGTCA TGGACTGGGT GGCCTGGTCA CGTTGGAGAG TGTCGAAGGG 1080

TGGTATGGCA AGGGGACTAT GAGTTGGGGC GGCGGGCATA CATTGGTTTG GTTTATCGAT 1140

CGGGAGAATG ACCTGTGTGG AATCTGTCCG TTGCAGGCGA AGTTGCCGGT TACGGAGATA 1200

CAAAAGATTG CGGATGTGAA GCAGTGCTTT AGGAGGGATA TTTATCGGGT TAGAGAGGCT 1260

TGGAAGGCTA GTGGGGGTGG GAAGGAGGAG 1290

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 430 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-gt11

(B) CLONE: zl780-ml65

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Pro Pro Pro Ser Gly Ala Gly Ser Ile Thr Ile Glu Ile Gin Ser 1 5 10 15

Ala Ile Ser Lys Gly Val Leu Asn Gly Ala Ile Leu Leu Ala Thr Asp

20 25 30

Ser Thr Ser Ser Phe Thr Phe Ser Ser Ala Ala Gly Thr Arg Thr Leu

35 40 45

Leu Ser Gly Glu Ala Val Pro Gln Ala Leu Asp Asp Val Leu Tyr Leu 50 55 60

Ala Ser Ala Thr Lys Leu Leu Thr Ser Ile Ala Ala Leu Gln Val Val 65 70 75 80

Glu Asp Gly Leu Leu Thr Leu Thr Ser Asp Leu Ser Ile Ile Val Pro

85 90 95

Glu Leu Thr Ser Lya Lya Val Phe Lys Gly Trp Ser Asp Ala Thr Ser

100 105 110

Asp Pro Pro Val Ala Ila Leu Glu Asp Gln Phe Pro Asp Asn Gln Pro

115 120 125 Ile Thr Leu Arg Ser Leu Leu Thr His Ser Ser Gly Met Ile Tyr Asp 130 135 140

Phe Phe Asp Pro Gly Gly Leu Val Lys Trp Asn Gly Lys Phe Asn Pro 145 150 155 160 Ile Glu Thr Leu Pro Asp Gly Lys Pro Lys Pro Arg Pro Val Glu Lys

165 170 175

Ala Phe Ala Tyr Pro Leu Ala Phe Gln Pro Asn Thr Ser Trp Met Tyr

180 185 190

Gly Pro Ser Ile Asp Trp Ala Gly Leu Ile Val Glu Arg Leu Thr Gly

195 200 205

Arg Ser Leu Gly Asp His Ile Arg Glu Arg Ile Ile Lys Ala Val Gly 210 215 220

Gly Asn Pro Ala Asp Ala Glu Phe Tyr Pro Pro Lys Asn Glu Asp Val 225 230 235 240

Arg Lys Arg Leu Ile Asp Leu His Pro Asp Asp Pro Leu Ala Thr Gly

245 250 255

Lys Gln Val Leu Ala Gly Gly Gly Asn Met Asn Leu Val Ala Asp Gly

260 265 270

Asp Pha Gly Gly His Gly Met Phe Thr Thr Gly Glu Asn Tyr Leu Lys

275 280 285

Val Leu Lys Ser Leu Leu Ala Asn Asp Gly Lys Leu Leu Ser Pro Glu 290 295 300

Met Val λan Leu Met Phe Glu Asp His Leu Thr Gly Gly Ala Lys Lys 305 310 315 320

Gly His Glu Asp Ala Leu Asn Gly Pro Leu Gly Ser Phe Phe Pro Val

325 330 335

Gly Thr Asp Glu Phe Gly Met Lys Val Gly His Gly Leu Gly Gly Leu

340 345 350

Val Thr Leu Glu Ser Val Glu Gly Trp Tyr Gly Lys Gly Thr Mat Ser

355 360 365

Trp Gly Gly Gly His Thr Leu Val Trp Phe Ile Asp Arg Glu Asn Asp

370 375 380

Leu Cys Gly Ile Cys Pro Leu Gln Ala Lys Leu Pro Val Thr Glu Ile 385 390 395 400 Gln Lya Ile Ala Asp Val Lys Gln Cys Phe Arg Arg Asp Ile Tyr Arg 405 410 415

Val Arg Glu Ala Trp Lys Ala Ser Gly Gly Gly Lys Glu Glu

420 425 430

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1584 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lamda-gt11

(B) CLONE: zl780-m210

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

ATGACCATGA TTACGCCAAG CTATTTAGGT GACACTATAG AATACTCAAG CTTGGGCCAA 60

GTCGGCCAGA ATTCCGTTGC TGTCGAATCA TCACAGCATT CTTCAGTCAA ACCCCAAACC 120

ATCACTTCCA TCACCACCAT CAAGATGCCT CCACCGTCCG GCGCCGGCTC CATCACCATC 180

GAGATCCAAT CCGCTATCTC AAAAGGCGTC CTCAATGGTG CCATCCTCCT CGCCACTGAC 240

TCAACCTCCT CCTTCACTTT CTCCTCCGCC GCGGGCACTC GAACTCTTCT CTCAGGAGAA 300

GCCGTCCCTC AGGCCCTCGA CGACGTCCTC TACCTCGCCT CCGCCACCAA ACTCCTGACC 360

TCCATCGCAG CCCTGCAAGT CGTCGAAGAT GGTCTTCTAA CCCTCACCTC CGACCTATCC 420

ATCATCGTCC CGGAATTGAC CTCCAAGAAA GTCTTCAAAG GCTGGTCCGA CGCCACCTCC 480

GATCCCCCGG TCGCCATCCT TGAAGACCAA TTCCCCGACA ACCAACCCAT CACTCTCAGG 540

TCCCTCCTGA CTCACTCCTC GGGAATGATC TACGATTTCT TCGACCCCGG CGGGCTCGTC 600

AAATGGAACG GCAAGTTCAA TCCTATCGAG ACTCTCCCCG ACGGGAAACC CAAGCCCCGC 660

CCCGTCGAAA AAGCCTTTGC TTATCCACTA GCTTTTCAGC CCAACACAAG CTGGATGTAT 720 GGTCCCTCAA TCGACTGGGC GGGCCTGATC GTGGAACGTC TCACGGGGCG CAGACTAGGC 780

GATCATATCC GCGAGAGAAT CATCAAGGCC GTTGGCGGGA ACCCTGCCGA TGCGGAGTTT 840

TACCCGCCCA AGAATGAAGA CGTCCGGAAG AGACTGATTG ACTTGCACCC TGACGACCCT 900

CTCGCTACAG GGAAACAGGT ACTCGCGGGT GGCGGGAATA TGAACCTTGT TGCGGATGGT 960

GATTTCGGTG GACACGGGAT GTTCACCACC GGCGAGAATT ACCTCAAGGT GTTGAAGAGT 1020

TTGCTGGCTA ATGATGGGAA ACTCCTCAGC CCCGAGATGG TCAACCTCAT GTTTGAAGAT 1080

CATCTCACGG GGGGAGCTAA AAAGGGTCAC GAAGACGCGC TGAATGGGCC GTTGGGATCA 1140

TTCTTTCCCG TGGGGACTGA TGAGTTTGGC ATGAAGGTGG GTCATGGACT GGGTGGCCTG 1200

GTCACGTTGG AGAGTGTCGA AGGGTGGTAT GGCAAGGGGA CTATGAGTTG GGGCGGCGGG 1260

CATACATTGG TTTGGTTTAT CGATCGGGAG AATGACCTGT GTGGAATCTG TCCGTTGCAG 1320

GCGAAGTTGC CGGTTACGGA GATACAAAAG ATTGCGGATG TGAAGCAGTG CTTTAGGAGG 1380

GATATTTATC GGGTTAGAGA GGCTTGGAAG GCTAGTGGGG GTGGGAAGGA GGAGTAAGTA 1440

CGAGGATTTG GGGCTAGGGA TGTTATTATA TGGTTCTTTT TGATGTGATG AATAATAATG 1500

GAGATTGTAG AAGGCGGGAA GCAGGCGAGT TATTAGAATA GTTATTATTC AGATACATTC 1560

CCCACATTGA AAAAAAAAAA AAAA 1584

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1290 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-gt11

(B) CLONE: 780-M210

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

ATGCCTCCAC CGTCCGGCGC CGGCTCCATC ACCATCGAGA TCCAATCCGC TATCTCAAAA 60

GGCGTCCTCA ATGGTGCCAT CCTCCTCGCC ACTGACTCAA CCTCCTCCTT CACTTTCTCC 120

TCCGCCGCGG GCACTCGAAC TCTTCTCTCA GGAGAAGCCG TCCCTCAGGC CCTCGACGAC 180

GTCCTCTACC TCGCCTCCGC CACCAAACTC CTGACCTCCA TCGCAGCCCT GCAAGTCGTC 240

GAAGATGGTC TTCTAACCCT CACCTCCGAC CTATCCATCA TCGTCCCGGA ATTGACCTCC 300 AAGAAAGTCT TCAAAGGCTG GTCCGACGCC ACCTCCGATC CCCCGGTCGC CATCCTTGAA 360

GACCAATTCC CCGACAACCA ACCCATCACT CTCAGGTCCC TCCTGACTCA CTCCTCGGGA 420

ATGATCTACG ATTTCTTCGA CCCCGGCGGG CTCGTCAAAT GGAACGGCAA GTTCAATCCT 480

ATCGAGACTC TCCCCGACGG GAAACCCAAG CCCCGCCCCG TCGAAAAAGC CTTTGCTTAT 540

CCACTAGCTT TTCAGCCCAA CACAAGCTGG ATGTATGGTC CCTCAATCGA CTGGGCGGGC 600

CTGATCGTGG AACGTCTCAC GGGGCGCAGA CTAGGCGATC ATATCCGCGA GAGAATCATC 660

AAGGCCGTTG GCGGGAACCC TGCCGATGCG GAGTTTTACC CGCCCAAGAA TGAAGACGTC 720 CGGAAGAGAC TGATTGACTT GCACCCTGAC GACCCTCTCG CTACAGGGAA ACAGGTACTC 780

GCGGGTGGCG GGAATATGAA CCTTGTTGCG GATGGTGATT TCGGTGGACA CGGGATGTTC 840

ACCACCGGCG AGAATTACCT CAAGGTGTTG AAGAGTTTGC TGGCTAATGA TGGGAAACTC 900

CTCAGCCCCG AGATGGTCAA CCTCATGTTT GAAGATCATC TCACGGGGCG AGCTAAAAAG 960

GGTCACGAAG ACGCGCTGAA TGGGCCGTTG GGATCATTCT TTCCCGTGGG GACTGATGAG 1020

TTTGGCATGA AGGTGGGTCA TGGACTGGGT GGCCTGGTCA CGTTGGAGAG TGTCGAAGGG 1080

TGGTATGGCA AGGGGACTAT GAGTTGGGGC GGCGGGCATA CATTGGTTTG GTTTATCGAT 1140

CGGGAGAATG ACCTGTGTGG AATCTGTCCG TTGCAGGCGA AGTTGCCGGT TACGGAGATA 1200

CAAAAGATTG CGGATGTGAA GCAGTGCTTT AGGAGGGATA TTTATCGGGT TAGAGAGGCT 1260

TGGAAGGCTA GTGGGGGTGG GAAGGAGGAG 1290

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 430 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Zopfiella latipes

(B) STRAIN: 780

(C) INDIVIDUAL ISOLATE: ATCC #44575

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA in lambda-gtll

(B) CLONE: zl780-m210

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Met Pro Pro Pro Ser Gly Ala Gly Ser Ile Thr Ile Glu Ile Gln Ser 1 5 10 15 Ala Ha Ser Lya Gly Val Leu Asn Gly Ala Ile Leu Leu Ala Thr Asp

20 25 30

Ser Thr Ser Ser Phe Thr Phe Ser Ser Ala Ala Gly Thr Arg Thr Leu

35 40 45

Leu Ser Gly Glu Ala Val Pro Gln Ala Leu Asp Asp Val Leu Tyr Leu 50 55 60

Ala Ser Ala Thr Lys Leu Leu Thr Ser Ile Ala Ala Leu Gln Val Val

65 70 75 80

Glu Asp Gly Leu Leu Thr Leu Thr Ser Asp Leu Ser Ile Ile Val Pro

85 90 95

Glu Leu Thr Ser Lys Lys Val Phe Lys Gly Trp Ser Asp Ala Thr Ser

100 105 110

Aap Pro Pro Val Ala Ila Leu Glu Aep Gln Phe Pro Asp Asn Gln Pro

115 120 125 Ile Thr Leu Arg Ser Leu Leu Thr His Ser Ser Gly Met Ile Tyr Asp 130 135 140

Phe Phe Asp Pro Gly Gly Leu Val Lys Trp Asn Gly Lys Phe Asn Pro 145 150 155 160 Ile Glu Thr Leu Pro Asp Gly Lya Pro Lys Pro Arg Pro Val Glu Lys

165 170 175

Ala Phe Ala Tyr Pro Leu Ala Phe Gln Pro Asn Thr Ser Trp Met Tyr

180 185 190

Gly Pro Ser Ile Asp Trp Ala Gly Leu Ile Val Glu Arg Leu Thr Gly

195 200 205

Arg Arg Leu Gly Asp His Ile Arg Glu Arg Ile Ile Lys Ala Val Gly 210 215 220

Gly Asn Pro Ala Asp Ala Glu Phe Tyr Pro Pro Lys Asn Glu Asp Val 225 230 235 240 Arg Lys Arg Leu Ile Asp Leu His Pro Asp Asp Pro Leu Ala Thr Gly

245 250 255

Lys Gln Val Leu Ala Gly Gly Gly Asn Met Asn Leu Val Ala Asp Gly

260 265 270

Asp Phe Gly Gly His Gly Mat Phe Thr Thr Gly Glu Asn Tyr Leu Lys

275 280 285

Val Leu Lya Ser Leu Leu Ala Asn Asp Gly Lys Leu Leu Ser Pro Glu 290 295 300

Met Val Asn Leu Met Phe Glu Asp His Leu Thr Gly Gly Ala Lys Lys 305 310 315 320

Gly His Glu Asp Ala Leu Asn Gly Pro Leu Gly Ser Phe Phe Pro Val

325 330 335

Gly Thr Asp Glu Phe Gly Met Lys Val Gly His Gly Leu Gly Gly Leu

340 345 350

Val Thr Leu Glu Ser Val Glu Gly Trp Tyr Gly Lys Gly Thr Met Ser

355 360 365

Trp Gly Gly Gly His Thr Leu Val Trp Phe Ile Asp Arg Glu Asn Asp 370 375 380

Leu Cys Gly Ile Cys Pro Leu Gln Ala Lys Leu Pro Val Thr Glu Ile 385 390 395 400 Gln Lya Ile Ala Asp Val Lys Gln Cys Phe Arg Arg Asp Ile Tyr Arg 405 410 415

Val Arg Glu Ala Trp Lya Ala Ser Gly Gly Gly Lys Glu Glu

420 425 430

Claims

WHAT IS CLAIMED IS:

1. A process for the production of S-naproxen comprising the enantioselective hydrolysis of R,S-naproxen ester by an ester hydrolase selected from ester hydrolases produced by a microorganism of the group Absidia griseola, Aspergillus sydowii, Doratomyces stemonitis,

Eupenicillium baarnenses, Graphium sp. , Heterocephalum aurantiacum, Pencillium roguefortii and Zopfiella latipes .

2. The process of Claim 1 in which the ester hydrolase is produced by Zopfiella latipes.

3. The process of Claim 2 wherein the ester hydrolase is produced by Zopfiella latipes Strain #511 (ATCC #26183) or Zopfiella latipes Strain #780 (ATCC #44575).

4. The process of Claim 3 wherein the ester hydrolase is produced by by recombinant methods in a heterologous host.

5. The process of Claim 1 or 2 comprising presenting said ester hydrolase to said R,S-naproxen ester either in an immobilized form or in a free soluble form.

6. The process of Claim 5 wherein S-naproxen is produced in an enantiomeric excess of greater than 95%, preferably an enantiomeric excess of greater than 98%.

7. The process of Claim 6 wherein the ester hydrolase is rec511.

8. The process of Claim 6 wherein the ester hydrolase is rec780.

9. The process of Claim 6 wherein the ester hydrolase is rec780-m10.

10. The process of Claim 6 wherein the ester hydrolase is rec780-m165.

11. The process of Claim 6 wherein the ester hydrolase is rec780-m165r210.

12. The process of Claim 6 wherein the hydrolysis of R,S-naproxen ester occurs at a temperature range from about 30°C to about 65°C.

13. The process of Claim 6 wherein said R,S-naproxen ester is a lower alkyl ester, preferably the ethyl ester or n-propyl ester.

14. The process of Claim 1 that comprises conducting said hydrolysis in an aqueous solution of a pharmaceutically acceptable cation, and the further step of isolating S-naproxen from said aqueous solution.

15. S-naproxen produced by the process of Claim 1 containing a detectable amount of said ester hydrolase.

16. A coding region of a gene encoding for an ester hydrolase capable of enantioselective hydrolysis of an R,S-naproxen ester, which region comprise the nucleotide sequence as set forth in Sequence I.D. No. 2, Sequence I.D. No. 5, Sequence I.D. No. 8, Sequence I.D. No. 11 or Sequence I.D. No. 14, or a sequence that hybridizes thereto.

17. The coding region of Claim 16 comprising the nucleotide sequence as set forth in Sequence I.D. No. 2.

18. The coding region of Claim 16 wherein said nucleotide sequence encodes an amino acid sequence as set forth in Sequence I.D. No. 3.

19. The coding region of Claim 16 comprising the nucleotide sequence as set forth in Sequence I.D. No. 5.

20. The coding region of Claim 16 wherein said nucleotide sequence encodes an amino acid sequence as set forth in Sequence I.D. No. 6.

21. The coding region of Claim 16 comprising the nucleotide sequence as set forth in Sequence I.D. No. 8.

22. The coding region of Claim 16 wherein said nucleotide sequence encodes an amino acid sequence as set forth in Sequence I.D. No. 9.

23. The coding region of Claim 16 comprising the nucleotide sequence as set forth in Sequence I.D. No. 11.

24. The coding region of Claim 16 wherein said nucleotide sequence encodes an amino acid sequence as set forth in Sequence I.D. No. 12.

25. The coding region of Claim 16 comprising the nucleotide sequence as set forth in Sequence I.D. No. 14.

26. The coding region of Claim 16 wherein said nucleotide sequence encodes an amino acid sequence as set forth in Sequence I.D. No. 15.

27. An ester hydrolase capable of the enantioselective hydrolysis of an R,S-naproxen ester to S-naproxen wherein said ester hydrolase hydrolyzes the reaction of R,S-naproxen ester at a temperature range from about 30°C to about 65°C, preferably at a temperature range from about 40°C to about 55°C.

28. The ester hydrolase of Claim 27 selected from ester hydrolases produced by a microorganism of the group Absidia griseola, Aspergillus sydowii, Doratomyces stemonitis, Eupenicillium baarnenses, Graphium sp. , Heterocephalum aurantiacum, Pencillium roguefortii and Zopfiella latipes.

29. The ester hydrolase of Claim 28 produced by Zopfiella latipes.

30. The ester hydrolase of Claim 29 produced by Zopfiella latipes Strain #511 (ATCC #26183) or Zopfiella latipes Strain #780 (ATCC #44575).

31. The ester hydrolase of Claim 27 comprising the capability of producing S-naproxen in an enantiomeric excess of greater that 95%, preferably an enantiomeric excess of greater that 98%.

32. The ester hydrolase of Claim 27 wherein the enantioselective hydrolysis is selective for lower akyl R,S-naproxen esters.

33. The ester hydrolase of Claim 27 wherein said ester hydrolase has a stability to S-naproxen inactivation of greater than 30g/L of S-naproxen in the reaction mixture.

34. The ester hydrolase of Claim 27 having the amino acid sequence as set forth in Sequence I.D. No. 3.

35. The ester hydrolase of Claim 27 having the amino acid sequence as set forth in Sequence I.D. No. 6.

36. The ester hydrolase of Claim 27 having the amino acid sequence as set forth in Sequence I.D. No. 9.

37. The ester hydrolase of Claim 27 having the amino acid sequence as set forth in Sequence I.D. No. 12.

38. The ester hydrolase of Claim 27 having the amino acid sequence as set forth in Sequence I.D. No. 15.

39. An ester hydrolase capable of the enantioselective hydrolysis of ethyl R,S-naproxen ester to S-naproxen, which hydrolase comprises an amino acid sequence as set forth in Sequence I.D. No. 3, Sequence I.D. No. 6, Sequence I.D. No. 9, Sequence I.D. No. 12 or Sequence I.D. No. 15.

40. An ester hydrolase capable of the enantioselective hydrolysis of n-propyl R,S-naproxen ester to S-naproxen, which hydrolase comprises an amino acid sequence as set forth in Sequence I.D. No. 3, Sequence I.D. No. 6, Sequence I.D. No. 9, Sequence I.D. No. 12 or Sequence I.D. No. 15.