AU3913093A

AU3913093A - Delignifying preparation exhibiting alpha-L-arabinofuranosidase activity, production and application thereof

Info

Publication number: AU3913093A
Application number: AU39130/93A
Authority: AU
Inventors: Eugene Rosenberg; Yuval Shoham
Original assignee: Ramot at Tel Aviv University Ltd; Technion Research and Development Foundation Ltd; Korsnas AB
Current assignee: Ramot at Tel Aviv University Ltd; Technion Research and Development Foundation Ltd; Korsnas AB
Priority date: 1992-03-31
Filing date: 1993-03-30
Publication date: 1993-11-08
Also published as: EP0633932A1; SE9201006D0; HU9402800D0; SE501096C2; CZ239494A3; CA2132335A1; BR9306158A; FI944501A0; NO943612D0; HUT70460A; JPH07508162A; SK118594A3; SE9201006L; NO943612L; KR950701381A; FI944501A

Description

* 5 DELIGNIFYING PREPARATION EXHIBITING g-L-ARABINOFURANOSIDASE ACTIVITY. PRODUCTION AND APPLICATION THEREOF

The invention relates to a preparation exhibiting enzymatic activity comprising a substantial portion of α-L-arabinofurano-

10 sidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C. Further, it relates to a method of producing said preparation by aerobic fermenta¬ tion of a selected Bacillus stearothermophilus strain. The invention also comprises said selected strain. Moreover, it

15 relates to a process comprising treatment of wood pulp with a preparation of the invention and also wood pulp that has been treated with said preparation. Additionally, it relates to the use of an enzyme expressed by the deposited Bacillus stearother¬ mophilus strain NCIMB 40494 in the treatment of wood pulp. Also,

20 it relates to α-L-arabinofuranosidase having an amino acid sequence which comprises one or two specified partial amino acid sequences or homologues thereof.

BACKGROUND

25

Due to environmental reasons, the pulp and paper industry has shown increasing interest in bleaching sequences aiming at delignification and bleaching of pulp without use of any chlorine-containing compounds.

30

To that end, many patent applications disclosing new enzymes for delignification of pulp have been filed recently. One such application is our WO 91/10724 (Swedish priority application was ^* issued as patent on December 19, 1991), which i.a. claims a 35 preparation exhibiting enzymatic activity and having the capability of delignifying wood pulp at a temperature of at least 65°C and a pH of at least 9. Said preparation is obtain¬ able by aerobic fermentation of a Bacillus stearothermophilus strain having the capability of producing said preparation, such as one of the two deposited strains NCIMB 40221 or NCIMB 40222. Purification of the enzymatic activity resulted in a xylanase of MW 41 000 - 42 000 Da.

Efforts were made to isolate another strain of Bacillus s earo hermophilus which could be used for purification of other delignifying enzymatic activities than xylanase activity. It was envisaged that e.g. an α-L-arabinofuranosidase being capable of delignifying wood pulp at a high temperature and a high pH would be an additional useful tool in the development of a bleaching sequence without any chlorine-containing bleaching chemicals.

Recently WO 91/18976 (filed by Novo Nordis A/S) was published. Claim 14 thereof is directed to an arabinofuranosidase enzyme produced by Bacillus stearothermophilus. On page 9, lines 11 and 12, is stated that the literature does not contain any reference for an arabinofuranosidase from thermophilic Bacillus. Even thoughWO 91/18976 claims an arabinofuranosidase enzyme produced by Bacillus stearothermophilus, it fails to disclose how it can be obtained.

Three strains have been deposited, two isolated and one mutated. However, none of these have been shown in said application to produce an arabinofuranosidase.

The only strain shown to produce an arabinofuranosidase is a non-deposited mutant strain designated BPS-3-H-17-4. It should further be mentioned that the preparations used in the experiments shown in the application (cf. top of page 17) have been conducted with fermentation products obtained from said non-deposited strain. On page 8, lines 12-16 is disclosed that said strain derives from one of the deposited strains after mutagenesis with ethylmethansulfonate. This mutagen is very unspecific and no conditions of how to arrive at an arabinofura- nosidase-producing strain is given. Thus, there is still not disclosed in the art how to obtain an arabinofuranosidase from a thermophilic Bacillus.

DESCRIPTION OF THE INVENTION

The present invention provides a preparation exhibiting enzymatic activity comprising a substantial portion of α-L- arabinofuranosidase activity having the capabality of deligni¬ fying wood pulp at a pH of 8 - 9 and a temperature of 65°C.

In the present specification and the appended claims the expression "wood pulp" is to be interpreted broadly, and thus it is intended to comprise all kinds of lignocellulosic materials.

One aspect of the invention is directed to a preparation exhibiting enzymatic activity, which is obtainable by aerobic fermentation, in a suitable medium, of a Bacillus stearother¬ mophilus strain selected from the strain NCIMB 40494 and mutants and variants thereof having substantially the same capability of producing said preparation as said strain NCIMB 40494, the enzymatic activity of said preparation comprising a substantial portion of α-L-arabinofuranosidase activity having the capabili¬ ty of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C.

In one embodiment of this aspect of the invention the prepara¬ tion is a clarified culture broth. In another embodiment of this aspect of the invention the preparation is a concentrated fraction of said culture broth exhibiting α-L-arabinofuranosida¬ se activity in a wood pulp medium. In yet another embodiment of this aspect of the invention the α-L-arabinofuranosidase activity derives from an α-L-arabinofuranosidase which is composed of two sub-units having the approximative molecular weights of 52 500 Da and 57 500 Da, respectively. In another aspect of the invention the preparation according to the above disclosed aspect of the invention is in association with a preparation exhibiting enzymatic activity produced by another microbial strain and having the capability of deligni- fying wood pulp at a temperature of at least 65°C and a pH of at least 9. In a specific embodiment of this aspect of the invention the preparation comprises an α-L-arabinofuranosidase produced by the Bacillus stearothermophilus strain NCIMB 40494 and a xylanase produced by the Bacillus stearothermophilus strain NCIMB 40222.

One additional aspect of the invention is directed to an α-L- arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence SEQ ID NO: 1:

Met? Gin Pro Tyr Arg Pro? Glu Glu Leu or a homologue thereof.

Another additional aspect of the invention is directed to an α-L-arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence SEQ ID

NO: 2:

Ser Met Lys Lys Ala Thr Met lie lie Glu Lys Asp Phe Lys lie Ala Glu lie Asp Lys Arg lie Tyr or a homologue thereof.

In the context of "an α-L-arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence SEQ ID NO: 1: Met? Gin Pro Tyr Arg Pro? Glu Glu Leu or a homologue thereof" and/or "an α-L-arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence SEQ ID NO: 2: Ser Met Lys Lys Ala Thr Met lie lie Glu Lys Asp Phe Lys lie Ala Glu lie Asp Lys Arg lie Tyr or a homologue thereof", such a homologue is intended to have an amino acid sequence which, in relation to the sequence SEQ ID NO: 1 and /or the sequence SEQ ID NO: 2, has some amino acid substitutions, extensions or deletions which do not lead to the elimimation of the α-L-arabinofuranosidase activity of the entire enzyme, e.g. in a wood pulp medium, preferably at a temperature of 65°C and a pH of 8 - 9. Further, such a homologue may e.g. have a sequence which has 80% or more amino acids in common with the sequence SEQ ID NO: 1 and /or the sequence SEQ ID NO: 2.

An example of a homologue of the sequence SEQ ID NO: 2 is the sequence SEQ ID NO: 3:

Ala Thr Lys Lys Ala Thr Met lie lie Glu Lys Asp Phe Lys lie Ala Glu lie Asp Lys Arg lie Tyr Gly Ser Phe lie Glu His Leu Gly Arg Ala Val Tyr Gly Gly lie Tyr Glu Pro Gly His Pro Gin Ala Asp Glu Asn Gly which is the N-terminal sequence of an α-L-arabinofuranosidase produced by the Bacillus stearothermophilus strain NCIMB 40222. Said α-L-arabinofuranosidase is composed of two identical sub- units having an approximate molecular weight of 64 000 Da each (the native enzyme, 128 000 Da as judged by SDS gel).

Also, a homologue of the sequence SEQ ID NO: 1 and /or the sequence SEQ ID NO: 2 is any amino acid sequence which is sufficiently homologous on the nucleotide level to be recognized by any DNA or RNA probe derived from said sequence(s).

Yet another aspect of the invention is directed to a method of producing a preparation exhibiting enzymatic activity, whereby a Bacillus stearothermophilus strain selected from the strain NCIMB 40494 and mutants and variants thereof having substantial¬ ly the same capability of producing said preparation as said strain NCIMB 40494, is subjected to aerobic fermentation, in a suitable medium, the enzymatic activity of said preparation comprising a substantial portion of α-L-arabinofuranosidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C. Still another aspect of the invention is directed to the isolated Bacillus stearothermophilus strain NCIMB 40494 and mutants and variants thereof, said mutants and variants having substantially the same capability of producing a preparation exhibiting enzymatic activity as said strain NCIMB 40494, said enzymatic activity comprising a substantial portion of α-L- arabinofuranosidase activity having the capability of deligni¬ fying wood pulp at a pH of 8 - 9 and a temperature of 65°C.

A further aspect of the invention is directed to the use of an enzyme expressed by the B_;_ stearothermophilus strain NCIMB 40494 for treatment of wood pulp. As is evident from Table 8 of the specification, said strain produces several types of extra- cellular carbohydrate-degrading enzymatic activities which derive from the expression of different genes comprised by the genome of said bacterium.

Even though these different activities have not all been investigated separately yet, it is believed that the different activities, separately or in different combinations, will be useful in the treatment of wood pulp.

An additional aspect of the invention is directed to a process comprising treatment of wood pulp, whereby wood pulp is treated in at least one- step with a preparation according to the invention. In one embodiment of this aspect of the invention, the wood pulp to be treated is sulphate pulp. In another embodiment the sulphate pulp to be treated is a partially delignified sulphate pulp. In yet another embodiment of this aspect of the invention the partially delignified sulphate pulp to be treated is an oxygen-delignified sulphate pulp.

A further aspect of the invention is directed to wood pulp which has been treated in at least one step with a preparation^' according to the invention. Still another aspect of the invention is directed to a DNA or RNA probe which recognizes the nucleotide sequence coding for the amino acid sequence SEQ ID NO: 1: Met? Gin Pro Tyr Arg Pro? Glu Glu Leu and/or the amino acid sequence SEQ ID NO: 2:

Ser Met Lys Lys Ala Thr Met lie lie Glu Lys Asp Phe Lys lie Ala Glu lie Asp Lys Arg lie Tyr.

The last mentioned aspect of the invention is useful in the finding of α-L-arabinofuranosidases having the capability of delignifying wood pulp at a temperature of at least 65°C and a pH of at least 8 - 9.

DEPOSITION OF MICROORGANISMS

The Bacillus sterothermophilus strain LI was deposited under the Budapest Treaty at the National Collections of Industrial and Marine Bacteria Ltd (NCIMB), Aberdeen, on March 24, 1992 and was given the accession number NCIMB 40494.

The Bacillus stearothermophilus strain T-6, given the accession number NCIMB 40222 by the same depository, was deposited earlier in connection with the filing of the priority application for WO 91/10724.

Isolation of thermostable α-L-arabinofuranosidase producing Bacillus stearothermophilus . strain LI

A. Preparation of alkaline extracted pulp (AKP) .

Suspensions of K15, oxygen semi-bleached soft wood sulphate pulp, (6-10% dry weight fiber) in 0.02 N NaOH (pH 11.7) were incubated at 60-62°C for 18-20 hours. While still hot, the fluids were separated from the fibers using a sinter-glass funnel under suction. The resulting solution was neutralized to pH 7.0 and concentrated by ultrafiltration (10,000 MW cut off) . The retentative (AKP-1) contained 5.2 mg/ml lignin, 1.85 mg/ml carbohydrate (by phenol-sulphuric acid and 0.83 mg/ml pentoses (by orcinol) .

B. Enrichment culture.

AKP-1 was supplemented with salts (0.1% MgS0₄.7H₂0, 10 mM K₂HP0₄, 0.1% urea and trace elements) and inoculated with mud and water samples taken from the water treatment pools at Korsnas. The flasks were incubated with shaking at 65°C and pH 9.0 for 48 h. A drop of turbid culture was then inoculated into a flask containing 15 ml of 0.2% galactomannan (Locust bean gum, Sigma) medium. After incubation for 24 hours at 65°C and pH 9.0, the culture was streaked onto LB agar. After incubation, approxima- tely 20 different colony types were isolated and obtained in pure culture. After preliminary testing, one of the strains was studied in detail: strain LI that contained a thermophilic α-L- arabinofuranosidase.

Strain LI was a Gram-positive, thermophilic bacterium; it grew well at 60-65°C, but did not grow at less than 40°C. It was rod- shaped with a terminal spore, aerobic and oxidase-positive. Strain LI grew on the following carbon sources: D-glucose, D- xylose, L-arabinose, D-mannose and xylan. It did not grow on melobiose and galactose as carbon sources. Based on these properties LI was classified as a strain of the heterogenous group. Bacillus stearothermophilus.

Initial experiments (with K15 pulp)

C. Thermophilic α-L-arabinofuranosidase from strain LI

Strain LI was selected for further study because the crude extracellular fluid of culture grown on galactomannan medium was active on delignification of K15 pulp. For the estimation of how much lignin is made extractable by the enzymatic preparation, a sensitive spectrophotometric assay was used. The absorbance values at 350 nm(A₃₅₀) were measured on the supernatant liquids of the samples, and measured values are presented as % lignin released. The growth and delignification activity of strain LI grown on different carbon sources is summarized in Table 1. The strain grew to some extent on the yeast extract and casamino acids without addition of another carbon source and yielded a net delignification of 5.1%. Additional growth was observed with all the sugars added except the galactose, which also inhibited the production of delignification activity. The best delignifi- cation activities were obtained on cultures grown on xylose and arabinose, 7.1% and 7.6%, respectively.

The extracellular enzymatic activities of strain LI grown on xylose and arabinose media are summarized in Table 2. Xylanase activity was determined by incubating a fresh solution of xylan with extracellular supernatant fluid and assaying for increase in reducing sugars by the ferricyanide method (Spiro, R.G. 1966, Methods in Enzymology 8_.: 7-9) . Assay buffer was 50 mM Tris. Cl, pH 7.0 and 0.5% xylan (oat spelts, Sigma). Units of activity for xylanase are μmol reducing sugar generated per minute at 65°C.

The arabinofuranosidase assay is a modification of the standard assay for β-galactosidase (G. Tabolt and Syguson, Appl. & Enviro. Microbiol, vol 56, No-. 11, 1990). Test tube contains 0.5 ml of 40 mM Tris-buffer, pH 7.0. 0.4 ml of the enzyme sample and 0.1 ml 10 mM p-nitrophenyl-α-L-arabinofuranoside (Sigma Chemicals) was incubated at 65°C for 15 min. The reaction was terminated by putting the test tube in ice water bath. The release of p-nitrophenol is determined spectrophotometricaly at 401 nm. 10 micromole of p-nitro-phenol per ml has an absorbance of 18.4. A unit of enzyme acting is defined as micromole of p- nitrophenyl release per min.

On xylose medium, only two activities were detected: 1.3 units per ml xylanase and 0.004 units per ml of α-L-arabinofurano¬ sidase. There were no detectable α-L-arabinopyranosidase, mannanase, α-D-galactosidase or α-L-mannosidase activities. It appeared, therefore, that the delignification activity of LI was due to xylanase activity when the cells were grown on xylose medium. However, when the cells were grown on arabinose medium, the major activity was α-L-arabinofuranosidase (0.5 units per ml) . It appeared, therefore, that the α-L-arabinofuranosidase was responsible for delignification in A-medium.

The α-L-arabinofuranosidase (AF) activity was concentrated from a ten liter culture of LI grown on A-medium (Table 3). The activity was precipitated with 80% saturated ammonium sulfate, yielding a crude enzyme preparation with 11 units per ml AF and 0.96 units per ml of xylanase. The AF activity did not bind to carboxyl-methyl cellulose (CMC), but did adsorb completely to DEAE cellulose or DEAE-Sephacel.

The concentrated AF demonstrated significant delignification activity (Table 4) . It should be pointed out that we have not yet optimized the conditions for using AF to delignify pulp. A potentially useful property of AF is that it should break the bond close to lignin, thereby leaving most of the hemi-cellulose with the cellulose fibers. This should give a higher yield of delignified pulp. In addition, it appears that the delignifica¬ tion activity of AF plus xylanase T-6 is more than additive.

Tables 5-7 describe some of the enzymatic properties of the purified AF, using PNP-α-L-Ara as substrate. The enzyme is most active between pH 6.5-8.0, with an optimum at pH 7.0. The enzyme has low activity at pH 9.0 at 70°C. The temperature optimum was 70°C at both pH 7.0 and pH 8.0. The enzyme was most active at 20 mM Na₂S0₄, pH 7.5 in 10 mM phosphate buffer at 70°C. At pH 7.0, the enzyme was very stable at 60°C, but lost about 50% of its activity at 70°C during 75 min and was completely inacti¬ vated at 80°C for 15 min. Extended experiments (with K20 pulp)

Extracellular carbohydrate-degrading enzymatic activities of Bacillus stearothermophilus - LI

Specific extracellular carbohydrate-degrading enzymatic activities were tested following growth in different media (Table 8). The cells were grown on different carbon sources, namely: p-nitrophenyl-β-D-mannopyranoside, p-nitrophenyl-α-D- galactopyranoside and p-nitrophenyl-α-L-arabinopyranoside.

Xylanase and two other endohemicellulases (mannase and arabina- nase) were assayed in 10 mM phosphate buffer, pH 8.0 at 60°C in 1 ml total assay volume. An appropriately diluted enzyme sample (0.1 ml) was added to 0.25 ml substrate, 0.1 ml 100 mM buffer and 0.55 ml water. The reaction was terminated by transferring to an ice water bath. Reducing sugar was determined by 3,5- dinitrosalicylic acid method as described by Miller (Miller, GL, (1959) Anal Chem 31:426-428). The substrates were 4% Oat splet xylan, 0.4% Locust Bean Gum-galactomannan, 4% arabinogalactan from Larchwood and 4% mannan from Saccharomyces cerevisiae.

The enzymatic release of lignin from softwood pulp was determi¬ ned by adding 200 mg wet weight of K-20 pulp (from the Korsnas paper mill Gavle, Sweden) to 3 ml of enzyme solution, adjusting to pH 8.0 or 9.0 with a concentrated NaOH solution, and incubating at 65°C with shaking. After 2 h, 1.5 ml of the liquid was removed and centrifuged at 10,000 x g for 5 min in a minifuge to remove residual pulp fibers. The clear liquid (0.5 ml) was diluted with 1.0 ml of 0.1 N NaOH for determination of absorbance at 350 nm. The control for each assay was incubation of the pulp under the same conditions without enzyme. Since 1 mg lignin per ml had an A₃₅₀ of 9.1, and pulp used in these experiments had a lignin content of 1.3% (Klason, determined by Z. Zosim) , the

% lignin released = ΔA₃₅₀ x 3 x 100

P x 0.013 where P is the pulp dry weight and ΔA_35Q is the absorbance after incubation minus absorbance before incubation. The net lignin released is the total minus the no enzyme control.

The two significant activities that were found were xylanase and α-L-arabinofuranosidase. Xylanasewas found in low concentration when the carbon sources in the growth media were locust bean gum (LBG) , D-glucose and L-arabinose. The xylanase activity was amplified with D-xylose as the carbon source, reaching 1.23 U/ml. Mannose inhibited production of xylanase activity. α-L- arabinofuranosidase activity was low, but significant on D- xylose medium and high on L-arabinose medium, reaching 1.5 U/ml. No activities of mannase, α-D-galactosidase, β-D-mannosidase or α-L-arabinopyranosidase were found.

Purification of α-L-arabinofuranosidase from Bacillus stearo¬ thermophilus.

Following growth of Bacillus stearothermophilus LI for 24 h at 60°C on an L-arabinose-containing medium, the cell free extracellular fluid contained 1.2 U/ml of α-L-arabinofurano¬ sidase activity with a specific activity of 1.7 U/mg (Table 9). Preliminary experiments indicated that the enzyme could be concentrated by precipitation at 90% ammonium sulfate satura- tion. However, there was only a 68% recovery of activity and essentially no increase in specific activity. Thus, ammonium sulfate precipitation was not used, and the crude enzyme was adsorbed directly to a DEAE Sephacel column (Table 9, anion exchanger) . After rinsing the column with 10 mM potassium phosphate buffer, pH 8.0, elution was performed with a linear gradient from 0.1 to 0.9 M NaCl. The α-L-arabinofuranosidase activity eluted as a sharp peak between 0.53 and 0.57 M NaCl. Following concentration and desalting of the active fractions by dialysis against polyethylene glycol, the specific activity increased 38-fold with 100% recovery of the activity. This material was applied directly to a Sephadex G-100 column. The active material eluted as a single sharp peak with an apparent molecular weight of 108,000. The overall purification was 59 times with a yield of 80%. The purified enzyme was examined by FPLC Superose 12 gel filtration in 100 mM potassium phosphate buffer, pH 7.0, and 100 mM NaCl. Over 95% of the activity and protein eluted as a single sharp peak with an apparent molecular weight of 114,800. By SDS PAGE, the purified enzyme showed two bands with molecular weights of 57,500 and 52,500. Analysis of these two bands revealed, after blotting on PVDF membrane and sequencing on an Applied Biosystems model 475A gas phase sequencer, the two N-terminal sequences SEQ ID NO: 1: and SEQ ID NO: 2.

Characterization of Bacillus stearothermophilus LI α-L-arabino¬ furanosidase.

At pH 7.0, the purified enzyme was completely stable at 60°C for at least 80 min, retained 50% of its maximum activity after incubation at 70°C for 75 min, and lost all its activity after 15 min at 80°C. At 70°C, the optimum pH for activity was 7.0; at pH 6.5 and pH 8.0, the activities were 55% and 50% of the optimum activity, respectively. At pH 7.0 and pH 8.0, the optimum temperature for activity was 70°C. The enzyme showed maximum activity in 20-50 mM Na₂S0₄; at 100 mM and 150 mM Na₂S0₄, the activity decreased 10% and 50%, respectively.

The kinetic parameters of the enzyme were measured using pNP-α- L-ara-f as the substrate. At pH 7.0 and 65°C, K_m and V_maχ were 2.2 x 10^" M and 101 μ ol min^~1mg^-1, respectively. The enzyme showed only low activity on high molecular weight substrates, such as arabinoxylan and arabinogalactan.

Delignification activity of Bacillus stearothermophilus LI α-L- arabinofuranosidase.

During the purification of the enzyme (Table 9), column eluents were examined routinely for delignification activity using semi- bleached Kraft pulp as the substrate. Delignification activity was associated with (a) the peak of α-L-arabinofuranosidase activity, (b) the peak of a lower molecular weight endoxylanase activity and (c) fractions which contained low amounts of both activities. Since the highest specific delignification activity occurred when both enzymes were present, it was decided to examine the possible synergistic activities of the α-L-arabino¬ furanosidase and a previous purified heat-stable xylanase T6 disclosed in WO 91/10724.

Table 10 summarizes a typical experiment in which delignifica¬ tion activity was examined with pure α-L-arabinofuranosidase, pure xylanase and a mixture of the two enzymes. At pH 8.0 and 65°C for 2 h, the mixture of 38 U/ml α-L-arabinofuranosidase and 5 U/ml xylanase T6 released a net of 19.2% of the lignin from the pulp, whereas the sum of each enzyme acting separately was only 16.5%. To achieve 16.5% net release of lignin using only xylanase T6 under these conditions required 50 U/ml of the enzyme. At pH 9.0 and 65°C for 2 h, the mixture of enzymes released 18.4% lignin, compared to only 13.7% for the sum of the two activities acting separately. Clearly, the two enzymes acted synergistically in releasing lignin from the pulp.

An apparent paradox is that the α-L-arabinofuranosidase enzyme showed less than 1% of its optimal activity at pH 9.0, using the model substrate P-nitrophenol α-L-arabinofuranosidase. However, in the biobleaching process, the enzyme was almost as effective at pH 9.0 as at pH 8.0. It is possible that the pulp somehow protected and conserved the enzyme activity at higher pH values. These results are encouraging since the industrial enzymatic delignification process is more easily performed at pH 9.0 and 65°C than at lower temperatures and pH values. Table 1

Growth and delignification activity of strain LI on different media.

The growth media consisted of carbon source (0.2%), 0.1% yeast extract, 0.5% casamino acids and E. salts (0.1% urea, 0.02% MgS0₄'7H₂0, 5mM KH₂P0₄, pH 9.0, 20 mM TrisΗCl, pH 9.0, 0.1% NaCl and standard trace elements mixture). All of the sugars were the D-configuration, except for L- arabinose; LB is locust bean galactomannane. Delignification of K15 was carried out with the extra¬ cellular supernatant (adjusted to pH 9.0), at 65°C for 2h.

Table 2

Extracellular enzymatic activities of LI

X-medium^a A-medium^b

Substrate Act. (U/ml) Act. (U/ml)

Xylan 1.3 0.05

PNP-α-L-AF 0.004 0.5

PNP-α-Gal < 0.001

PNP-α-Man < 0.001

PNP-α-AP < 0.001

LB-GM < 0.001

Xylan and LB-GM (locust bean galactomannan) degrading activities were determined by production of reducing su¬ gars and PNP-X activities were measured by liberation of PNP (p-nitrophenol) .

Cells were grown for 24 hrs in either X-medium or A-medium (xylose or arabinose medium described in Table 1) and centrifuged to remove the cells.

Table 3

Preliminary purification of α-L-arabinofuranosidase from LI grown on A-medium

Vol Protein³ AF Xylanase

Fraction (ml) (mg/ml) (U/ml) (U/ml)

Crude supernatant 8100 (NH₄)₂S0₄ ppt 400 DEAE-Sephacel 20

Protein determined by BioRad. Table 4

Delignification of K15 by concentrated α-L-arabinofuranosidase

% Lignin release³

Enzyme Total Net

10 mM phosphate buffer, pH 8.0, 65°C, 2h.

Concentrated by DEAE-Sepharel chromatography; contains

<0.1 U/ml xylanase.

Xylanase T-6 is produced by NCIMB 40222, disclosed in WO

91/10724, and is capable of delignifying wood pulp at a pH of at least 9 and a temperature of at least 65°C.

Table 5

Effect of temperature on the activity of AF at pH 7 and 8

Temperature pH 7 pH 8 AF (U/ml)^a AF (U/ml)^a

40 2.1 1.0 50 5.0 2.4 60 8.3 5.2 65 10.1 6 70 10.3 6 75 9.9 5 80 4.2 0 90 0.8 ,3 100 0

In 20 mM Tris buffer. Table 6

Effect of salt concentration on the activity of AF.

Na₂S0₄

(mM) AF (U/ml)^f

0 3.1

20 3.6

50 3.4

100 3.0

150 1.0

^a In 10 mM phosphate buffer, pH 7.5, at 70'

Table 7

Stability of α-L-Arabinofuranosidase (AF) activity.

^a In 20 mM Tris, pH 7.0, Table 8

Extracellular carbohydrate-degrading enzymatic activities of Bacillus stearothermophilus LI³

10

15

20

25

30

L-Araninose Arabinogalactan α-L-arabinosidase <0.01 L-Arabinose Xylan Xylanase 0.11 L-Arabinose pNP-α-L-ara-f α-L-arabino-furanosidase 1.5

35 Cells were grown for 24 h at 60°C and pOH 98.0 in E salts containing 0.1% yeast extract, 0.1% casamino acids and the carbon source at 0.2%.

After incubation for 24 h, the culture was centrifuged and

40 the extracellular carbohydrate-degrading enzymes assayed as described in the specification.

LGB is Locust Bean Gum, a galactomannan; pNP is p-nitro- phenol.

^*45 Table 9

Summary of α-L-arabinofuranosidase purification procedure

A 1.4 liter culture of strain LI was prepared as described in the specification. The culture was centrifuged for 30 min at 10,000 x g. The supernatant fluid was then passed through a 0.8 μm filter to remove residual cells. This crude supernatant was the starting material for the purification procedure.

DEAE sephacel column followed by dialysis against poly¬ ethylene glycol.

Sephadex G-100.

Table 10

Delignification activity of α-L-arabinofuranosidase and xylanase

T6 on semi-bleached pulp

Delignification (%)'

Enzyme⁵ pH Total Net

No enzyme

No enzyme α-L-arabinofuranosidase α-L-arabinofuranosidase

Xylanase

Xylanase α-L-arabinofuranosidase+xylanase α-L-arabinofuranosidase+xylanase

Purified α-L-arabinofuranosidase (38 units per ml, speci¬ fic activity 126 U/mg) and xylanase T6 (5 U/ml, 192 U/mg) were used. The enzymes were in 10 mM phosphate buffer.

The conditions used were as follows: 10 mM each of Na₂S0₄ and (NH₄)₂S0₄, 65°C, 2h at pH 8.0 or 9.0.

SEQUENCE LISTING

( 1) GENERAL INFORMATION:

(i) APPLICANT:

[A) NAME: Korsnas AB

^'B) STREET: none

C) CITY: Gavle

Ε) COUNTRY: SWEDEN

^;F) POSTAL CODE (ZIP): S-801 81

^;G) TELEPHONE: 026-151000

Η) TELEFAX: 026-195253

A) NAME: The Technion Research and Development Foundation Ltd

B) STREET: Technion City

C) CITY: Haifa

E) COUNTRY: Israel

F) POSTAL CODE (ZIP): none

A) NAME: Ramot - University Authority for Applied Res.&Ind.De . Ltd

B) STREET: 32 H. Levanon Street

C) CITY: Tel-Aviv

E) COUNTRY: Israel

F) POSTAL CODE (ZIP): none

(ii) TITLE OF INVENTION: Delignifying Preparation Exhibiting Alpha-L-Arabino-furanosidase activity, Production and Application Thereof

(iii) NUMBER OF SEQUENCES: 3

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus stearothermophilus

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

Xaa Gin Pro Tyr Arg Xaa Glu Glu Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus stearothermophilus

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

Ser Met Lys Lys Ala Thr Met lie lie Glu Lys Asp Phe Lys lie Ala 1 5 10 15

Glu lie Asp Lys Arg lie Tyr 20

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus stearothermophilus

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

Ala Thr Lys Lys Ala Thr Met lie lie Glu Lys Asp Phe Lys lie Ala 1 5 10 15

Glu lie Asp Lys Arg lie Tyr Gly Ser Phe lie Glu His Leu Gly Arg 20 25 30

Ala Val Tyr Gly Gly lie Tyr Glu Pro Gly His Pro Gin Ala Asp Glu 35 40 45

Asn Gly 50

Claims

1. A preparation exhibiting enzymatic activity, c h ar a c t e r i s e d in that it is obtainable by aerobic fermentation, in a suitable medium, of a Bacillus stearother- mophilus strain selected from the strain NCIMB 40494 and mutants and variants thereof having substantially the same capability of producing said preparation as said strain NCIMB 40494, the enzymatic activity of said preparation comprising a substantial portion of α-L-arabinofuranosidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C.

2. A preparation according to claim 1, wherein said preparation is a clarified culture broth.

3. A preparation according to claim 2, wherein said preparation is a concentrated fraction of said culture broth exhibiting α- L-arabinofuranosidase activity in a wood pulp medium.

4. A preparation according to claim 3, wherein said α-L- arabinofuranosidase activity derives from an α-L-arabino¬ furanosidase which is composed of two subunits having the approximative molecular weights of 52 500 Da and 57 500 Da, respectively.

5. A preparation according to any one of claims 1-4 in asso¬ ciation with a preparation exhibiting enzymatic activity produced by another microbial strain and having the capability of delignifymg wood pulp at a temperature of at least 65°C and a pH of at least 9.

6. A preparation according to claim 5, wherein said preparation comprises an α-L-arabinofuranosidase produced by the Bacillus stearothermophilus strain NCIMB 40494 and a xylanase produced by the Bacillus stearothermophilus strain NCIMB 40222.

7. An α-L-arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence (SEQ ID N0:1) Met? Gin Pro Tyr Arg Pro? Glu Glu Leu or a homologue thereof.

8. An α-L-arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence (SEQ ID N0:2)

9. A method of producing a preparation exhibiting enzymatic activity, c h a r a c t e r i s e d in that a Bacillus stearothermophilus strain selected from the strain NCIMB 40494 and mutants and variants thereof having substantially the same capability of producing said preparation as said strain NCIMB 40494, is subjected to aerobic fermentation, in a suitable medium, the enzymatic activity of said preparation comprising a substantial portion of α-L-arabinofuranosidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C.

10. The isolated Bacillus stearothermophilus strain NCIMB 40494 and mutants and variants thereof, said mutants and variants having substantially the same capability of producing a preparation exhibiting enzymatic activity as said strain NCIMB 40494, said enzymatic activity comprising a substantial portion of α-L-arabinofuranosidase activity having the capabi¬ lity of delignifying wood pulp at a pH of 8 - 9 and a tempera¬ ture of 65°C.

11. Use of an enzyme expressed by the Bacillus stearothermophi¬ lus strain NCIMB 40494 in the treatment of wood pulp.

12. A process comprising treatment of wood pulp, c h a r a c t e r i s e d in that wood pulp is treated in at least one step with a preparation according to any one of claims 1-6.

13. A process according to claim 12, wherein said wood pulp is sulphate pulp.

14. A process according to claim 13, wherein said wood pulp is a partially delignified sulphate pulp.

15. A process according to claim 14, wherein said partially delignified sulphate pulp is an oxygen-delignified sulphate pulp.

16. A wood pulp, c h a r a c t e r i s e d in that it has been treated in at least one step with a preparation according to any one of claims 1-6.

17. A DNA or RNA probe which recognizes the nucleotide sequence coding for the amino acid sequence (SEQ ID NO: 1)

Met? Gin Pro Tyr Arg Pro? Glu Glu Leu and/or the amino acid sequence (SEQ ID NO: 2) Ser Met Lys Lys Ala Thr Met lie lie Glu Lys Asp Phe Lys lie Ala Glu lie Asp Lys Arg lie Tyr.