IE62566B1 - A process for the enzymatic hydrolysis of alpha-a minodipinyl-monoamino compounds. - Google Patents

Abstract

gamma -Glutamyltranspeptidase, which can be prepared by fermentation, can be used to hydrolyse alpha -aminoadipinyl-monoamino compounds, especially D-( delta )- alpha -aminoadipinyl-7-aminocephalosporanic acid.

Description

J-GIutamyLtranspeptidases (^p-GTP hereinafter) play an important part in amino acid metabolism and in the glutathione cycle in animal tissues and in microorganisms Cfieth. Enzymol. 77, 237 ( 1981 )3» They are responsible for the transport of various amino acids in the form of their ^-glutamyl derivatives, the formation of polyglutamic acid in Bacilli, and the breakdown of glutathione C^-glutamylcysteinyl-glyc ine), It has already been proposed (EP 0,275,901) to use ^-GTP to hydrolyze ad i p i nyI - or glut aryl-monoamino compounds.

It has now been found, surprisingly, that y^GTP catalyzes the hydrolysis of e-aminoadipinyl-monoamino compounds of the formula I HOOC-CH-(CH2)3-C-NH-R1 (ϊ) NH, 0 in which 1 R denotes amino acids, dipeptides, cephems, cephams or derivatives thereof.

This is all the more surprising because it has hitherto been assumed that neither Q nor C$ side-chains are accepted by the active center of j^GTP (see Agric. Biol. Chem. 42 , 1978, pages 371 - 81).

Hence the invention relates to: 1» A ^'-glutamyltranspept idase having the following properties - a molecular weight of 40,000 to 80,000, - an isoelectric point at pH 4.4 to 5.9, - for L-J-gIutamyI paranitroaniIide as substrate a pH optimum in the range 6.5 to 10, and a Km of 9 to 36 μϊ·ί at pH 8, and - hydrolysis of G-aminoadipinyI-monoamino compounds of the formula I HOOC-CH-(CH?)o-C-NH-R1 (I) I Z J II nh2 O in which 1 R denotes amino acids, dipeptides, cephems, cephams or- derivatives thereof. e> A process for the preparation of the ^ =g I u t amy 11 r ans peptidase having the properties mentioned in 1., which comprises cultivation of bacteria of the genera Pseudomonas, Proteus, Arthrobacter and Bacillus in a nutrient medium until the said^GTP accumulates in the nutrient medium. 3. The use of the Jp-gIutamy11ranspeptidase having the properties mentioned in 1. for the hydrolysis of tf-aminoadipinyI-monoamino compounds of the formula I.

The invention is described in detail hereinafter, especially in its preferred embodiments. The invention is furthermore defined in the patent claims.

The J^-glutamyltranspeptidase (J^GTP) catalyzes the hydrolysis of α-aminoadipinyI-monoamino compounds of the formula I, as defined above, to give the corresponding acid and the monoamino compound. 7-Aminocephalosporanic acid derivatives are preferably used as substrate.

T; G> The enzyme occurs in the periplasm of microorganisms and extracellularly and can be characterized by a molecular weight of 40,000 to 80,000, preferably 50,000 to 70,000, especially 55,000 to 65,000, and by an isoelectric point which is at a pH of 4.4 to 5.9, preferably 4.8 to 5.5.

The pH optimum for L-/)-g I u t amy I p a r an i t r o an i I i de as substrate is in the pH range 6.5 to 10. The transpeptidase according to the invention has a Km for the same substrate of 9 to 36 μΜ, preferably 15 to 20 μΜ, in particular 17.8 μΗ, at pH 8.

The jo-GTP according to the invention is irreversibly in10 hibited in the presence of azaserine or iodoacetamide.

The enzyme shows reversible inhibition in the presence of copper, mercury and a mixture of serine and borate, as well as in the presence of 7-aminocepha I osporanic acid.

The ^-GTP is prepared with the aid of microorganisms, as also described in European Patent Application EP 0,275,901. In this process, bacteria, especially of the genera Pseudomonas, Proteus, Arthrobacter and Bacillus, are cultivated in a nutrient medium until ^-GTP accumulates in the nutrient medium. Suitable examples are: Pseudomonas putida ATCC 17390, Pseudomonas aeruginosa MCTC 10701, Proteus vulgaris ATCC 9634, Arthrobacter parafineus ATCC 31917 ss well as Pseudomonas fragi D SM 3881 and Bacillus subtilis IFO 3025. The enzyme is particularly preferably obtained from Bac. subtilis IFO 3025. Mutants and variants of the said microorganisms are also suitable.

The microorganisms are cultured aerobically, singly or in mixed culture, for example submerged with shaking or stirring in shaken flasks or fermenters, where appropriate with air or oxygen being passed in. The fermentation can take place in a temperature range from about 20 to 37°C, preferably at about 25 to 30°C, in particular at 28 to 30°C. Fermentation is carried out in a pH range between and 8.5, preferably between 5.5 and 8.0. Under these conditions, the culture broth shows considerable accumulation of the enzyme in general after 1 to 3 days. Syn thesis of the -GTP starts in the late log phase and - 4 reaches its maximum in the stationary phase of growth.

The production of the periplasmic enzyme can be followed with the aid of activity assays by HPLC analysis or photometry.

The nutrient solution used to produce the/^GTP contains 0.2 to 5 %, preferably 0.5 to 2 %, organic nitrogen compounds and inorganic salts. Suitable organic nitrogen compounds are: amino acids, peptones, furthermore meat extracts, milled seeds, for example of corn, wheat, beans, soybeans or the cotton plant, distillation residues from the manufacture of alcohol, meat meals or yeast extracts. Examples of inorganic salts which the nutrient solution can contain are chlorides, carbonates, sulfates or phosphates of the alkali metal or alkaline earth metals, iron, zinc and manganese, but also ammonium salts and n i trates .

The addition of assimilable carbohydrates increases the yield of biomass. Carbohydrates are also added in the abovementioned concentrations. It is possible to add as preferred carbon source for example sugars, such as glucose or sucrose, as well as carbohydrate-containing natural products such as malt extract, to the nutrient solution.

Although the optimal fermentation conditions differ for each microorganism, either they are already known to those skilled in the art or they can be established in easy preliminary tests.

Purification can be carried out by classical processes via lysozyme digestion, ammonium sulfate precipitation, and ion exchange and gel permeation chrotaatography. The enzyme can be coupled by conventional methods (Colowick and Kaplan, Meth. Enzymol., vol. XLIV).

It is possible to use for the enzymatic reaction both - 5 whole cells in free or immobilized form, with the addition of β-lactamase inactivators, for example clavulanic acid or thienamycin, and the isolated enzyme which can also be carrier-bound . Examples of suitable materials for the immobilization of whole cells are chitosan, alginate, K-carrageenan , poIyacryIohydrazides and other known substances from processes known from the literature (K. Venkatsubramanian, Immob. Cells (1979), ACS Symposium Series, page 106). carried out at f about 28 to 38° is that in which The hydrolysis reaction is most suitably about pH ό.ό to 8 and at a temperature o The preferred compound of the formula I A R 1 denotes the CH2R in which R^ is hydrogen, OH or "O-C-CH3. ί The ^-GTP has industrial importance in particular for obtaining 7-aminocephaIosporanic acid from cephalosporin C. To date however, a yeast (Trigonopsis variabilis) has always been used to generate from cephalosporin C glutaryl7-aminocephalosporanic acid which only then could be hydro lyzed enzymatically, in a second reaction step, to give 70®inocephaIosporanic acid. It is now possible with the process according to the invention to prepare 7-aminocephaIosporanic acid from cephalosporin C in a single step The invention is described in more detail in the examples which follow. Unless otherwise indicated, percentage data relate to weight.

The J/-GTP-producing microorganism strains are maintained on agar slants of the following composition; Glucose 1 X Casein peptone 0.4 X Meat extract 0.4 X Yeas t extract 0.05 X Liver extract 0.05 X 1* HaC I 0.25 «!/ pH 7.2 e The slant tubes are incubated at 28°C for 2 days. The celts are then rinsed off with 10 ml of physiological saline, and 1 ml of this suspension is used to inoculate a 50 ml preculture of the following composition in an Erlemneyer flask of capacity 300 mis Peptone Halt extract pH 7.0 0.5 The flask is incubated shaker for 24 hours „ inoculum for 50 ml of at 30°C and 190 rpm in 2.5 ml of this culture main culture: a are rotary used as Bacilli Peptone 0.12 X Yeast extract 0.12 X Glucose 0.25 X Ha lactate (60 %) 5.6 m I HH4CI 0.12 X k2hpo4 0.12 X KH2PO4 0.034 X HgSO4 x 7 H20 0.025 X HaCl 0.5 X KCl 0.5 Of fa CaCl2 x 2 H20 0.0015 w fa HnC15 x 4 HpO 0.0007 oy Λ» Fe(NH4)citrate 0.00015 X 28°C The culture is incubated at and a shaking frequency of 190 rpm for 24 hours and is then harvested by centr i fugat ion. (fGTP which activities of some strains are listed follows: in the table Strain B . s ub t i I i s II II IFO 3025 IF0 3013 IFO 3335 ^•GTP (mU/ml culture solution) Exaraple 2 a Z-GTP titer of 150 mU/ml of culture solution is reached, A preculture with Bac. subtilis IFO 3025 is cultured in analogy to Example 1. 50 ml of this culture are used as inoculum for 2 I of main culture solution in a 5 I fermenter. The strain is cultured at 34°C and a partial pressure of oxygen of 70 X. The formation of the /p-GTP is followed by photometry, and the culture is harvested at the maximum enzyme titer. Under the given conditions, VQ Example solution are separated by means of cross(exclusion limit 300,000 dalton) into e and biomass. The culture filtrate obway contains a /p-GTP activity of 1350 U. tL of culture flow filtration culture filtrat tained in this The enzyme is precipitated by addition of ammonium sulfate to 70 X saturation and is taken up again in 1/10 of the volume. After dialysis against 20 mM tris, pH 8.0, the enzyme is further purified on a DEAE-cellulose column (DE 52, Whatman). The active eluates are combined and concentrated. A /p-GTP product obtained in this way (containing about 25 U of Λ-GTP/ml) is used for the conversions Example 4 The following mixture is chosen for preparative conversion of deacetyl-CPC: 100 μΐ of enzyme concentrate prepared as in Example 3, and 100 μΐ of 40 mM deacetyl-CPC dissolved in 20 mM potassium phosphate buffer, pH 7.3, are incubated at a temperature of 33°C. lip to 16 % deacetyI-7-aminocepha I osporanic acid are produced under the chosen conditions.

E x a 39 p L e 5 With incubation conditions analogous to those Example 4, 3 % 7-aminocephaLosporanic acid is from CPC . detailed in I iberated Example 6 Determination activity a) HPLC assay μΐ of 80 mM deacetyl-CPC are mixed with 100 to 140 μΐ of 250 mM potassium phosphate buffer, pH 5.0, and 10 to 50 μΐ of enzyme solution and incubated at 33°C.

A 20 μΙ-sample is taken every 10 minutes. The reaction is stopped with 20 μΐ of methanol. It is centrifuged and diluted with water in the ratio 1:10. A 10 μΐ sample is investigated by HPLC for the 7-aminocephalosporanic acid content.

Stationary phase: C-18 silica gel Mobile phase: ΚΗρΡθ4 50 mM in HgO/MeOH (80:20) + 0.001 % tetrabutylammonium sulfate b) Photometric assay 600 μΐ of utamyl-p-nitroanilide (166 μΜ) 300 yl of potassium phosphate buffer, pH 5.7, 50 mM 100 yl of culture solution are mixed together and incubated at 37°C.

Claims (7)

1. Patent cleiais

1. GIuta my 11ranspeptidase having the following propert ies - a molecular weight of 40,000 to 80,000, - an isoelectric point at pH 4.4 to 5.9, - for L-J^gIutamy I paranitroaniIide as substrate a pH optimum in the range 6.5 to 10, and a Km of 9 to 36 ym at pH 8, and - hydrolysis of G~aminoadipinyI-mono amino compounds of the formula I HOOC-CH- (CH ? ) ,-C-NH-R 1 1 z II NH 2 o (I) i n which R 1 denotes amino acids, dipeptides, cephems, cephams Ο r derivatives thereof. A process for the preparation of the J-gIutamyItrans peptidase as claimed in claim 1, which comprises cultivation of bacteria of the genera Pseudomonas, Proteus, Arthrobacter and Bacillus in a nutrient medium until GTP as claimed in claim 1 accumulates in the nutrient medium. 3. The process glutamylt r a n s p e p subtilis IFO 302 4 „ The use of in claim 1 for t compounds of the HOOC- C HOOC-CH- (Ci^-C-NH-R 1 NH O O (I) in which R denotes amino acids, dipeptides, cephems, cephams or derivatives thereof. The use as claimed in claim 4 , wherein the compound 1 the formula I in which R denotes the radical COOH in which R c is hydrogen, OH or -O-C-CHj is hydrolyzed, 0

2. 6» The use as claimed in claim 4 or 5, wherein the hydrolysis is carried out at a pH of 6.0 to 8.0. 10

3. 7 - The use as claimed in one or more of claims 4 to 6, wherein the hydrolysis is carried out at 28 to 38°C.

4. 8. 7> -Glutamyltranspeptidase according to claim 1, substantially as hereinbefore described.

5. 9. A process according to claim 2 for the preparation of 15 ^'-glutamyltranspept idase p substantially as hereinbefore described and exemplified.

6. 10. X-Glutamyltranspeptidase whenever prepared by a process claimed in a preceding claim.

7. 11. Use according to claim 4 f substantially as hereinbefore 20 described.