AU616450B2

AU616450B2 - A process for the enzymatic hydrolysis of alpha- aminoadipinyl-monoamino compounds

Info

Publication number: AU616450B2
Application number: AU27323/88A
Authority: AU
Inventors: Werner Aretz; Klaus Sauber
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1987-12-21
Filing date: 1988-12-20
Publication date: 1991-10-31
Anticipated expiration: 2008-12-20
Also published as: JP2816167B2; HU202917B; AU2732388A; DK710588A; CZ847388A3; DK710588D0; DE3743323A1; EP0321849A3; KR0149656B1; EP0321849B1; IE883807L; PT89282A; KR890010211A; DE3882503D1; CZ283529B6; PT89282B; CA1317246C; ZA889442B; EP0321849A2; HUT49168A

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

4; Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION (ORIGINAL) 616450 616450 Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: *Priority: p Related Art:

C

bO C SName of Applicant: ,Address of Applicant: HOECHST AKTIENGESELLSCHAFT Bruningstrasse, D-6230 Frankfurt/Main Federal Republic of Germany.

Actual Inventor: WERNER ARETZ and KLAUS SAUBER C 0 Address for Service EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: A PROCESS FOR THE ENZYMATIC HYDROLYSIS OF (c-AMINOADIPINYL- MONOAMINO COMPOUNDS The following statement is a full description of this invention, including the best method of performing it known to 1.

Ata.;^

L~;

la A PROCESS FOR THE ENZYMATIC HYDROLYSIS OF Oa-AMINOADIPINYL- MONOAMINO COMPOUNDS Description A process for the enzymatic hydrolysis of (-aminoadipinyl-monoamino compounds y-Glutamyltranspeptidases (y-GTP hereinafter) play an important part in amino acid metabolism and in the glutathione cycle in animal tissues and in microorganisms [Meth. Enzymol. 77, 237 (1981)]. They are responsible for the transport of various amino acids in the form of their y-glutamyl derivatives, the 1 0 formation of polyglutamic acid in Bacilli, and the breakdown of glutathione (y-glutamylcysteinyl-glycine).

It has already been proposed (EP 0,275,901) to use y-GTP to hydrolyze adipinyl- or glutaryl-monoamino compounds.

It has now been found, surprisingly, that the y-GTP the subject of this invention 1 5 catalyzes the hydrolysis of a-aminoadipinyl-monoamino compounds of the formula I HOOC- CH-(CH 2 3 C-NH-R1 (I) I I I

NH

2

O

S 2 in which R1 denotes amino acids, dipeptides, cephems, cephams or derivatives thereof.

20 This is all the more surprising because it has hitherto been assumed that neither C4 nor C6 side-chains are accepted by the active center of prior art y-GTP (see Agric.

Biol. Chem. 42, 1978, pages 371-81). The y-GTP of the instant invention is able to accept C4 C6 side-chains and moreover C 4 C6 side-chains wherein R1, as defined below, is an amino acid, a dipeptide, cephem, cepham or derivatives thereof.

2 5 Hence the invention relates to: S: 25 1. A y-glutamyltranspeptidase having the following properties o e oo il

I

2 a molecular weight of 40,000 to 80,00, an isoelectric point at pH 4.4 to 5.9, for gLutamyLparanitroanilide as substrate a pH optimum in the range 6.5 to 10, and a Km of 9 to 36 pM at pH 8, and hydrolysis of f.-aminoadipinyl-monoamino compounds of the formuLa I

HOOC-CH-(CH

2 3

-C-NH-R

1

(I)

NH

2

O

in which R1 denotes amino acids, dipeptides, cephems, cephams 2* A or derivatives thereof, 15 2. A process for the preparation of the /J-glutamyltranspeptidase 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 20 the nutrient medium.

3. The use of the J-glutamyltranspeptidase having the properties mentioned in 1. for the hydrolysis of *-aminoadipinyl-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 4/-glutamyLtranspeptidase 'GTP) catalyzes the hy- Sdrolysis of a-aminoadipinyl-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.

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, C: i-i i 3 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- y-glutamylparanitroanilide 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 Im, preferably 15 to 20 pm, in particular 17.8 gim, at pH 8.

The y-GTP according to the invention is irreversibly inhibited 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-aminocephalosporanic acid.

The y-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 cultivaied in a nutrient medium until y-GTP accumulates in the nutrient medium. suitable examples are: Pseudomonas putida ATCC 17390, Pseudomonas aeruginosa NCTC 10701, Proteus 15 vulgaris ATCC 9634, Arthrobacter parafineus ATCC 31917 as well as Pseudomonas fragi o. DSM 3881 and Bacillus subtilis IFO 3025. The enzyme is particularly preferably obtained from Bac. subitlis IFO 3025. Mutants and variants of the said microorganisms Sare also suitable. All the organisms mentioned hereinbefore are reasonably available to the skilled addressee from the institutions mentioned.

20 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 S temperature range from about 20 to 370C, preferably at about 25 to 30 0 C, in particular S0* at 28 to 300C. Fermentation is carried out in a pH range between 5 and 8.5, preferably 25 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. Synthesis of the y-GTP starts

O@

in the late log phase and *Goo 4

W

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 15 solution can contain are chlorides, carbonates, sulfates or phosphates of the alkali metal or alkaline earth metals, Siron, zinc and manganese, but also ammonium salts and nitrates.

20 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- 25 products such as malt extract, to the nutrient solution.

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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 chromatography. 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 B-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, polyacryLohydrazides and other known substances fiom processes known from the literature (K.

Venkatsubramanian, Immob. Cells (1979), ACS Symposium Series, page 106).

The hydrolysis reaction is most suitably carried out at *0 about pH 6.6 to 8 and at a temperature of about 28 to 38 0

C.

The preferred compound of the formula I is that in which S 1 R denotes the radical 0•2 1. :CH2 R

COOH

in which R is hydrogen, OH or -O-C-CH 3 The -GTP has industrial importance in particular for obtaining 7-aminocephalosporanic acid from cephalosporin C.

To date however, a yeast (Trigonopsis variabilis) has al- S* ways been used to generate from cephalosporin C glutaryl- 25 7-aminocephalosporanic acid which only then could be hydrolyzed enzymatically, in a second reaction step, to give 7aminocephalosporanic acid. It is now possible with the process according to the invention to prepare 7-aminocephalosporanic acid from cephalosporin C in a single step.

SThe invention is described in more detail in the examples which follow. Unless otherwise indicated, percentage data relate to weight.

Example 1 The -GTP-producing microorganism strains are maintained on agar slants of the following composition:

~I

6 Glucose 1 Casein peptone 0.4 Meat extract 0.4 Yeast extract 0.05 Liver extract 0.05 NaCL 0.25 pH 7.2 The slant tubes are incubated at 28 C for 2 days. The cells 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 I Erlenmeyer flask of capacity 300 ml: 15 Peptone 1 Malt extract 0.5 S" pH 0 The flask is incubated at 30 0 C and 190 rpm in a rotary 20 shaker for 24 hours. 2.5 ml of this culture are used as inoculum for 50 ml of main culture: i Bacilli Peptone 0.12 '.25 Yeast extract 0.12 Glucose 0.25 Na lactate (60 5.6 ml

NH

4 CI 0.12

K

2 HP04 0.12

KH

2

PO

4 0.034 A* MgS04 x 7 H 2 0 0.025 NaCL 0.5 KCL 0.5 CaCI2 x 2 H 2 0 0.0015 i 35 MnCL 2 x 4 H 2 0 0.0007 Fe(NH 4 )citrate 0.00015 The culture is incubated at 28 0 C and a shaking frequency i- -1 @0 0

S

0

S

00 0@ 5.

0S S S @6

S

S S

S.

0 0 0 50 S 40 0 7of 190 rpm for 24 hours and is then harvested by centrifugation.

-fGTP activities of some strains are listed in the table which follows: Strain r'-GTP (mU/ml culture solution) B. subtiLis IFO 3025 IFO 3013 IFO 3335 Example 2 A preculture with Bac. subtilis IFO 3025 is cultured in 15 analogy to Example 1. 50 ml of this culture are used as inoculum for 2 L of main culture solution in a 5 L fermenter. The strain is cultured at 340C and a partial pressure of oxygen of 70 The formation of the -GTP is followed by photometry, and the culture is harvested 20 at the maximum enzyme titer. Under the given conditions, a -GTP titer of 150 mU/ml of culture solution is reached.

Example 3 25 9 L of culture solution are separated by means of crossflow filtration (exclusion limit 300,000 dalton) into culture filtrate and biomass. The culture filtrate obtained in this way contains a P-GTP activity of 1350 U.

The enzyme is precipitated by addition of ammonium sulfate to 70 saturation and is taken up again in 1/10 of the volume. After dialysis against 20 mM tris, pH the enzyme is further purified on a DEAE-cellulose column (DE 52, Whatman). The active eluates are combined and concentrated. A f-GTP product obtained in this way (containing about 25 U of -GTP/ml) is used for the conversions.

4a a 8 Example 4 The following mixture is chosen for preparative conversion of deacetyl-CPC: 100 pL of enzyme concentrate prepared as in Example 3, and 100 pl of 40 mM deacetyl-CPC dissolved in 20 mM potassium phosphate buffer, pH 7.3, are incubated at a temperature of 33 0

C.

Up to 16 deacetyl-7-aminocephalosporanic acid are produced under the chosen conditions.

Example 15 With incubation conditions analogous to those detailed in J o• Example 4, 3 7-aminocephalosporanic acid is liberated from CPC.

Example 6 Determination of -GTP activity 6 S a) HPLC assay 25 50 Lh of 80 mM deacetyl-CPC are mixed with 100 to 140 o pl of 250 mM potassium phosphate buffer, pH 5.0, and 10 to 50 pL of enzyme solution and incubated at 33 C.

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

i Stationary phase: C-18 silica gel Mobile phase: KH 2

PO

4 50 mM in H 2 0/MeOH (80:20) 0.001 tetrabutylammonium sulfate b) Photometric assay 600 pl of glutamyl-p-nitroaniLide (166 pM) 9- 300 p.L of potassium phosphate buffer, pH 5.7, 50 mM and 100 piL of cuLture solution are mixed together and incubated at 37 0

C.

E 405 9620 1 mo L .c m *4 p

S

S. S a.

S *S

S.

d S

S

**S

55 0

S.

S 0 5* a 54

S

4.* 0 S 55 0

S

OSS.

'55.

S. S 4 0.

t

Claims

1. Substantially purified y-Glutamyltranspeptidase (y-GTP) having the following characteristics a molecular weight of 40,000 to 80,000, an isoelectric point at pH 4.4 to 5.9, for L- y-glutamylparanitroanilide as substrate a pH optimum in the range 6.5 to 10, and a Km of 9 to 36 ipm at pH 8, and the ability to hydrolyse a-aminoadipinyl-monoamino compounds of the formula I HOOC-CH-(CH 2 3 C-NH-R1 (I) I II NH 2 0 in which R1 denotes amino acids, dipeptides, cephems, cephams or derivatives thereof.

2. y-GTP obtained by fermentation of Bacillus subtilis IFO 3025 having a molecular weight of 40,000 to 80,000, an isoelectric point at pH 4.4 to 5.9, a pH S. optimum of 6.5 to 10 with L-y-Glutamylparanitroanilide as substrate, a Km of 9 to 36 p.m at pH 8 and the ability to catalyse the hydrolysis of a-aminoadipinyl- monoamino compounds of the formula I HOOC-CH-(CH 2 3 C-NH-R1 (I) S|I i II NH 2 O in which R denotes amino acids, dipeptides, cephems, cephams or derivatives thereof.

3. A process for the preparation of the y-GTP as claimed in claim 1 or 2 which comprises cultivation of Bacillus subtilis IFO 3025 in a nutrient medium to elicit production of said y-GTP followed by isolation of said y-GTP. j I 11

4. The use of the y-glutamyltranspeptidase as claimed in claim 1 or 2 for the catalysis of the hydrolysis of a-aminoadipinyl-monoamino compounds of the formula I HOOC-CH-(CH 2 3 C-NH-R1 NH 2 in which R1 denotes amino acids, dipeptides, cephems, cephams or derivatives thereof. The use as claimed in claim 4, wherein the compound of the formula I in which R 1 denotes the radical S N C r F ip o CH R 2 COOH in which R2 is hydrogen, OH or C-CH 3 is hydrolyzed.

I I 0 O

6. The use as claimed in claim 4 or 5, wherein the hydrolysis is carried out at a pH of 6.6 to II.~ 12

7. The use as claimed in one or more of claims 4 to 6, wherein the hydrolysis is carried out at 28 to 380C. DATED this 23rd day of July, 1991. HOECHST AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA DBM/KJS/ML o e