IE883807L - A process for the enzymatic hydrolysis of ó-aminoadipinyl-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

9 ■; 6 6 - 1 - /f-Glutamyltranspeptidases (GTP hereinafter) play an i m — portant part in amino acid metabolism and in the glutathione cycle in animal tissues and in microorganisms C^eth. Enzymol. 77 , 237 (1981)D. They are responsible tor 5 the transport of various amino acids in the form of their (j -glutamyl derivatives,, the formation of polyglutamic acid in Bacilli,, and the breakdown of glutathione (^-glutamyl-cysteinyl-glyc ine), It has already been proposed (EP 0,275,901) to use GTP 10 to hydrolyze adipinyl- or glutaryl-monoamino compounds™ It has now been found,, surprisingly, that ^GTP catalyzes the hydrolys i s of c-aminoadipinyl-monoamino compounds of the formula I HOOC-CH-(CH2)3-C-NH-R1 (I) NH2 0 15 in which 1 6? denotes amino acids, dipept ides, cephems, cephams or derivatives thereof.

This is all the more surprising because it has hitherto been assumed that neither C 4 nor C § side-chains ere 20 accepted by the active center of GTP (see Agric. Biol. Chen. 42, 1978, pages 371 - 81).

Hence the invention relates to: 1. A ^"-glutamyltranspept idase having the following properties - 2 - - a molecular weight of 40,000 to 80^000, - an isoelectric point at pH 4.4 to 5.9, - for L-ff-glutamylparanitroanilide as substrate a pH optimum in the range 6-5 to 10, and a Km of 9 to 36 iiM at pH 8, and - hydrolysis of cs-aminoadipinyl-monoamino compounds of the formula I HOOC-CH-(CH?)o-C-NH-R1 (I) I ^ J II NH2 O in which 1 R denotes amino acids,, dipeptides, cephems, cephams or- derivatives thereof- 15 2. A process for the preparation of the ^glutamyltrans-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 20 the nutrient medium. 10 3. The use of the jT~glutamyltranspeptidase having the properties mentioned in 1 „ for the hydrolysis of <*-aminoadipinyl-monoamino compounds of the formuLa I. 25 The invention is described in detail hereinafter^ especially in its preferred embodiments. The invention is furthermore defined in the patent claims* 30 The glucernyltranspept idase (/p?GTP) catalyzes the hydrolysis of a-aminoadipinyl-monoamino compounds of the formula I, as defined above^ to give the corresponding acid and the monoamino compound* 7-Aminocephalosporanic T> acid derivatives are preferably used as substrate. 35 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,.00Q, 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-^-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 y M , preferably 15 to 20 y H , in particular 17.8 yH, at pH 8.

The .J"GTP according to the invention is irreversibly inhibited in the presence of azaserine or iodoacetamide. The enzyme shows reversible inhibition in the presence of copperf mercury and a mixture of serine and borate,, as well as in the presence of 7-aminocephalosporanic 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 paraf ineus ATCC 31917 cs well as Pseudomonas fregi DSffl 3881 and Bacillus subtilis I F0 3025. The ensyme is particularly preferably obtained from Bac. subtilis IFO 3025. Mutants and variants of the said microorganisms are also suitable.

The microorganisms are cultured aerob i c a 11 y, 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» Fermentat ion is carried out in a pH range between 5 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 A -GTP starts in the late log phase and 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 /j^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 nitrates.

The addition of assimilable carbohydrates increases the yield of biomass. Carbohydrates are also added in the abovement ioned 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 chromatography. The enzyme can be coupled by conventional methods (Colowick and Kaplan, Heth. Enzymol., vol. XLIV).

It is possible to use for the enzymatic reaction both 10 15 20 25 30 - 5 - whole cells in free or immobilized form, with the addition of 8-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-carrsgsenan, polyacrylohydrazides-and other known substances from processes known from the literature (K, Venkatsubraraanian, Immob. Cells (19 79), ACS Symposium Series,, page 106) .

The hydrolysis reaction is most suitably carried out at about pH 6.6 to 8 and at a temperature of about 28 to 38°C The preferred compound of the formula I is that in which A R1 denotes the radical COOH in which R ^ is hydrogen, OH or -0-C-CH-s.

I The (y-GTP has industrial importance in particular for obtaining 7-aminocepha Iosporanic acid from cephalosporin C . To date however,, a yeast (Trigonopsis variabilis) has always been used to generate from cephalosporin C glutaryl-7-aainocephalosporanic acid which only then could be hydro ly^ed enzymatically,, in a second reaction step,, to give 7 -aesinocephalosporanic acid. It is now possible with the process according to the invention to prepare 7-am i no-cepha Iosporanic 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.

Example 1 The GTP-produc ing microorganism strains are maintained on agar slants of the following compositions - 6 - Glucose 1 % Casein peptone 0. 4 % Heat extract 0. 4 % Yeas t extract 0. 05 % Liver extract 0. 05 % NaCl 0. 25 «!/ A pH 7.2 Ths slant tubes are incubated at 28°C for 2 days. The 10 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 Erlen in eyer flask of capacity 300 mis 15 Peptone Malt extract pH 7.0 The flask is incubated at 30°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: Bacilli Peptone 0.12 % 25 Yeast extract 0.12 % Glucose 0.25 % Ha I ac t at e (60 %) 5.6 m I iH^Cl 0.12 X K2HPO4 0.12 % 30 KH2PO4 0.034 % Mgso4 X 7 h2o 0.025 % MaC I 0.5 % KCL 0.5 cy AJ CaC12 x Z h2o 0.0015 w /o 35 HnCIp x 4 h2o 0.0007 o* FeCNH^Jci t r a t e 0.00015 % I fo 0.5 % The culture is incubated at 28°C and a shaking frequency of 190 rpm for 2 4 hours and is then harvested by centrifugal ion.

(^-G TP activities of some strains are Listed in the table which follows: Strain ^-GTP (mil/ml culture solution) B. subtilis IFO 3025 60 IF0 3013 15 IFO 3335 25 Example 2 A preculturs with B a c „ subtilis IFO 3025 is cultured in an a logy to Example 1- 50 ml of this culture are used as inoculum for 2 I of main culture solution in a 5 I fer-raenter. The strain is cultured at 34°C and a partial pressure of oxygen of 70 % „ The formation of the /j-GTP is followed by photometry, and the culture is harvested at the maximum enzyme titer. Under the given conditions, a ^-GTP titer of 150 mU/ml of culture solution is reached.

Example 3 9 1 of culture solution are separated by means of cross-flow filtration (exclusion limit 300,000 dalton) into culture filtrate and biomass. The culture filtrate obtained in this way contains a 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 mini 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 fp-GTP product obtained in this way (con-taining about 25 U of A -GTP/ml) is used for the conversions Example 4 - 8 - The following mixture is chosen for preparative conversion of deacetyL-CPC: 100 y I of 40 mM deacetyl-CPC dissolved in 20 m M potassium phosphate buffere pH 7.3,, are incubated at a temperature of 33°C . 10 Up to 16 % deacetyI-7-aminocepha Iosporanic acid are produced under the chosen conditions.

Example S 15 With incubation conditions analogous to those detailed in Example 4 3 % 7-aminocephalosporanic acid is liberated from CPC.

E x ampIe 6 5 100 yl of enzyme concentrate prepared as in Example 3,, and 20 Determination of -GTP activity a) HPLC assay 25 50 y I of 80 m M deacetyl-CPC are mixed with 100 to 140 Ul of 250 mH potassium phosphate buffer^ pH 5.0^ and 10 to 50 yl of enzyme solution and incubated at 33°C„ A 20 yl-sample is taken every 10 minutes. The reaction 30 is stopped with 20 y I of methanol. It is centrifuged and diluted with water in the ratio 1:10. A 10 y I sample is investigated by HPLC for the 7-aminocephalosporanic acid content. 35 Stationary phases C — 18 silica gel Mobile phases KHgPO^ 50 mfi in h^O/MeOH (80:20) + 0.001 % tetrabutyIammonium sulfate b) Photoniet r i c assay 600 yl of L- -gIutamy I-p-nitroaniIide (166 yH) - 9 - 300 yl of potassium phosphate buffer, pH 5„ 7, 50 mM and 100 pi of culture solution are mixed together and incubated at 37°C „ E 1 405 = 9620 mo I -cm

Claims (1)

1. Patent claims - 10 - Glutamyltranspeptidase 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"1glutamylparanitroanilide as substrate a pH optimum in the range 6.5 to 10, and a Km of 9 to 36 pin at pH 8,, and - hydrolysis of a~aminoadipinyI-monoamino compounds of the formula I HOOC-CH- (CH?) -,-C-NH-R1 (I) I ^ J l| NH2 o 1 R denotes amino acids, dipeptides, cephems, cephams in which 1 R denot or derivatives thereof. A process for the preparation of the ^-glutamyltranspeptidase as claimed in claim 1, which comprises cultivation of bacteria of the genera Pseudomonas, Proteus, Arthrobacter and Bacillus in a nutrient medium until ^p-GTP as claimed in claim 1 accumulates in the nutrient medium. The process as claimed in claim 2 , wherein the glutamyltranspeptidase is obtained by cultivation of B. subtilis IFO 3025. The use of the ^glutamyltranspeptidase as claimed in claim 1 for the hydrolysis of ct-aminoadipinyl-monoa#ino compounds of the formula I hooc-ch-(ch2)3-c-nh-r1 (i) nh2 o - 11 1 R denotes amino acids, dipeptides, cephesns, cephams or in which 1 R denot derivatives thereof The use as claimed in claim 4r wherein the compound i of the formula I in which R denotes the radical 2 in which R is hydrogen^ OH or -O-C-GHj is hydrolyzed. 0 6- The use as claimed in claim 4 or 5, wherein the hy drolysis is carried out at a pH of 6.6 to 8.0. 10 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. 8. 7> -Glutamyltranspeptidase according to claim 1, substantially as hereinbefore described. 9. h process according to claim 2 for the preparation of 15 ^-glutamyltranspeptidase, substantially as hereinbefore described and exemplified. 10. #-GlutamyItranspeptidase whenever prepared by a process claimed in a preceding claim. 11. Use according to claim 4, substantially as hereinbefore 20 described. f. r. kelly & co., agents for the applicants.