CA1317246C - Process for the enzymatic hydrolysis of -aminoadipinyl-monoamino compounds - Google Patents
Process for the enzymatic hydrolysis of -aminoadipinyl-monoamino compoundsInfo
- Publication number
- CA1317246C CA1317246C CA000586515A CA586515A CA1317246C CA 1317246 C CA1317246 C CA 1317246C CA 000586515 A CA000586515 A CA 000586515A CA 586515 A CA586515 A CA 586515A CA 1317246 C CA1317246 C CA 1317246C
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- Prior art keywords
- aminoadipinyl
- gtp
- hydrolysis
- glutamyltranspeptidase
- monoamino compounds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/104—Aminoacyltransferases (2.3.2)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
- C12P35/02—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by desacylation of the substituent in the 7 position
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- Enzymes And Modification Thereof (AREA)
Abstract
Abstract of the disclosure A process for the enzymatic hydrolysis of .alpha.-aminodipinyl-monoamino compounds ?-Glutamyltranspeptidase which can be prepared by fermen-tation can be used to hydrolyze .alpha.-aminoadipinyl-monoamino compounds, especially D-(?)-.alpha.-aminoadipinyl-7-amino-cephalosporanic acid.
Description
13~72~
HOECHST AKTIENGESELLSCHAFT HOE 87tF 383 Dr. KH~AP
Description A process for the enzymatic hydrolysis of ~-aminoadipinyl-monoamino compounds ~-Glutamyltranspeptidases (~-GTP hereinafter) play an im-portant part in amino acid metabolism and in the gluta-thione cycLe ;n animal tissues and in m;croorganisms ~[Meth. Enzymol. 77, 237 (1981)]. They are responsible for -the transport of various amino acids in the ~orm of their ~-glutamyl derivatives, the formation of polyglutamic acid in Bacilli, and the breakdown of glutathione (~-glutamyl-cysteinyl-glycine).
It has already been proposed (EP 0,275,901) to use ~-GTP
to hydrolyze adipinyl- or glutaryl-monoamino compouncls.
lt has now been found, surprisingly, that ~GTP catalyzes the hydrolysis of ~-aminoadipinyl-monoamino compounds of the formula I
HOOC- ~CH- (CH2)3-C-NH-R
in which R1 denotes amino acids, dipeptides, cephems, cephams or derivat;ves thereof.
3~ 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 ~ GTP (see Agric. ~iol.
Chem. 42, 1978, pages 371 - 81).
Hence the invention relates to:
1. A ~-glutamyltranspeptidase having the following properties :~3~72~6 - a molecular weight of 40,000 to 80,000, - an isoelectric point at pH 4.4 to 5.9, - for L-~-glutamyLparanitroanilide 3s substrate a pH optimum in the range 6.5 to 10, and a Km of 9 to 36 ~M at pH 8, and - hydroLysis of ~-aminoadipinyl-monoamino compounds of the formula I
HOOC-CH- (CH2)3-C-NH-Rl (I) in which R1 denotes amino acids, dipeptides, cephems, cephams or derivatives thereof~
HOECHST AKTIENGESELLSCHAFT HOE 87tF 383 Dr. KH~AP
Description A process for the enzymatic hydrolysis of ~-aminoadipinyl-monoamino compounds ~-Glutamyltranspeptidases (~-GTP hereinafter) play an im-portant part in amino acid metabolism and in the gluta-thione cycLe ;n animal tissues and in m;croorganisms ~[Meth. Enzymol. 77, 237 (1981)]. They are responsible for -the transport of various amino acids in the ~orm of their ~-glutamyl derivatives, the formation of polyglutamic acid in Bacilli, and the breakdown of glutathione (~-glutamyl-cysteinyl-glycine).
It has already been proposed (EP 0,275,901) to use ~-GTP
to hydrolyze adipinyl- or glutaryl-monoamino compouncls.
lt has now been found, surprisingly, that ~GTP catalyzes the hydrolysis of ~-aminoadipinyl-monoamino compounds of the formula I
HOOC- ~CH- (CH2)3-C-NH-R
in which R1 denotes amino acids, dipeptides, cephems, cephams or derivat;ves thereof.
3~ 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 ~ GTP (see Agric. ~iol.
Chem. 42, 1978, pages 371 - 81).
Hence the invention relates to:
1. A ~-glutamyltranspeptidase having the following properties :~3~72~6 - a molecular weight of 40,000 to 80,000, - an isoelectric point at pH 4.4 to 5.9, - for L-~-glutamyLparanitroanilide 3s substrate a pH optimum in the range 6.5 to 10, and a Km of 9 to 36 ~M at pH 8, and - hydroLysis of ~-aminoadipinyl-monoamino compounds of the formula I
HOOC-CH- (CH2)3-C-NH-Rl (I) in which R1 denotes amino acids, dipeptides, cephems, cephams or derivatives thereof~
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 8acillus in a nutrient medium until the said ~-GTP accumulates in the nutrient medium.
3. The use of the ~-glutamyltranspeptidase having the properties mentioned in 1. for the hydrolysis of ~-aminoad;pinyl-monoamino compounds of the formula I.
The invention is described in detail hereinafter, especial-ly in its preferred embodiments. The invention is further-more defined in the patent claims.
The ~-glutamyltranspeptidase ( ~ GTP) catalyzes the hy-drolys;s of ~-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, _ 3 _ ~3~72~
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- ~ glutamylparanitroan;lide as sub-strate 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 ~M, preferably 15 to 20 uM, in particular 17.
~M, at pH 8.
The ~-GTP according to the invention is irreversibly in-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-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 cul-tivated in a nutrient medium until ~-GTP accumulates in the nutrient medium. Suitable examples are: Pseudomonas putida ATCC 17390, Pseudomonas aeruginosa NCTC 10701, Proteus vulgaris ATCC 9634, Arthrobacter parafineus ATCC
31917 as well as Pseudomonas fragi DSM 38~1 and Bacillus subtilis IF0 3025. The enzyme is particularly preferably obtained from ~ac. subtilis IF0 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 ~ermenters, where appropriate with air or oxygen being passed in. The fermentation can take place in a temperature range from about 20 to 37C~
preferably at about 25 to 30C, in particular at 2~ to 30C. Fermentation is carried out in a pH range between 5 and ~.5, preferably between 5.5 and 8Ø Under these conditions, the culture broth shows considerable accumu-lation of the enzyme in general after 1 tG 3 days~ Syn-thesis of the ~-GTP starts in the late log phase and _ 4 _ ~ 3 ~ 7 2lI~
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 pho-tometry.
The nutrient solution used to produce the ~ GTP contains 0.2 to 5 %, preferably 0.5 to 2 %, organic nitrogen com-pounds and inorganic salts. Suitable organic nitrogen compounds are: amino acids, peptones, furthermore meat ex-tracts, m;lled seeds, for example of corn, wheat, beans, soybeans or the cotton plant, distillation residues from the manufacture of alcohol, meat meals or yeast ex-tracts. Exa~ples of inorganic salts which the nutrient solution can conta;n are chlorides, carbonates, sulfates or phosphates of the alkali metal or alkaline earth metals~
;ron, z;nc and manganese, but also ammonium salts and nitrates.
The addition of assimilable carbohydrates increases the y;eld of biomass. Carbohydrates are also added in the abovementioned concentrations. It is possible to add as preferred carbon source for example sugars, such as glu-cose 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 pre~
l;minary 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 (Colo~ick and Kaplan, Meth. Enzymol., vol. XLIV).
It is possible to use for the enzymatic reaction both ~3~'72'~
whole cells in free or immobil;zed form, with the addition of ~-lactamase inactivators, for example clavulanic acid or thienamycin, and the isolated enzyme ~hich can also be carrier-bound. Examples of ~uitable materials for the immobilization of whole cells are ch;tosan~ algin-ate, K-carrageenan, polyacrylohydrazides and other known substances from processes known from the literature (K.
Yenkatsubramanian, Immob. Cells (197~), ACS Symposium Series, page 106).
The hydrolysis reaction is most suitably carried out at about pH 6.6 to ~ and at a temperature of about 28 to 38C.
The preferred compound of the formula I is that in which R1 denotes the radical ~ S
~N~CH2R
COOH
in which R2 ;5 hydrogen~ OH or -0-8-tH3.
The ~-GTP has industrial importance in particular for ob-taining 7-aminocephalosporanic acid from cephalosporin C.
To date however, a yeast (Trigonopsis variabilis) has al-~ays been used to generate- from cephalosporin C glutaryl-7-aminocephalosporanic acid which only then could be hydro-lyzed enzymatically, in a second reaction step, to give 7-aminocephalosporanic acid. It is now possible with the process according to the invention to prepare 7-amino-cephalosporanic acid from cephalosporin C in a single step.
The invention is described in more detail in the examples which follow. Unless otherwise indicated, percentage clata relate to we;ght.
Exa~ple 1 The ~-GTP-producing microorganism strains are maintained on agar slants of the follow;ng composition:
- 6 - ~3 6lucose Casein peptone0.4 %
Meat extract 0.4 %
Yeast extract 0.05 5 Liver extract 0.05 %
NaCl 0.25 %
pH 7.2 The sLant tubes are incubated at 28C for 2 days. The cells are then rinsed off with 10 ml of phys;ological saliner and l ml of this suspension ;s used to inoculate a 50 ml preculture of the following composition in an Erlenmeyer fLask of capacity 300 ml:
Peptone 1 %
Malt extract 0.5 %
pH 7.0 The flask is incubated at 30C and 190 rpm in a rotary shaker for 24 hours. 2.5 ml of this culture are used as inoculum for 50 ml of ma;n culture:
Bacilli -Peptone 0.12 %
25 Yeast extract 0.12 %
Glucose 0.25 %
: Na lactate (60 %) 5.6 ml NH4Cl 0.12 %
K2HPo4 0.12 %
KH2P04 0.034 %
MgS04 x 7 H20 0.025 %
NaCl 0.5 %
KCl 0~5 %
CaCl2 x 2 H20 0.0015 %
MnCl2 x 4 H20 O.Oû07 %
Fe(NH4)c;trate 0~00015 %
The culture is incubated at 28C and a shaking frequency _ 7 _ ~ 3 ~
of 190 rpm for 24 hours and is then harvested by cent-rifugation.
~ GTP activities of some strains are listed in the table which follows:
Strain _ -GTP ~mU/ml culture solution) 8. subtilis IF0 3025 S0 " " IF0 3013 15 10 " " IF0 3335 25 Examp(e 2 A preculture with Pac. subtilis lF0 3025 is cultured in analogy to Example 1. 50 ml of this culture are used as inoculum for 2 l of main culture solution in a 5 l fer-menter. The strain is cultured at 34C and a partial pressure of oxygen of 70 %. The formation of the ~-GTP
;s 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.
Exa~ple 3 9 L of culture solution are separated by means of cross-flow filtration ~exclusion limit 300,000 dalton) into culture filtrate and biomass. The culture filtrate ob-tained in this way contains a ~-GTP activity of 1350 Un The enzyme is precipitated by addition of ammonium sul ; fate to 70 % saturation and is taken up again in 1/10 of the volume. After dialysis against 20 mM tris, pH 8~0r the enzyme is further purified on a DEAE-cellulose column (DE 52, Whatman). The active eluates are combined and concentrated. A ~-GTP product obtained in this way (con-taining about 25 U of ~-GTP/ml) is used for the conversions.
~3~72~
Example 4 The following mixture is chosen for preparative conversion of deacetyl-CPC:
100 ~l of enzyme concentrate prepared as in Example 3, and 100 ~l of 40 mM deacetyl-CPC dissolved in 20 mM potassium phosphate buffer, pH 7.3, are incubated at a temperature of 33C.
Up to 16 % deacetyl-7-aminocephalosporanic acid are pro-duced under the chosen conditions.
Exa~ple S
With incubation conditions analogous to those detailed in Example 4, 3 % 7-aminocephalosporanic acid is liberated from CPC.
Example 6 Determination of ~-GTP activity a) HPLC assay 50 ~l of 80 mM deacetyl-CPC are mixed with 100 to 140 ~l of 250 mM potassium phosphate buffer, pH 5.0, and 10 to 50 ~l of enzyme solution and incubated at 33C.
A 20 ~l-sample is taken every 10 minutes. The reaction is stopped with 20 ~l of methanol. It is centrifuged and diLuted with water in the ratio 1:10. A 10 ~l sample is investigated by HPLC for the 7-aminocephalo-sporanic acid content.
Stationary phase: C-18 silica gel Mobile phase: KH2P04 50 mM in H20/MeOH ~80:20) ~ 0.001 % tetrabutylammoniu~ sulfate b) Photometric assay 600 ~l of L-~-glutamyl-p-nitroanilide (166 ~M) - 9 13~l ~2~
300 ~l of potassium phosphate bu~fer, pH 5.7, 5Q mM and 100 ~l of culture solution are mixed together and incubated at 37C.
405 = 96Z0 mol . cm
The invention is described in detail hereinafter, especial-ly in its preferred embodiments. The invention is further-more defined in the patent claims.
The ~-glutamyltranspeptidase ( ~ GTP) catalyzes the hy-drolys;s of ~-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, _ 3 _ ~3~72~
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- ~ glutamylparanitroan;lide as sub-strate 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 ~M, preferably 15 to 20 uM, in particular 17.
~M, at pH 8.
The ~-GTP according to the invention is irreversibly in-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-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 cul-tivated in a nutrient medium until ~-GTP accumulates in the nutrient medium. Suitable examples are: Pseudomonas putida ATCC 17390, Pseudomonas aeruginosa NCTC 10701, Proteus vulgaris ATCC 9634, Arthrobacter parafineus ATCC
31917 as well as Pseudomonas fragi DSM 38~1 and Bacillus subtilis IF0 3025. The enzyme is particularly preferably obtained from ~ac. subtilis IF0 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 ~ermenters, where appropriate with air or oxygen being passed in. The fermentation can take place in a temperature range from about 20 to 37C~
preferably at about 25 to 30C, in particular at 2~ to 30C. Fermentation is carried out in a pH range between 5 and ~.5, preferably between 5.5 and 8Ø Under these conditions, the culture broth shows considerable accumu-lation of the enzyme in general after 1 tG 3 days~ Syn-thesis of the ~-GTP starts in the late log phase and _ 4 _ ~ 3 ~ 7 2lI~
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 pho-tometry.
The nutrient solution used to produce the ~ GTP contains 0.2 to 5 %, preferably 0.5 to 2 %, organic nitrogen com-pounds and inorganic salts. Suitable organic nitrogen compounds are: amino acids, peptones, furthermore meat ex-tracts, m;lled seeds, for example of corn, wheat, beans, soybeans or the cotton plant, distillation residues from the manufacture of alcohol, meat meals or yeast ex-tracts. Exa~ples of inorganic salts which the nutrient solution can conta;n are chlorides, carbonates, sulfates or phosphates of the alkali metal or alkaline earth metals~
;ron, z;nc and manganese, but also ammonium salts and nitrates.
The addition of assimilable carbohydrates increases the y;eld of biomass. Carbohydrates are also added in the abovementioned concentrations. It is possible to add as preferred carbon source for example sugars, such as glu-cose 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 pre~
l;minary 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 (Colo~ick and Kaplan, Meth. Enzymol., vol. XLIV).
It is possible to use for the enzymatic reaction both ~3~'72'~
whole cells in free or immobil;zed form, with the addition of ~-lactamase inactivators, for example clavulanic acid or thienamycin, and the isolated enzyme ~hich can also be carrier-bound. Examples of ~uitable materials for the immobilization of whole cells are ch;tosan~ algin-ate, K-carrageenan, polyacrylohydrazides and other known substances from processes known from the literature (K.
Yenkatsubramanian, Immob. Cells (197~), ACS Symposium Series, page 106).
The hydrolysis reaction is most suitably carried out at about pH 6.6 to ~ and at a temperature of about 28 to 38C.
The preferred compound of the formula I is that in which R1 denotes the radical ~ S
~N~CH2R
COOH
in which R2 ;5 hydrogen~ OH or -0-8-tH3.
The ~-GTP has industrial importance in particular for ob-taining 7-aminocephalosporanic acid from cephalosporin C.
To date however, a yeast (Trigonopsis variabilis) has al-~ays been used to generate- from cephalosporin C glutaryl-7-aminocephalosporanic acid which only then could be hydro-lyzed enzymatically, in a second reaction step, to give 7-aminocephalosporanic acid. It is now possible with the process according to the invention to prepare 7-amino-cephalosporanic acid from cephalosporin C in a single step.
The invention is described in more detail in the examples which follow. Unless otherwise indicated, percentage clata relate to we;ght.
Exa~ple 1 The ~-GTP-producing microorganism strains are maintained on agar slants of the follow;ng composition:
- 6 - ~3 6lucose Casein peptone0.4 %
Meat extract 0.4 %
Yeast extract 0.05 5 Liver extract 0.05 %
NaCl 0.25 %
pH 7.2 The sLant tubes are incubated at 28C for 2 days. The cells are then rinsed off with 10 ml of phys;ological saliner and l ml of this suspension ;s used to inoculate a 50 ml preculture of the following composition in an Erlenmeyer fLask of capacity 300 ml:
Peptone 1 %
Malt extract 0.5 %
pH 7.0 The flask is incubated at 30C and 190 rpm in a rotary shaker for 24 hours. 2.5 ml of this culture are used as inoculum for 50 ml of ma;n culture:
Bacilli -Peptone 0.12 %
25 Yeast extract 0.12 %
Glucose 0.25 %
: Na lactate (60 %) 5.6 ml NH4Cl 0.12 %
K2HPo4 0.12 %
KH2P04 0.034 %
MgS04 x 7 H20 0.025 %
NaCl 0.5 %
KCl 0~5 %
CaCl2 x 2 H20 0.0015 %
MnCl2 x 4 H20 O.Oû07 %
Fe(NH4)c;trate 0~00015 %
The culture is incubated at 28C and a shaking frequency _ 7 _ ~ 3 ~
of 190 rpm for 24 hours and is then harvested by cent-rifugation.
~ GTP activities of some strains are listed in the table which follows:
Strain _ -GTP ~mU/ml culture solution) 8. subtilis IF0 3025 S0 " " IF0 3013 15 10 " " IF0 3335 25 Examp(e 2 A preculture with Pac. subtilis lF0 3025 is cultured in analogy to Example 1. 50 ml of this culture are used as inoculum for 2 l of main culture solution in a 5 l fer-menter. The strain is cultured at 34C and a partial pressure of oxygen of 70 %. The formation of the ~-GTP
;s 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.
Exa~ple 3 9 L of culture solution are separated by means of cross-flow filtration ~exclusion limit 300,000 dalton) into culture filtrate and biomass. The culture filtrate ob-tained in this way contains a ~-GTP activity of 1350 Un The enzyme is precipitated by addition of ammonium sul ; fate to 70 % saturation and is taken up again in 1/10 of the volume. After dialysis against 20 mM tris, pH 8~0r the enzyme is further purified on a DEAE-cellulose column (DE 52, Whatman). The active eluates are combined and concentrated. A ~-GTP product obtained in this way (con-taining about 25 U of ~-GTP/ml) is used for the conversions.
~3~72~
Example 4 The following mixture is chosen for preparative conversion of deacetyl-CPC:
100 ~l of enzyme concentrate prepared as in Example 3, and 100 ~l of 40 mM deacetyl-CPC dissolved in 20 mM potassium phosphate buffer, pH 7.3, are incubated at a temperature of 33C.
Up to 16 % deacetyl-7-aminocephalosporanic acid are pro-duced under the chosen conditions.
Exa~ple S
With incubation conditions analogous to those detailed in Example 4, 3 % 7-aminocephalosporanic acid is liberated from CPC.
Example 6 Determination of ~-GTP activity a) HPLC assay 50 ~l of 80 mM deacetyl-CPC are mixed with 100 to 140 ~l of 250 mM potassium phosphate buffer, pH 5.0, and 10 to 50 ~l of enzyme solution and incubated at 33C.
A 20 ~l-sample is taken every 10 minutes. The reaction is stopped with 20 ~l of methanol. It is centrifuged and diLuted with water in the ratio 1:10. A 10 ~l sample is investigated by HPLC for the 7-aminocephalo-sporanic acid content.
Stationary phase: C-18 silica gel Mobile phase: KH2P04 50 mM in H20/MeOH ~80:20) ~ 0.001 % tetrabutylammoniu~ sulfate b) Photometric assay 600 ~l of L-~-glutamyl-p-nitroanilide (166 ~M) - 9 13~l ~2~
300 ~l of potassium phosphate bu~fer, pH 5.7, 5Q mM and 100 ~l of culture solution are mixed together and incubated at 37C.
405 = 96Z0 mol . cm
Claims (7)
1. ?-Glutamyltranspeptidase 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-?-glutamylparanitroanilide as substrate a pH optimum in the range 6.5 to 10, and a Km of 9 to 36 µm at pH 8, and - hydrolysis of .alpha.-aminoadipinyl-monoamino compounds of the formula I
(I) in which R1 denotes amino acids, dipeptides, cephems, cephams or derivatives thereof.
(I) in which R1 denotes amino acids, dipeptides, cephems, cephams or derivatives thereof.
2. A process for the preparation of the ?-glutamyltrans-peptidase as claimed in claim 1, which comprises culti-vation 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 as claimed in claim 2, wherein the ?-glutamyltranspeptidase is obtained by cultivation of B.
subtilis IF0 3025.
subtilis IF0 3025.
4. The use of the ?-glutamyltranspeptidase as claimed in claim 1 for the hydrolysis of .alpha.-aminoadipinyl-monoamino compounds of the formula I
(I) in which R1 denotes amino acids, dipeptides, cephems, cephams or derivatives thereof.
(I) in which R1 denotes amino acids, dipeptides, cephems, cephams or derivatives thereof.
5. The use as claimed in claim 4, wherein the compound of the formula I in which R1 denotes the radical in which R2 is hydrogen, OH or -O-?-CH3 is hydrolyzed.
6. The use as claimed in claim 4, wherein the hydrolysis is carried out at a pH of 6.6 to 8Ø
7. The use as claimed in any one of claims 4 to 6, wherein the hydrolysis is carried out at 28 to 38°C.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873743323 DE3743323A1 (en) | 1987-12-21 | 1987-12-21 | PROCESS FOR ENZYMATIC HYDROLYSIS OF (ALPHA) -AMINOADIPINYL-MONOAMINO COMPOUNDS |
| DEP3743323.7 | 1987-12-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1317246C true CA1317246C (en) | 1993-05-04 |
Family
ID=6343136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000586515A Expired - Fee Related CA1317246C (en) | 1987-12-21 | 1988-12-20 | Process for the enzymatic hydrolysis of -aminoadipinyl-monoamino compounds |
Country Status (14)
| Country | Link |
|---|---|
| EP (1) | EP0321849B1 (en) |
| JP (1) | JP2816167B2 (en) |
| KR (1) | KR0149656B1 (en) |
| AT (1) | ATE91711T1 (en) |
| AU (1) | AU616450B2 (en) |
| CA (1) | CA1317246C (en) |
| CZ (1) | CZ283529B6 (en) |
| DE (2) | DE3743323A1 (en) |
| DK (1) | DK710588A (en) |
| ES (1) | ES2058224T3 (en) |
| HU (1) | HU202917B (en) |
| IE (1) | IE62566B1 (en) |
| PT (1) | PT89282B (en) |
| ZA (1) | ZA889442B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE90382T1 (en) * | 1987-01-17 | 1993-06-15 | Hoechst Ag | USE OF GAMMA GLUTAMYL TRANSPEPTIDASE. |
| US5104800A (en) * | 1989-06-27 | 1992-04-14 | Merck & Co., Inc. | One-step cephalosporin c amidase enzyme |
| US5229274A (en) * | 1989-06-27 | 1993-07-20 | Merck & Co., Inc. | Gene encoding one step cephalosporin C amidase and expression thereof in recombinant bacillus |
| JP3036775B2 (en) * | 1990-02-07 | 2000-04-24 | 協和醗酵工業株式会社 | Method for producing r-glutamyl transpeptidase |
| JPH04281787A (en) * | 1991-03-04 | 1992-10-07 | Yoshihiro Asada | Method for producing γ-glutamyl transpeptidase |
| HU219370B (en) * | 1991-10-15 | 2001-03-28 | Gist Brocades Bv | Novel bioprocesses for preparing 7-aca and 7-adac |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6121097A (en) * | 1984-07-10 | 1986-01-29 | Asahi Chem Ind Co Ltd | Preparation of 7-aminocephalosporanic acid and derivative thereof |
| ATE90382T1 (en) * | 1987-01-17 | 1993-06-15 | Hoechst Ag | USE OF GAMMA GLUTAMYL TRANSPEPTIDASE. |
-
1987
- 1987-12-21 DE DE19873743323 patent/DE3743323A1/en not_active Withdrawn
-
1988
- 1988-12-15 DE DE8888120945T patent/DE3882503D1/en not_active Expired - Fee Related
- 1988-12-15 ES ES88120945T patent/ES2058224T3/en not_active Expired - Lifetime
- 1988-12-15 EP EP88120945A patent/EP0321849B1/en not_active Expired - Lifetime
- 1988-12-15 AT AT88120945T patent/ATE91711T1/en not_active IP Right Cessation
- 1988-12-19 ZA ZA889442A patent/ZA889442B/en unknown
- 1988-12-20 JP JP63319704A patent/JP2816167B2/en not_active Expired - Lifetime
- 1988-12-20 KR KR1019880017019A patent/KR0149656B1/en not_active IP Right Cessation
- 1988-12-20 CA CA000586515A patent/CA1317246C/en not_active Expired - Fee Related
- 1988-12-20 PT PT89282A patent/PT89282B/en not_active IP Right Cessation
- 1988-12-20 DK DK710588A patent/DK710588A/en not_active Application Discontinuation
- 1988-12-20 HU HU886507A patent/HU202917B/en not_active IP Right Cessation
- 1988-12-20 AU AU27323/88A patent/AU616450B2/en not_active Ceased
- 1988-12-20 IE IE380788A patent/IE62566B1/en not_active IP Right Cessation
- 1988-12-20 CZ CS888473A patent/CZ283529B6/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0321849A2 (en) | 1989-06-28 |
| DE3743323A1 (en) | 1989-06-29 |
| AU616450B2 (en) | 1991-10-31 |
| PT89282A (en) | 1989-12-29 |
| AU2732388A (en) | 1989-06-22 |
| DK710588A (en) | 1989-06-22 |
| IE883807L (en) | 1989-06-21 |
| ATE91711T1 (en) | 1993-08-15 |
| DK710588D0 (en) | 1988-12-20 |
| HU202917B (en) | 1991-04-29 |
| IE62566B1 (en) | 1995-02-08 |
| CZ847388A3 (en) | 1997-12-17 |
| CZ283529B6 (en) | 1998-04-15 |
| KR890010211A (en) | 1989-08-07 |
| HUT49168A (en) | 1989-08-28 |
| PT89282B (en) | 1993-07-30 |
| JP2816167B2 (en) | 1998-10-27 |
| ZA889442B (en) | 1989-08-30 |
| ES2058224T3 (en) | 1994-11-01 |
| JPH01199576A (en) | 1989-08-10 |
| EP0321849B1 (en) | 1993-07-21 |
| EP0321849A3 (en) | 1990-09-12 |
| KR0149656B1 (en) | 1998-08-17 |
| DE3882503D1 (en) | 1993-08-26 |
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| MKLA | Lapsed |