CN1428424A - Preparation method of glutaryl-7-aminocefaphytanic acid acyltransferase - Google Patents
Preparation method of glutaryl-7-aminocefaphytanic acid acyltransferase Download PDFInfo
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- CN1428424A CN1428424A CN 02139886 CN02139886A CN1428424A CN 1428424 A CN1428424 A CN 1428424A CN 02139886 CN02139886 CN 02139886 CN 02139886 A CN02139886 A CN 02139886A CN 1428424 A CN1428424 A CN 1428424A
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Abstract
The method for preparing glutaryl-7-aminocephaphylanic acid acylase is characterized by that it utilizes colibacillus gene engineering bacterium to make shake-flask strain culture in culture medium, fermentation strain culture and fermentation culture, then utilizes centrifuge to collect thallus, soakes to extract enzyme liquor, makes the enzyme liquor undergo the processes of plate-frame filtration, ultrafiltration concentration, purification and fixation so as to obtain the invented product GL-7-ACA ACY.
Description
Technical field:
The present invention relates to the preparation method of the Glularyl-7-amino-cephalo-alkanoic acid acylase that a kind of semi-synthetic cynnematin synthetic key intermediate 7-amino-cephalosporanic acid uses.
Background technology:
Cynnematin is the microbiotic that important curative effect is arranged, and semi-synthetic cynnematin then has stronger antibacterial ability maybe can make pharmacokinetic property improve.And the key intermediate of semi-synthetic cynnematin is 7-amino-cephalosporanic acid (7-ACA), and the method for traditional mode of production in the past mainly is by chemical process, and four big steps are arranged, the process complexity, and the agents useful for same costliness, seriously polluted, and yield is lower, and second-rate.And bioconversion method is a kind of method preferably.Studies show that; cephalosporin can be by D-amino-acid oxidase oxidative deamination; and further decarboxylic reaction generates GL-7-ACA; and the 7-position side chain of GL-7-ACA can be obtained 7-ACA by the hydrolysis of Glularyl-7-amino-cephalo-alkanoic acid acylase (GL-7-ACA ACY) institute, thereby Glularyl-7-amino-cephalo-alkanoic acid acylase (GL-7-ACA ACY) is the key enzyme that generates 7-ACA.
Summary of the invention:
Purpose of the present invention aims to provide a kind of by utilizing the production of bacillus coli gene engineering bacterium fermentation, extracts enzyme liquid, Plate Filtration by centrifugal collection thalline, immersion again, gets product after ultrafiltration and concentration, the purifying immobilization; Can make product that the preparation method of the Glularyl-7-amino-cephalo-alkanoic acid acylase (GL-7-ACA ACY) of higher yields is arranged.
The bacillus coli gene engineering bacteria is carried out shake-flask seed cultivate in substratum, ferment-seeded is cultivated, fermentation culture, and wherein the composition of shake-flask seed stage substratum and content are:
Tryptones 1% yeast extract paste 0.5% sodium-chlor 1.0%
The composition of ferment-seeded substratum and content:
Yeast extract paste 1.5% ammonium chloride 0.24% oxysuccinic acid 0.22% potassium primary phosphate 0.68%
Fermentation culture: medium component and content:
Ammonium chloride 0.6% yeast extract paste 2.4% lactose 0.5% bubble enemy 0.05%
Suitable shake-flask seed, ferment-seeded culture condition are that temperature is 25 ℃, and pH value is 6.1-7.0, and incubation time respectively is 10-14 hour.
Air flow quantity when ferment-seeded is cultivated is 1: 1V/V/M.
The condition of fermentation culture is that temperature is 25 ± 1 ℃, and pH value is 6.5-8.2, and air flow quantity is 1: 1-1.5V/V/M, incubation time are 22-26 hour.
Bacterium used in the present invention is the bacillus coli gene engineering bacteria; the GL-7-ACA acylase produces bacterium (code name: E.coli-GLAA2002); entering U.S. company limited from Korea S buys; producing bacterium is RecombinantEscherichia coli BL21 (DE3), and GL-7-ACA acylase gene source is Pseudomonas sp.
It is characterized by 1, the um of cell size: 0.6um * (1-4), length has bigger variation in different growing stages, it is typically shaft-like that cell is; 2, bacterium colony smooth surface, moistening is glossy; 3, be leather Lan Shi negative bacterium; 4, do not produce proteolytic enzyme;
The extraction of GL-7-ACA acylase, purifying and immobilization process are: at first with dilute hydrochloric acid fermented liquid PH is transferred to 7.0-7.2; reduce PH to 6.6-6.8 with 20% calcium chloride again; add the chitosan of biomass 2% then, with diluted alkaline PH is transferred to 7.6-7.8 at last.With supercentrifuge centrifugal centrifugal bacterium liquid, control bacterium liquid vigor 5-8u/ml adds the tensio-active agent that is equivalent to bacterium liquid 2% (g/l), stirs immersion 3-5 hour naturally.Plate Filtration is collected clear liquid, and the clear liquid ultrafiltration and concentration concentrates enzyme with 30% (W/V) ammonium sulfate purifying twice, and purifying enzyme liquid ultrafiltration desalination wash water to the electricity of elutant is led below the 40us/cm, then with immobilization with carrier immobilized.
Product is described
Immobilization GL-7-ACA acylase
White or off-white color spherical particle
Particle diameter (100um) :≤1%
Water content≤70%
Enzyme activity 40-100 (u/g, 25 ℃)
Transform down at 20 ℃ with 1500u immobilization D-amino-acid oxidase; can use more than 120 batches continuously; 2300g cephalosporin sylvite can be transformed and obtain 1616g GL-7-ACA; use 1200u immobilization GL-7-ACA acylase 20 ℃ of conversions again; can use continuously more than 160 batches, can get 1035g7-ACA.
Embodiment:
Embodiment
The GL-7-ACA acylase produces bacterium bacillus coli gene engineering bacteria (code name: E.coli-GLAA2002); entering U.S. company limited from Korea S buys; producing bacterium is RecombinantEscherichia coli BL21 (DE3), and GL-7-ACA acylase gene source is Pseudomonas sp.This bacterium is seeded in the 500ml triangular flask of the shake-flask seed substratum that 100ml is housed these substratum Tryptones 1.0 grams, yeast extract paste 0.5 gram, sodium-chlor 1.0 grams.Culture temperature is controlled at 25 ℃, and pH value is 6.5, cultivates 12 hours.Again 400ml is shaken bottle mycelia and move into and carry out ferment-seeded in the fermentor tank that 100 liters of substratum are housed and cultivate, yeast extract paste 1500 grams wherein,, ammonium chloride 240 grams, oxysuccinic acid 220 grams, potassium primary phosphate 680 grams.Culture temperature is 25 ℃, and pH value is 6.5, and air flow quantity is 1: 1V/V/M, cultivated 12 hours.In fermentation culture stage, in the fermentor tank of 2500 liters of substratum, substratum is 15 kilograms of ammonium chlorides, 60 kilograms of yeast extract pastes, 12.5 kilograms of lactose, 1.25 kilograms of bubble enemies.Sterilizing earlier afterwards is chilled to 60 ℃, adds aseptic KH
2PO
4-K
2HPO
4Damping fluid (KH
2PO
423.1g/l, K
2HPO
4164.3g/l) control sterilization back pH value is 6.8.Fermentation seed liquid is moved in 2500 liters of fermentor tanks, and the control culture temperature is 25 ℃, and air flow quantity is 1: 1.5V/V/M, pH value are 6.5-8.2, cultivate 25 hours.The fermentation ends enzyme activity can reach 2.5-4u/ml, and 2500 liters of fermented liquid total activities are 6.25-10 1,000,000 units (volumetry, 25 ℃).
The extraction of GL-7-ACA acylase, purifying and immobilization process are: at first with dilute hydrochloric acid fermented liquid PH is transferred to 7.0-7.2; reduce PH to 6.6-6.8 with 20% calcium chloride again; add the chitosan of biomass 2% then, with diluted alkaline PH is transferred to 7.6-7.8 at last.With supercentrifuge centrifugal centrifugal bacterium liquid, control bacterium liquid vigor 5-8u/ml adds the tensio-active agent cetyl trimethylammonium bromide that is equivalent to bacterium liquid 2% (g/l), stirs immersion 3-5 hour naturally.Plate Filtration, collect clear liquid, the clear liquid ultrafiltration and concentration, concentrate enzyme with 30% (W/V) ammonium sulfate purifying twice, purifying enzyme liquid ultrafiltration desalination wash water to the electricity of elutant is led below the 40us/cm, then with immobilization with carrier immobilized, the immobilized enzyme vigor is 40-100 (u/g), 2500 liters of fermented liquids can get immobilized enzyme 0.8-1.4 1,000,000, total recovery 12%-14% (volumetry, 25 ℃).
Claims (3)
1, a kind of preparation method of Glularyl-7-amino-cephalo-alkanoic acid acylase, the bacillus coli gene engineering bacteria is carried out shake-flask seed in substratum cultivate, ferment-seeded is cultivated, fermentation culture, again by centrifugal collection thalline, soak and extract enzyme liquid, Plate Filtration, get product after ultrafiltration and concentration, the purifying immobilization; The bacillus coli gene engineering bacteria that uses, GL-7-ACA acylase produce bacterium, and (code name: E.coli-GLAA2002), producing bacterium is Recombinant Escherichia coli BL21 (DE3), and GL-7-ACA acylase gene source is Pseudomonas sp;
Wherein the composition of shake-flask seed stage substratum and content are:
Tryptones 1% yeast extract paste 0.5% sodium-chlor 1.0%
The composition of ferment-seeded substratum and content:
Yeast extract paste 1.5% ammonium chloride 0.24% oxysuccinic acid 0.22% potassium primary phosphate 0.68%
Fermentation culture: medium component and content:
Ammonium chloride 0.6% yeast extract paste 2.4% lactose 0.5% bubble enemy 0.05%
2, the preparation method of a kind of Glularyl-7-amino-cephalo-alkanoic acid acylase according to claim 1, suitable shake-flask seed, ferment-seeded culture condition are that temperature is 25 ℃, and pH value is 6.1-7.0, and incubation time respectively is 10-14 hour.Air flow quantity when ferment-seeded is cultivated is 1: 1V/V/M.
3, the preparation method of a kind of Glularyl-7-amino-cephalo-alkanoic acid acylase according to claim 1, the condition of fermentation culture are that temperature is 25 ± 1 ℃, and pH value is 6.5-8.2, and air flow quantity is 1: 1-1.5V/V/M, incubation time are 22-26 hour.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02139886 CN1428424A (en) | 2002-12-30 | 2002-12-30 | Preparation method of glutaryl-7-aminocefaphytanic acid acyltransferase |
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|---|---|---|---|
| CN 02139886 CN1428424A (en) | 2002-12-30 | 2002-12-30 | Preparation method of glutaryl-7-aminocefaphytanic acid acyltransferase |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100372931C (en) * | 2006-04-18 | 2008-03-05 | 北京科技大学 | Process for immobilization of glutaryl-7-amino cephalosporanic acid acylase |
| CN100448997C (en) * | 2004-10-13 | 2009-01-07 | 清华大学 | Expression vector and application |
| US8003358B2 (en) | 2005-08-08 | 2011-08-23 | Bioright Worldwide Company Limited | Two-step enzyme method for preparing 7-aminocephalosporanic acid |
-
2002
- 2002-12-30 CN CN 02139886 patent/CN1428424A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100448997C (en) * | 2004-10-13 | 2009-01-07 | 清华大学 | Expression vector and application |
| US8003358B2 (en) | 2005-08-08 | 2011-08-23 | Bioright Worldwide Company Limited | Two-step enzyme method for preparing 7-aminocephalosporanic acid |
| CN100372931C (en) * | 2006-04-18 | 2008-03-05 | 北京科技大学 | Process for immobilization of glutaryl-7-amino cephalosporanic acid acylase |
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