CN1844383A

CN1844383A - Process for immobilization of glutaryl-7-amino cephalosporanic acid acylase

Info

Publication number: CN1844383A
Application number: CNA200610011742XA
Authority: CN
Inventors: 罗晖; 史芫芫; 于慧敏; 李强; 沈忠耀
Original assignee: Tsinghua University; University of Science and Technology Beijing USTB
Current assignee: Tsinghua University; University of Science and Technology Beijing USTB
Priority date: 2006-04-18
Filing date: 2006-04-18
Publication date: 2006-10-11
Anticipated expiration: 2026-04-18
Also published as: CN100372931C

Abstract

This invention provides an immobilization method for glutaryl-7-aminocephalosporanic acid acylase, belonging to the biological enzymology engineering field. Processing steps comprise: cultivating and collecting the free cells with glutaryl-7-aminocephalosporanic acid acylase activity, redissloving the cells in pH 6-9 buffer, adding cross linking agent at the final concentration of 5-100mmol/L, cross linking for 1-4 hours at 0-45deg C, centrifuging, washing the pellets with buffer, and then acquiring the immobilized glutaryl-7-aminocephalosporanic acid acylase with the free state bacteria as vectors. The invention has dramatic advantages compared to the conventional methods of immobilized enzymes and cells, and shows brilliant prospects in industial application in combination with membrane separating process.

Description

A kind of process for fixation of Glularyl-7-amino-cephalo-alkanoic acid acylase

Technical field

The invention belongs to the bio-enzyme engineering technical field; a kind of process for fixation of Glularyl-7-amino-cephalo-alkanoic acid acylase particularly is provided, has adopted one of key enzyme of Production by Enzymes 7-amino-cephalosporanic acid (7-ACA) usefulness---a kind of fixation support and the method for Glularyl-7-amino-cephalo-alkanoic acid acylase.

Background technology

7-amino-cephalosporanic acid (7-aminocephalosporanic acid, 7-ACA) be the important source material of present semi-synthetic cynnematin, mainly (Cephalosporin C, CPC) the D-alpha-amino group hexanedioyl side chain of sloughing on the 7-position by chemical method or enzymatic cleavage obtains by cephalosporin.

Enzyme process substitutes traditional chemical method cracking cephalosporin and produces 7-ACA, has that technology is simple, efficient is high, cost is low, an advantage such as safe, pollution-free and constant product quality, all has very big competitive power in economy and environmental protection.In the last few years, people had dropped into substantial contribution and strength to the research and development of Production by Enzymes semisynthetic antibiotics.Produce 7-ACA with enzymatic cleavage, at first, (D-Amino acid oxidase under effect DAAO), produces the intermediate of a tool ketone group to CPC, and this intermediate is unstable, is easy to the H that is produced simultaneously at the D-amino-acid oxidase ₂O ₂The chemical oxidation decarboxylation, be transformed into glutaryl--7-amino-cephalosporanic acid (Glutaryl-7-amidocephalosporanic acid, GL-7-ACA); Under the effect of Glularyl-7-amino-cephalo-alkanoic acid acylase (Glutaryl-7-amidocephalosporanic acid acylase, GL-7-ACA acylase), slough its side chain then, generate 7-ACA.

At present, the technology industrialization abroad that utilizes two step enzyme method catalytic pyrolysis CPC to make 7-ACA, domestic also introduction of foreign technology carry out the 7-ACA trial production, but used immobilized enzyme all needs import.This in addition method has certain disadvantages, for example: needed enzyme is carried out the multistep separation and purification before the preparation immobilized enzyme, can cause a large amount of losses of enzyme; Immobilized enzyme and immobilized cell all can increase the diffusional resistance of substrate and product, and rate of catalysis reaction is descended; The enzyme of immobilized enzyme and immobilized cell yield alive is all lower, generally is lower than 50%.At the 7-ACA production field, although existing many enterprises are from external Production by Enzymes line and the production technology introduced, but because can not the autonomous production immobilized enzyme, and the immobilized enzyme of import costs an arm and a leg, and adopts the 7-ACA cost of Production by Enzymes even is higher than the cost of chemical method.Therefore, seek that price is low, energy consumption is low, to the little novel carriers of loss of enzyme activity and new process for fixation to reduce immobilized cost, particularly important to realizing that at home Production by Enzymes replaces chemical method to produce 7-ACA.

In the modern biological project field, membrane separation technique is more and more paid close attention to.Use membrane separation plant, macromolecular substance (or cell) well can be separated with small-molecule substance, and less as the macromolecular substance (enzyme molecule) and the cell activity loss of catalyzer, can use repeatedly.Membrane separation technique is simple to operate, and cost is lower, realizes continuous operation easily.The preparation that the method for the bacterium catalyzed combination membrane sepn of free state is carried out 7-ACA has been proved to be feasible (Chinese patent, application number: 200410039573.1).But directly the bacterium with undressed free state carries out catalysis, and common catalysis several batches enzyme promptly loses activity, and the enzyme catalysis stability that how to improve the free state bacterium is crucial (modern chemical industry, 2005,25 volume supplementary issues, 138～141).

Along with China's entry into the WTO, the cynnematin that advanced foreign technology is produced will pour in China, and domestic microbiotic industry will face severe situation.Therefore, step up to develop the technology of the Production by Enzymes 7-ACA with advanced level, satisfy domestic needs and strengthen competitiveness in the international market extremely urgent.

Summary of the invention

The object of the present invention is to provide a kind of process for fixation of Glularyl-7-amino-cephalo-alkanoic acid acylase; with the free state bacterium is the method for the immobilization GL-7-ACA acylase of carrier; so that the binding film isolation technique reduces production costs when simplifying production technique.

The present invention realizes by following method:

1, cultivation and collection have the active free cell of GL-7-ACA acylase,

2, be resuspended in pH6～9 buffered soln collecting the free cell that obtains in the step 1, add the linking agent that final concentration is 5～100mmol/l then; Under 0～45 ℃, crosslinked 1～4 hour, centrifugal, the gained solid washed with damping fluid, and obtaining with the free state bacterium is the immobilization Glularyl-7-amino-cephalo-alkanoic acid acylase of carrier.4 ℃ of preservations are standby.

3, the application in producing 7-amino-cephalosporanic acid; Separating and the repeated use of immobilized enzyme of described immobilized enzyme and catalysate, employing be centrifugal or the method for membrane sepn, reuse; Perhaps adopt the isolating method of hollow fiber ultrafiltration membrane that immobilized enzyme is separated with catalysate, make immobilized enzyme obtain reusing.

It is of the present invention that to have glutaryl--active free cell of 7-amino-cephalosporanic acid acylase be the recombination bacillus coli of GL-7-ACA acylase.Described buffered soln is Tutofusin tris (Tris)-HCl damping fluid, and concentration is 0～1mol/L, and pH is 6～9; Described buffered soln is preferably the 0.1mol/L Tris-HCl damping fluid of pH8.0.Described linking agent is a glutaraldehyde.

The invention has the advantages that after the immobilization, enzyme work has improved 20～40% by being that carrier carries out crosslinked immobilization to the GL-7-ACA acylase with the free state bacterium, catalytic stability has increased by 10～20 times.

Embodiment

Embodiment 1

The immobilization process of GL-7-ACA acylase is: at first with the centrifugal collection thalline of fermented liquid of GL-7-ACA acylase recombination bacillus coli pET-ACY; be that 100mM/L, pH are that 8.0 Tris-HCl buffered soln is resuspended with concentration again; add the glutaraldehyde solution for preparing in advance at last; making its final concentration is 40mmol/l; put 4 ℃ centrifugal after crosslinked 4 hours; abandon supernatant liquor, the gained solid washes with the Tris-HCl damping fluid, and promptly obtaining with the free state bacterium is the immobilization GL-7-ACA acylase of carrier.Record the work of GL-7-ACA acidated enzyme after the immobilization and be 1.38 times before the immobilization.

Embodiment 2

The immobilization process of GL-7-ACA acylase is: at first with the centrifugal collection thalline of fermented liquid of GL-7-ACA acylase recombination bacillus coli pET-ACY; be that 100mM/L, pH are that 8.0 Tris-HCl buffered soln is resuspended with concentration again; add the glutaraldehyde solution for preparing in advance at last; making its final concentration is 70mmol/l; put 25 ℃ centrifugal after crosslinked 2 hours; abandon supernatant liquor, the gained solid washes with the Tris-HCl damping fluid, and promptly being able to the free state bacterium is the immobilization GL-7-ACA acylase of carrier.Record the work of GL-7-ACA acidated enzyme after the immobilization and be 1.36 times before the immobilization.

Embodiment 3

What utilize embodiment 1 preparation is the immobilization GL-7-ACA acylase of carrier with the free state bacterium, carries out the experiment that GL-7-ACA catalysis generates 7-ACA.In the test tube of immobilized enzyme is housed, adding with concentration is that 100mM/L, pH are the GL-7-ACA solution (5g/L) of 8.0 Tris-HCl buffer preparation, carry out catalyzed reaction under 37 ℃, catalysis 30min is one batch, and centrifugal collection immobilized enzyme carries out catalysis repeatedly.After testing, after 30 batches of the catalysis, the activity of immobilized enzyme has also kept 62%.

Embodiment 4

Earlier with the centrifugal collection thalline of fermented liquid of GL-7-ACA acylase recombination bacillus coli pET-ACY, be that 100mM/L, pH are that 8.0 Tris-HCl buffered soln is resuspended with concentration again.Utilize the thalline of the free state of Non-crosslinked processing to carry out the experiment that GL-7-ACA catalysis generates 7-ACA.In the test tube that the free state thalline is housed, adding with concentration is that 100mM/L, pH are the GL-7-ACA solution (5g/L) of 8.0 Tris-HCl buffer preparation, carry out catalyzed reaction under 37 ℃, catalysis 30min is one batch, and centrifugal collection thalline carries out catalysis repeatedly.After testing, after 3 batches of the catalysis, the activity of immobilized enzyme has also kept 36%.

Embodiment 5

What utilize embodiment 1 preparation is the immobilization GL-7-ACA acylase of carrier with the free state bacterium, carries out the experiment that GL-7-ACA catalysis generates 7-ACA.In the test tube of immobilized enzyme is housed, adding with concentration is that 100mM/L, pH are the GL-7-ACA solution (10g/L) of 8.0 Tris-HCl buffer preparation, carries out catalyzed reaction under 37 ℃, reacts 120min, GL-7-ACA substantially transforms and generates 7-ACA, and transformation efficiency is about 90.3%.The catalyzed reaction liquid that obtains adopts membrane filter method to carry out separating of immobilization GL-7-ACA enzyme and product.Select hollow fiber ultrafiltration membrane (molecular weight cut-off 30,000).Adopt the isolating reaction solution of membrane filter method, adopt the pH to 3.5 of 6N HCl conditioned reaction liquid, make 7-ACA carry out isoelectric point crystallization.Through filtration under diminished pressure and vacuum-drying, obtain the 7-ACA crystal of white, product purity is 92.5%.

Embodiment 6

With the immobilization GL-7-ACA acylase that obtains through membrane sepn among the embodiment 5, the condition of pressing embodiment 5 is catalysis GL-7-ACA solution (5g/L) once more.Through the catalyzed reaction of 120min, GL-7-ACA substantially transforms and generates 7-ACA, and transformation efficiency is about 91.2%.This explanation, the immobilization GL-7-ACA acylase of process catalyzed reaction and membrane sepn still has good catalytic activity, can repeatedly recycle.

Claims

1, a kind of process for fixation of Glularyl-7-amino-cephalo-alkanoic acid acylase is characterized in that:

A, cultivation and collection have the active free cell of Glularyl-7-amino-cephalo-alkanoic acid acylase;

B, the free cell that collection among the step a is obtained are resuspended in pH6.0～9.0 buffered soln, add the linking agent that final concentration is 5～100mmol/L then; Under 0～45 ℃, crosslinked 1～4 hour, centrifugal, the gained solid washed with damping fluid, and obtaining with the free state bacterium is the immobilization GL-7-ACA acylase of carrier;

C, the application in producing 7-amino-cephalosporanic acid; Separating and the repeated use of immobilized enzyme of described immobilized enzyme and catalysate, employing be that ultracentrifugal method is collected immobilized enzyme; Perhaps adopt the isolating method of hollow fiber ultrafiltration membrane that immobilized enzyme is separated with catalysate.

2, method according to claim 1 is characterized in that: described to have glutaryl--active free cell of 7-amino-cephalosporanic acid acylase be the recombination bacillus coli of Glularyl-7-amino-cephalo-alkanoic acid acylase.

3, method according to claim 1 is characterized in that: described buffered soln is the Tris-HCl damping fluid, and concentration is 0～1mol/L, and pH is 6～9.

4, according to claim 1 or 3 described methods, it is characterized in that: described buffered soln is selected the 0.1mol/L Tris-HCl damping fluid of pH8.0.