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Preparation method for cephalosporin C acylase

A cephalosporin and acylase technology, applied in the field of biochemistry, can solve the problems of difficult control of catalytic reactions and low conversion rates

Active Publication Date: 2015-12-02
ANHUI BBCA FERMENTATION TECH ENG RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the conversion rate from cephalosporin C to 7-ACA is low compared with the chemical method, and the reaction catalyzed by D-aminoacidoxidase (DAAO) is difficult to control

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] First spread the genetically engineered bacteria of Escherichia coli producing cephalosporin C acylase strain on the LB agar plate and cultivate until the colony diameter reaches 1mm.

[0031] Insert the cephalosporin C acylase-producing strain cultured on the plate into a ring containing peptone 16g / L, glycerol 2.5g / L, yeast extract powder 10g / L, and sodium chloride 5g in a sterile state. / L, 50ppm kanamycin in the seed medium Erlenmeyer flask, this seed medium is prepared with tap water, adjusts to pH7.0 with the solution of sodium hydroxide 10wt%.

[0032] Culture at 37°C with aeration and OD550 to 10. The cultivated mature seeds are inserted into the medium for fermentation culture according to the inoculum amount of 3% of the volume of the fermentation medium. The medium contains dipotassium hydrogen phosphate 6.5g / L, citric acid monohydrate 3g / L, calcium chloride monohydrate 0.05g / L, magnesium sulfate heptahydrate 2.5g / L, zinc chloride 0.003g / L, dihydrate Copper...

Embodiment 2

[0036]First spread the genetically engineered Escherichia coli CPCA2013, which produces cephalosporin C acylase strain, on an LB agar plate and cultivate until the diameter of the colony reaches 1mm.

[0037] Inject the cephalosporin C acylase producing strain CPCA2013 cultured on the plate into a ring containing peptone 16g / L, glycerol 2.5g / L, yeast extract powder 10g / L, sodium chloride 5g / L , 50ppm kanamycin, the pH is adjusted to 7.0 with sodium hydroxide, and the seed medium prepared with tap water is placed in a triangular flask, and the OD550 to 10 is ventilated at 37°C.

[0038] Insert the matured seeds into tap water containing 6.5g / L of dipotassium hydrogen phosphate, 3g / L of citric acid monohydrate, 0.05g / L of calcium chloride monohydrate, and sulfuric acid heptahydrate by 3% of the volume of the fermentation medium. Magnesium 2.5g / L, zinc chloride 0.003g / L, copper chloride dihydrate 0.003g / L, boric acid 0.005g / L, manganese chloride tetrahydrate 0.002g / L, cobalt chlo...

Embodiment 3

[0041] First spread the genetically engineered Escherichia coli CPCA2013, which produces cephalosporin C acylase strain, on an LB agar plate and cultivate until the diameter of the colony reaches 1 mm.

[0042] Inject the cephalosporin C acylase producing strain CPCA2013 cultured on the plate into a ring containing peptone 16g / L, glycerol 2.5g / L, yeast extract powder 10g / L, sodium chloride 5g / L , 50ppm kanamycin, adjust the pH to 7.0 with sodium hydroxide, and prepare the seed medium in a triangular flask prepared with tap water, and culture it with ventilation at 37°C for an OD550 to 10.

[0043] The mature seeds of cultivating are inserted in 5 liters of fermentors by 3% of the fermentation medium volume, and the medium wherein is prepared with tap water, containing dipotassium hydrogen phosphate 6.5g / L, citric acid monohydrate 3g / L, Calcium chloride monohydrate 0.05g / L, magnesium sulfate heptahydrate 2.5g / L, zinc chloride 0.003g / L, copper chloride dihydrate 0.003g / L, boric ...

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Abstract

The invention provides a preparation method for cephalosporin C acylase, which belongs to the field of biochemistry. The method comprises the following steps: 1) plate cultivation: a step of coating an LB agar plate medium with a cephalosporin C acylase producing strain genetically engineered Escherichia coli strain CPCA2013 and carrying out cultivation; 2) seed cultivation: a step of selecting and picking a single colony from the LB agar plate medium, inoculating the single colony into a seed medium and carrying out seed cultivation; and 3) ferment cultivation: a step of inoculating a seed liquid obtained after seed cultivation into a fermentation medium for cultivation, wherein the inoculation amount of the seed liquid is 3 to 4% of the volume of the fermentation medium. According to the cephalosporin C acylase produced in the invention, the enzyme activity of fermentation broth is about 5000 U / L, and large-scale industrial production of the cephalosporin C acylase may be carried out according to results of cost accounting; and the enzyme activity of the fermentation broth is 0.8 time higher compared with the enzyme activity of fermentation broth produced in the prior art, enzymatic hydrolysis effect is good, and enzyme activity stability is high.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a method for producing cephalosporin C acylase by using microorganisms. Background technique [0002] Because the allergic reaction of cephalosporin antibiotics is small, it is relatively stable to β-lactamase, has the advantages of broad antibacterial spectrum, good curative effect, low toxicity, no cross-resistance with other antibiotics, less cross-allergic reaction with penicillin, etc. , has become the most important effective drug against infection in recent years. Cephalosporin C (Cephalosporin C, CPC) is cleaved by chemical or enzymatic methods to obtain 7-ACA. [0003] Because of the long process, many steps, harsh reaction conditions, and the production of a large amount of three wastes, the chemical synthesis method has been eliminated. At present, a two-step enzymatic method is mainly used to prepare 7-ACA. First, cephalosporin C is catalyzed by D-amino acid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12R1/19
Inventor 李荣杰徐斌常珠侠汪本助
Owner ANHUI BBCA FERMENTATION TECH ENG RES
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