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Mutant cephalosporin C acylase, method for preparing same and method for converting 7-aminocephalosporin acid (ACA)

A cephalosporin and acylase technology, which is applied in the field of bioengineering, can solve the problems of low catalytic activity, large external influence on enzyme activity, product inhibition, etc., and achieves the effect of fast conversion rate, small external influence and small inhibition.

Active Publication Date: 2013-03-20
HUNAN FLAG BIOTECHNOLOGY CO LTD
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Problems solved by technology

[0005] The purpose of the present invention is to provide a mutant cephalosporin C acylase and its preparation method and application, to solve the problem that the mutant cephalosporin C acylase in the prior art has little catalytic activity, and the enzymatic activity is greatly affected by the outside world. Technical Issues of Product Inhibition

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  • Mutant cephalosporin C acylase, method for preparing same and method for converting 7-aminocephalosporin acid (ACA)
  • Mutant cephalosporin C acylase, method for preparing same and method for converting 7-aminocephalosporin acid (ACA)
  • Mutant cephalosporin C acylase, method for preparing same and method for converting 7-aminocephalosporin acid (ACA)

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preparation example Construction

[0052] Another aspect of the present invention also provides a kind of preparation method of aforementioned mutant cephalosporin C acylase, comprises the following steps:

[0053] a) Perform site-directed mutation, site-directed saturation mutation or multi-point mutation on the gene sequence having SEQ ID NO.2 in the sequence listing to obtain the gene sequence having SEQ ID NO.4 in the sequence listing; having SEQ ID NO.6 in the sequence listing The gene sequence; it has the gene sequence of SEQ ID NO.8 in the sequence table; under stringent conditions, it can hybridize with the gene sequence defined by any one of a, b or c and encode a protein with cephalosporin C acylase activity Gene sequence; a mutant gene that has more than 90% homology with the gene sequence defined in any one of a, b or c and encodes a gene sequence of a protein with cephalosporin C acylase activity;

[0054] b) inserting the mutant gene into the plasmid to obtain an expression vector;

[0055] c) tr...

Embodiment 1

[0092] a) Using the plasmid extracted from the Top10' strain containing the wild-type cephalosporin C acylase expression vector pET28-CPCA clone strain as the mutation template, the extracted plasmid has the gene sequence of SEQ ID NO.2 in the sequence table. Design mutation primers according to the gene sequence of the extracted plasmid, perform site-directed mutation at position 288 by reverse PCR in the reverse PCR reaction system, first pre-denature at 95°C for 5 minutes, then denature at 94°C for 30S, and anneal at 60°C 1 min, extended at 72°C for 8 min, repeated denaturation, annealing, and extension 18 times, and finally extended at 72°C for 10 min to obtain the PCR product. The preparation method of the reverse PCR reaction system is to mix 5 μl of 10*pfxbuffer, 1 μl of forward primer, 1 μl of reverse primer, and 1 μl of Mg 2+ , 1 μl of template DNA, 10 μl of 5*Enhancer Buffer, 2 μl of dNTPs, 0.5 μl of pfx high-fidelity DNA polymerase with an enzyme activity of 2.5U / μl...

Embodiment 2

[0099] a) Using the plasmid extracted from the Top10' strain containing the wild-type cephalosporin C acylase expression vector pET28-CPCA I clone strain as the mutation template, the extracted plasmid has the gene sequence of SEQ ID NO.2 in the sequence table. Design mutation primers according to the gene sequence of the extracted plasmid, perform reverse PCR reaction in the reverse PCR reaction system, and perform site-directed mutation at 417 again based on the mutation at 288. First, pre-denature at 95°C for 5 minutes, and then at 94°C Denature for 30S, anneal at 60°C for 1 min, extend at 72°C for 8 min, repeat denaturation, annealing, and extension 18 times, and finally extend at 72°C for 10 min to obtain the PCR product. The preparation method of the reverse PCR reaction system is to mix 5 μl of 10*pfx buffer, 1 μl of forward primer, 1 μl of reverse primer, 1 μl of Mg2+, 1 μl of template DNA, 10 μl of 5*Enhancer Buffer, 2 μl of dNTPs, 0.5 μl of pfx high-fidelity DNA poly...

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Abstract

The invention provides a mutant cephalosporin C acylase. One or a plurality of amino acid locus replacement is carried out on an amino acid sequence shown as SEQ ID NO.1 in a sequence table to obtain a mutant amino acid sequence; and one or more of 288-locus valine, 296-locus histidine and 417-locus histidine is or are adopted as amino acid locus(es). The technical problems that the mutant cephalosporin C acylase of the prior art has small catalytic activity, seriously influenced enzymatic activity by the outside and product inhibition are solved.

Description

technical field [0001] The present invention relates to the field of bioengineering, and in particular, relates to a mutant cephalosporin C acylase. Another aspect of the present invention also provides a preparation method and application of the aforementioned mutant cephalosporin C acylase. Background technique [0002] 7-aminocephalosporanic acid (7-ACA) is an important intermediate in the production of semi-synthetic cephalosporin antibiotics in the pharmaceutical industry. The industrial production of 7-ACA mainly uses chemical and enzymatic methods. At present, most domestic 7-ACA production still adopts chemical methods, but due to the disadvantages of using a large number of expensive chemical reagents, harsh reaction conditions, serious environmental pollution, and high cost, enzymatic production of 7-ACA has obvious advantages, simple process, and high reaction rate. The conditions are mild, the production is environmentally friendly, the cost is low, and the quali...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12N15/63C12P35/02
Inventor 许岗黄斌曾红宇
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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