Cephalosporin acylase mutant and encoding gene and application thereof

A technology of cephalosporin acylase and sporin acylase, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of low CPC activity

Active Publication Date: 2012-01-18
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The main substrate of cephalosporin acylase is GL-7ACA, but its activity on CPC is low

Method used

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  • Cephalosporin acylase mutant and encoding gene and application thereof
  • Cephalosporin acylase mutant and encoding gene and application thereof
  • Cephalosporin acylase mutant and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the acquisition of cephalosporin acylase mutant

[0038] 1. Expression vector pET30(a)-CA-WT containing wild-type cephalosporin acylase CA

[0039] The amino acid sequence of the cephalosporin acylase of Pseudomonas SE83 acyII (GenBank: AAA25690.1, the amino acid sequence is sequence 1) was queried by NCBI (www.ncbi.nlm.nih.gov / ), and the method of overlapping PCR was used to synthesize Nucleotide sequence of cephalosporin acylase gene suitable for expression in E. coli. Specifically, its full-length amino acid sequence is input into the DNAworks program (http: / / helixweb.nih.gov / dnaworks / ) online. By setting the codon preference, DNAWorks outputs the nucleotide sequence fragments suitable for the cephalosporin acylase gene expressed in E.coli, each length is about 45bp, a total of 78 overlapping oligonucleotide sequences . The synthesized oligonucleotide fragments were dissolved with 10 mM Tris-HCl, pH 8.0 buffer. The PCR reaction system is in a 0.5ml ...

Embodiment 2

[0088] Example 2, Activity detection and conversion rate of cephalosporin acylase mutant

[0089] 1. Activity detection of cephalosporin acylase mutants

[0090] Take 20 μL of the purified CA-1C and CA-113 obtained in Example 1, respectively, and mix with pH=8.0, 100 mM Tris-HCl containing 3% (mass volume ratio) cephalosporin C (CSPC) 180 μl of buffer solution were mixed to obtain a pre-reaction mixture, and after reacting at 25° C. for 10 min, 200 μl of 40% glacial acetic acid was added to terminate the reaction to obtain a reaction product. Purified CA (wild-type cephalosporin acylase) was used as a positive control to obtain a positive control reaction product.

[0091] The pre-reaction mixture and products were detected by high-performance liquid chromatography, wherein Dima C18 liquid chromatography column 250mm×4.6mm was used, and the mobile phase consisted of 15% chromatographic methanol, 7.5% chromatographic acetonitrile, and 1% glacial acetic acid. 7-Aminocephalospo...

Embodiment 3

[0102] Embodiment 3, the mutant of cephalosporin acylase (CA) undergoes the construction of the derivative protein of amino acid residue substitution and activity determination

[0103] The idea is to start from the pET30(a)-CA-1C obtained in step 3 of the above-mentioned embodiment 1, and use the technique of overlapping PCR to introduce the Serβ471Ala mutation on the basis of CA-1C.

[0104] Using the pET30(a)-CA-1C obtained in Step 3 of Example 1 as a template, first use CA-For (the sequence is the same as in Example 1, Step 2) and CA-β471Lower: 5'-CATAACG AGC CAGCGCGCCATA-3' (where the underlined part is the mutation introduction site) was amplified by PCR at an annealing temperature of 60°C to amplify the upstream fragment of the mutant Serβ471Ala site;

[0105] Again, using pET30(a)-CA-1C as a template, use CA-Rev (the sequence is the same as in Example 1, step 2) and CA-β471Upper: 5'-GGCGCGCTG GCT CGTTATGT 3' (where the underlined part is the mutation introduction si...

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Abstract

The invention discloses a cephalosporin acylase mutant and an encoding gene and an application thereof. The protein provided by the invention is any one of the following 1)-3): 1) protein formed by an amino acid sequence shown as sequence 4 in the sequence table; 2) protein formed by an amino acid sequence shown as sequence 3 in the sequence table; 3) protein which is obtained by substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid residue sequence of sequence 3 or sequence 4 in the sequence table, has the functions of cephalosporin acylase, andis derived from 1). Experiment of the invention shows that the invention provides a wild-type CPC acylase mutant; HPLC results show that the specific activity for CPC of the wild-type CPC acylase mutant of the invention is increased by 6.5 times when compared with that of wild-type enzymes. In one-step conversion, the conversion rate is up to above 98% within 3 hours.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cephalosporin acylase mutant, its coding gene and its application. Background technique [0002] Cephalosporins are an important class of β-lactam semi-synthetic broad-spectrum antibiotics. At present, the industry adopts fermentation technology to produce cephalosporin C (Cephalosporin C, abbreviated as CPC), and through chemical cracking or enzymatic cracking, an important intermediate compound 7-aminocephalospora-nic acid (7-aminocephalospora-nic acid, abbreviated for 7-ACA). Then use 7-aminocephalosporanic acid as the core to condense with different side chains to obtain semi-synthetic β-lactam antibiotics. At present, the production of 7-aminocephalosporanic acid by chemical method has been gradually eliminated due to environmental pollution, and it has been replaced by enzymatic production. Enzyme production is mainly divided into two-step enzymatic method and one-step enz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12N15/63C12N5/10C12N1/21C12P35/00C12R1/19
Inventor 林章凛肖瀛洲张艳马敦超
Owner TSINGHUA UNIV
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