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Cephalosporin C acylase mutant

A cephalosporin and acylase technology, applied in the field of genetic engineering, can solve the problem that the enzyme activity cannot meet the industrial production and the like

Active Publication Date: 2016-05-04
上海邦林生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, great progress has been made in the transformation of CPC acylase derived from the Pseudomonassp.GK16 strain. The 45th position of the β subunit of the derived CPC acylase was replaced by I to V, and the 58th position of the β subunit The position is replaced by F to V, the 153rd position of β subunit is replaced by Y to T, the 177th position of β subunit is replaced by F to L, the 382nd position of β subunit is replaced by V to L, the obtained mutant The CPC acylase enzyme activity has increased by 25.3 times, but the enzyme activity still cannot meet the requirements of industrial production

Method used

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  • Cephalosporin C acylase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of wild-type CPC acylase gene recombinant Escherichia coli

[0058]For the CPC acylase derived from Pseudomonassp.GK16, based on its published gene sequence SEQIDNO: 2 (Matsuda et. al., J.Bacteriol. 163: 1222-1228, 1985), the gene sequence of the whole gene was synthesized, and in Restriction endonuclease sites NdeI and XhoI were designed at both ends of the gene, and subcloned into the corresponding sites of the vector pET24a (Novagen) to obtain the recombinant plasmid pET24a-wt-CPC, which was transformed into the expression host Escherichia coli BL21 (DE3) to obtain the wild type Recombinant E. coli of CPC acylase.

Embodiment 2

[0059] Example 2 Error-prone PCR method constructs random mutation point library and screening

[0060] 2.1 Error-prone PCR method to construct random mutation point library

[0061] Using the CPC acylase wild-type gene SEQIDNO:2 as a template, an error-prone PCR technique was used to construct a random mutant library. The forward primer CPC-Nde-F is 5'- CATATG GAGCCGACCTCGAC-3', reverse primer CPC-Xho-R is 5'- CTCGAG TGGCTTGAAGTTGAAG-3'

[0062] 50μL error-prone PCR reaction system includes: 50ng plasmid template pET24a-wt-CPC, 30pmol pair of primers CPC-Nde-F and CPC-Xho-R, 1XTaqbuffer, 0.2mMdGTP, 0.2mMdATP, 1mMdCTP, 1mMdTTP, 7mMMgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl 2 , 2.5 units of Taq enzyme (fermentas). The PCR reaction conditions were: 95°C for 5 minutes; 94°C for 30s, 55°C for 30s, 72°C for 2min / kbp; 30 cycles; 72°C for 10min. The 2.0kb random mutation fragment was recovered from the gel as a large primer, and MegaPrimerPCR was performed with KOD-plu...

Embodiment 3

[0075] Embodiment 3 carries out directed evolution by site-directed mutagenesis technique

[0076] According to the four sites of V240F, A306T, R553L, and H623N screened in Example 2, degenerate primers were designed, and the pET24a-wtCPC plasmid was used as a template to construct a site-directed combinatorial mutation library. Then, by site-directed mutagenesis, using the plasmids of highly active strains screened from the combined mutation library as templates, five mutation points of I215V, F228V, Y323T, F347L, and V552L, namely I45βV, F58βV, Y153βT, F177βL, and V382βL of the β subunit, were added. The primers used in the construction process are listed in Table 3.

[0077] Table 3 List of directed evolution primers

[0078]

[0079] *where: N=A / G / C / T.

[0080] 3.1 Construction of directed evolution mutation library by site-directed mutagenesis

[0081] Using the pET24a-wtCPC plasmid as a template, three sets of primer pairs 240-F1 and 306-R1, 306-F2 and 553-R2, 553-...

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Abstract

The invention relates to a cephalosporin C acylase mutant. Cephalosporin C acylase is constructed through a point mutation method, in comparison with wild type cephalosporin C acylase coming from Pseudomonas sp.GK16, the activity of the cephalosporin C acylase is improved by 20.5-150 times, and the cephalosporin C acylase mutant can be used for producing 7-ACA through a one-step enzymatic method.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a cephalosporin C acylase constructed by a point mutation method and used for one-step enzymatic production of 7-ACA (7-aminocephalosporanic acid). Background technique [0002] Cephalosporin antibiotics are the most widely used β-lactam antibiotics, most of which are 7-ACA derivatives synthesized by 7-aminocephalosporanic acid (7-ACA). Antibiotics account for 40% of the global antibiotic market. [0003] 7-ACA is generally obtained by cleaving cephalosporin C (Cephalosporin C, referred to as CPC) chemically or enzymatically, and removing the side chain of the molecule. Due to the complex chemical process, high energy consumption and serious pollution, in recent years, industrial production of 7-ACA has basically been replaced by biological enzyme preparation. The biological enzymatic method currently used is divided into two-step enzymatic method and on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12N1/21C12N1/19C12P35/02C12R1/19C12R1/125C12R1/865C12R1/84
CPCC12N9/80C12P35/02C12Y305/01093C12N1/165C12N1/185C12N1/205C12R2001/125C12R2001/19C12R2001/84C12R2001/865
Inventor 王金刚梁岩陈舒明任亮
Owner 上海邦林生物科技有限公司
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