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Cephalosporin C acylase mutant with high thermal stability

A technology of acylase mutant and cephalosporin, applied in the field of genetic engineering, can solve the problems of affecting industrial application, low stability and high production cost of 7-ACA

Active Publication Date: 2020-05-19
SHANGHAI TAOYUSHENG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the large-scale production of 7-ACA, it was found that the stability of CPC acylase mutants such as 130-ED2 was not high, especially the thermal stability was poor, and the enzyme activity would be significantly reduced or even inactivated after a long period of reaction. Supplementary enzymes are used to maintain the reaction, resulting in high production costs of 7-ACA, which affects industrial applications

Method used

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  • Cephalosporin C acylase mutant with high thermal stability
  • Cephalosporin C acylase mutant with high thermal stability
  • Cephalosporin C acylase mutant with high thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of initial CPC acylase 130-ED2 expression strain

[0059] According to the CPC acylase 130-ED2 coding gene (formerly SEQ ID NO: 13) published in PCT / CN2017 / 076688, the whole gene synthesis is carried out, here is the sequence SEQ ID NO: 2. 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-130ED2, which was transformed into the expression host Escherichia coli BL21 (DE3), and the expression initial The recombinant Escherichia coli of CPC acylase is still numbered 130-ED2.

Embodiment 2

[0060] Example 2 Error-prone PCR method constructs random mutation point library and screening

[0061] 2.1 Error-prone PCR method to construct random mutation point library

[0062] Using SEQ ID NO:2 as a template, an error-prone PCR technique was used to construct a random mutant library.

[0063] Forward primer C-F: 5'- CATATG GAACCGACCTCCACCCCGCAG-3',

[0064] Reverse primer C-R: 5'- CTCGAG CGGTTTGAAGTTGAACGGGGTACGTTC-3'.

[0065] 50μL error-prone PCR reaction system includes: 50ng plasmid template pET24a-13ED2, 30pmol pair of primers C-F and C-R, 1X Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl 2 , 2.5 units of Taq enzyme (Fermentas). The PCR reaction conditions are: 95°C for 5min; 94°C for 30s, 55°C for 30s, 72°C

[0066] 2min / kbp; 30 cycles; 10min at 72°C. The 2.0kb random mutation fragment was recovered from the gel as a large primer, and MegaPrimer PCR was performed with KOD-plus DNA polymerase:...

Embodiment 3

[0082] Embodiment 3 The second round of error-prone PCR method constructs random mutation point library and screening

[0083] 3.1 Error-prone PCR method to construct random mutation point library

[0084] Referring to the method of Example 2, the plasmid of the CPC acylase mutant strain 130-V1 strain obtained in Example 2 was extracted as a template, and a random mutant library was constructed by using error-prone PCR technology. The error-prone PCR system is the same as step 2.1 in Example 2, and the error-prone PCR is continued with the forward primer C-F and the reverse primer C-R.

[0085] 3.2 High-throughput screening, heat treatment and activity determination of mutant library

[0086] The method is the same as step 2.2, step 2.3 and step 2.4 of embodiment 2.

[0087] Through the screening of the random mutation library, two strains 130-V3 and 130-V4 were screened out with significantly improved activity relative to the CPC sodium salt substrate, but no improvement in...

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Abstract

The invention discloses a cephalosporin C acylase mutant with a high thermal stability constructed by point mutation, which has an amino acid sequence selected from SEQ ID Nos: 3-7. Compared with initial cephalosporin C acylase with SEQ ID No: 1, the cephalosporin C acylase mutant disclosed by the invention has high thermal stability and higher enzyme activity, so that the cephalosporin C acylasemutant can be used for producing 7-aminocephalosporanic acid by adopting an 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 mutant with high thermal stability constructed by a point mutation method and its use in one-step enzymatic production of 7-aminocephalosporanic acid (7-ACA) application. Background technique [0002] Most of the cephalosporin antibiotics currently accounting for 40% of the global antibiotic market are 7-ACA derivatives synthesized from 7-aminocephalosporanic acid (7-ACA for short). 7-ACA is generally obtained by cleaving cephalosporin C (Cephalosporin C, referred to as CPC) by chemical or biological enzymatic methods, and removing the molecular side chain. The biological enzymatic method generally uses cephalosporin C acylase (CPC acylase) to catalyze the removal of the side chain of CPC to generate 7-ACA. [0003] See the patent document PCT / CN2017 / 076688. Through previous research, the inventors genetically engineered the CPC...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N1/21C12N1/19C12N1/15C12P35/02C12R1/125C12R1/84C12R1/865C12R1/19C12R1/75C12R1/82
CPCC12N9/80C12P35/02
Inventor 杨晟蒋宇王金刚梁岩
Owner SHANGHAI TAOYUSHENG BIOTECHNOLOGY CO LTD
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