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Cephalosporin C acylase mutant as well as preparation method and application thereof

A cephalosporin and acylase technology, applied in the field of bioengineering, can solve the problems of low enzyme activity and low industrial production efficiency

Active Publication Date: 2021-04-16
SHANDONG JINCHENG KERUI CHEMICAL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Although the enzyme activity of the cephalosporin C acylase mutant provided by the above two patents is 8.6 times and 52 times higher than that of the wild type cephalosporin C acylase, its enzyme activity is still low and the industrial production efficiency is low

Method used

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  • Cephalosporin C acylase mutant as well as preparation method and application thereof
  • Cephalosporin C acylase mutant as well as preparation method and application thereof
  • Cephalosporin C acylase mutant as well as preparation method and application thereof

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Embodiment 1

[0034] (1) The gene of interest was cloned using SEQ ID NO: 4, the wild-type gene of CPC acylase, as a template. The primer sequences were: upstream primer F1: GGGAATTCCATATGCT F2: GAGAGTTCTGCACCG downstream primer R1: CCCGGAATTCTCATGG R2: CTTGAAGTTGAAGTTGAAGG, wherein F1 and R2 contained 20bp identical Homology arms, F2 and R1 contain 20bp homology arms, so as to carry out double point mutations.

[0035] (2) The PCR amplification system is as follows:

[0036]

[0037] The PCR reaction conditions were pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 45°C for 5 s, extension at 72°C for 3 min, and 30 cycles; extension at 72°C for 10 min.

[0038] (3) Subsequent purification of the PCR product was performed using a purification kit, and the obtained target gene was double-digested with BamHI and HinDIII endonucleases. For the plasmid vector PET-28a + Carry out the same double digestion to obtain the recombinant plasmid PET-28a + -CPCA. Esche...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a cephalosporin C acylase mutant as well as a preparation method and application thereof. The cephalosporin C acylase mutant has an amino acid sequence as shown in SEQ ID NO: 2, wherein the amino acid sequence shown as SEQ ID NO: 2 is obtained by carrying out substitution mutation on the 245-247th amino acid of the amino acid sequence shown as SEQ ID NO: 1; the tyrosine at the 245th site is mutated into phenylalanine, the arginine at the 246th site is mutated into methionine, and the leucine at the 247th site is mutated into serine. The enzyme activity of the cephalosporin C acylase mutant disclosed by the invention is improved by 210 times or more, compared with that of wild type GL-7-ACA acylase SED ID NO: 1, and can be applied to one-step enzymatic production of 7-aminocephalosporanic acid (7-ACA).

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a cephalosporin C acylase mutant, a preparation method and application thereof. Background technique [0002] 7-Aminocephalosporanic acid (7-ACA) is the main precursor for the synthesis of β-lactam antibiotics, which is mainly synthesized by chemical and enzymatic methods in industry. However, due to the complex process, serious pollution and high energy consumption of the chemical synthesis method, it has been replaced by the biological enzymatic method in recent years. The biological enzymatic method has a simple process, mild reaction conditions, and stable product quality. The chemical method is mature, and the biological enzyme method has a lot of room for improvement compared with the chemical method. With the gradual improvement of the country's requirements for green environmental protection, the biological enzyme method will have a brighter future pro...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/70C12P35/02
Inventor 王辉崔倩倩王宾刘家栋
Owner SHANDONG JINCHENG KERUI CHEMICAL CO LTD
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