Penicillin V acylase mutant, encoding array, recombinant expression vector, genetically engineered bacterium and application

A technology of acylase mutants and penicillin V, applied in genetic engineering, application, plant gene improvement, etc., can solve the problems of less batches, long reaction time, and low catalytic activity, and achieve enhanced stability and catalytic activity Improved effect

Active Publication Date: 2019-01-08
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the process of pilot production of 6-APA by penicillin V acylase enzymatic method in raw material pharmaceutical companies, it is found that due to the shortcomings of wild-type penicillin V acylase, such as low catalytic activity, long reaction time,

Method used

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  • Penicillin V acylase mutant, encoding array, recombinant expression vector, genetically engineered bacterium and application
  • Penicillin V acylase mutant, encoding array, recombinant expression vector, genetically engineered bacterium and application
  • Penicillin V acylase mutant, encoding array, recombinant expression vector, genetically engineered bacterium and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Construction, expression and purification and immobilization of recombinant protein of wild-type PVA recombinant genetically engineered bacteria

[0069] 1-1 Construction of wild-type PVA prokaryotic expression vector and construction of recombinant genetically engineered bacteria

[0070] The wild-type PVA gene and amino acid sequence used in the present invention are derived from Bacillus sphaericus (GeneBank accession number: P12256.1). The coding gene is optimized through the codon bias of Escherichia coli and the whole gene synthesis is carried out, and the wild-type The penicillin V acylase is named PVA-WT, and its coding gene is named pva-wt. See SEQ ID NO: 1 and SEQ ID NO: 2 for the nucleotide sequence and amino acid sequence.

[0071] refer to figure 1 , the wild-type PVA coding gene pva-wt synthesized by the whole gene and the prokaryotic expression vector pET30a(+) were subjected to EcoR I and Xho I double enzyme digestion respectively, and after ...

Embodiment 2

[0084] Embodiment 2: the preparation of high activity PVA mutant

[0085] 2-1 Construction of PVA mutant library

[0086] In order to improve the activity of wild-type PVA, the inventors used the recombinant expression vector pET-pva-wt as a DNA template, wherein the primers were T7 universal primers (SEQ ID NO: 9 and 10), and constructed a random mutation by error-prone PCR body library, and by adjusting the error-prone PCR reaction system Mg 2+ and Mn 2+ Concentration and concentration of dCTP and dTTP oligonucleotides, so that the base mismatch rate of the mutant library is 5 / 1000, that is, to ensure that a mutant has 1 to 3 amino acid mutations, the specific process of constructing the mutant library is as follows .

[0087] Error-prone PCR reaction system:

[0088]

[0089]

[0090] The error-prone PCR product obtained above was subjected to electrophoresis and gel-cut recovery and purification, and the purified product and the prokaryotic expression vector pET3...

Embodiment 3

[0098] Example 3: Preparation of high substrate concentration tolerance PVA mutants

[0099] Since the activity of wild-type PVA is inhibited by the concentration of penicillin V, in order to obtain a mutant resistant to high concentration of penicillin V and improve its catalytic efficiency, the inventors performed directed evolution mutation again on the basis of the above-mentioned mutant PVA-1, according to the above-mentioned The method in the examples was used to construct a mutant library and perform screening (increasing the penicillin V potassium salt concentration (W / V) from 10% to 20%). About 20,000 clones were screened from the above-mentioned mutant library, and 5 mutants with obvious color changes and higher values ​​than PVA-1 were obtained. After re-screening by shaking flasks, a clone with 2 times higher activity than PVA-1 was obtained, which was named PVA-2. The sequencing results showed that PVA-2 had an amino acid at position 91 relative to wild-type penic...

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Abstract

The invention provides a penicillin V acylase mutant, an encoding array, a recombinant expression vector, a genetically engineered bacterium and application. Penicillin V acylase derived from Bacillussphaericus is mutated, the specific activity of the obtained mutant PVA-3 is improved by nine times, the immobilization activity is improved to 820U/g from 92U/g, and after being incubated for one hour under a condition of 30% penicillin V sylvite, the mutant has activity residue of 89.1%; the reaction time that the immobilized mutant PVA-3 is adopted to prepare 6-APA (6-Aminopenicillanic Acid) by splitting 25% penicillin V under a condition that the pH value is 6.0 at 25 DEG C is shortened to 60 minutes from 180 minutes, a substrate conversion rate is increased to 99.5% or greater, the activity is not greatly lost after continuous use for 600 times or greater, and good operation stability can be achieved. By adopting the mutated PVA-3, the production cost can be greatly reduced, the production efficiency can be improved, and the mutant is applicable to industrial application.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, in particular to a penicillin V acylase mutant, coding sequence, recombinant expression vector, genetic engineering bacteria and application. Background technique [0002] Penicillin V is a penicillin antibiotic used in the early stage. It has antibacterial activity against most Gram-positive bacteria, Gram-negative cocci, individual Gram-negative bacilli (such as Haemophilus), spirochetes, and actinomycetes. Strains producing β-lactamases have no antibacterial effect. Currently, penicillin V (PenV) produced by fermentation is mostly used as a raw material for the production of 6-amino-penicillanic acid (6-APA), an intermediate of β-lactam antibiotics. 6-APA is an important intermediate in the synthesis of amoxicillin and ampicillin. [0003] At present, the industrial production of 6-APA of β-lactam antibiotics is mainly obtained by using penicillin G acylase (penicill...

Claims

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Application Information

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IPC IPC(8): C12N9/84C12N15/55C12N11/02C12P37/06
CPCC12N9/84C12N11/02C12P37/06C12Y305/01011
Inventor 黄斌赵强周晶辉许岗
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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