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Method for obtaining enzyme mutant with high expression, high activity and high stability

A high-stability, mutant technology, applied in the fields of genetic engineering and enzyme engineering, can solve problems such as difficult to obtain thermal stability, and achieve the effect of fast speed, simple screening method and high precision

Inactive Publication Date: 2018-07-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The premise of site-directed mutagenesis is to obtain accurate information on the molecular structure of the enzyme, while in vitro directed evolution faces a large number of mutant screening, making it difficult to obtain mutants with significantly improved thermal stability in a short period of time

Method used

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  • Method for obtaining enzyme mutant with high expression, high activity and high stability
  • Method for obtaining enzyme mutant with high expression, high activity and high stability
  • Method for obtaining enzyme mutant with high expression, high activity and high stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Construction of a functional polypeptide library

[0081] According to the amino acid sequence of the parent short peptide 9S1 (AEAEAKAKAEAEAKAK) 9 , chemically synthesize its gene and connect it to the N-terminal of the gene sequence of the enzyme to be screened (PGL, LOX and ASN), and clone it into the plasmid pET-22b(+) / enzyme of the target enzyme or protein (enzyme is the protein to be screened The expression gene, here PGL, LOX and ASN) between the NdeI and NcoI restriction sites, was constructed as a pET-22b(+) / 9S1-enzyme plasmid. The GFP expression gene gfp was fused to the end of pET-22b(+) / 9S1-enzyme to construct pET-22b(+) / 9S1-enzyme-gfp plasmid.

[0082] Using pET-22b(+) / 9S1-enzyme-gfp plasmid as template, degenerate upstream primer nSAP-up and specific downstream primer nSAP-down enzyme The linearized gene fragments of pET-22b(+) / nSAP-enzyme-gfp containing different amino acid compositions and lengths of SAP were obtained by PCR.

[0083] The ...

Embodiment 2

[0084] Embodiment 2: initial screening

[0085] Primary screening: transform the recombinant plasmid and the recombinant plasmid library into the expression host Escherichia coli E.coliBL21(DE3), cultivate and induce in the seed medium, and continue to cultivate for a certain period of time (related to the expression of the enzyme to be screened), (related bacteria culture The method is: when OD 600When reaching 0.6, add IPTG induction (wherein the IPTG induction amount of PGL-GFP fusion enzyme is 0.04mM, and LOX-GFP is 1mM, and ASN-GFP is 1mM), and simultaneously adjusts temperature to cultivate under the most suitable induction temperature of this enzyme ( PGL-GFP was cultured at 30°C for 5h, LOX-GFP was cultured at 20°C for 10h, and ASN-GFP was cultured at 30°C for 5h). ) to dilute the cells to be screened, set the flow cytometer MoFlo XDP flow cytometry (Beckman Coulter, USA) nozzle size to 100 μm, 20 mM phosphate buffer of pH 7.4 as sheath fluid, cell OD 600 The detecti...

Embodiment 3

[0086] Embodiment 3: double screening identification

[0087] Re-screening and identification: The mutants with strong fluorescence intensity obtained after sorting by flow cytometry were cultured in shake flasks and measured for fluorescence intensity. Inoculate a single colony on the seed medium for overnight culture and then transfer to the fermentation medium, when OD 600 When reaching 0.6, add IPTG induction (wherein the IPTG induction amount of PGL-GFP fusion enzyme is 0.04mM, and LOX-GFP is 1mM, and ASN-GFP is 1mM), and simultaneously adjusts temperature to cultivate under the most suitable induction temperature of this enzyme ( PGL-GFP was cultured at 30°C for 48h, LOX-GFP was cultured at 20°C for 72h, and ASN-GFP was cultured at 30°C for 24h).

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Abstract

The invention discloses a method for obtaining an enzyme mutant with high expression, a high activity and high stability, and belongs to the technical field of genetic engineering. By means of a functional polypeptide library which is constructed on the basis of SAPs, the whole process of obtaining the enzyme mutant is quick, efficient and convenient; meanwhile, compared with wild-type alkaline pectinase, the activity of extracellular amylase of the fused enzyme mutant of alkaline pectinase is maximally improved by 15.32 times, the stability is maximally improved by 3.86 times, and the specific enzyme activity is improved by 2.55 times; the activity of intracellular amylase of the fused enzyme mutant of lipoxygenase is maximally improved by 2.49 times, the stability is maximally improved by 3.13 times, and the specific enzyme activity is improved by 0.9 time; the activity of extracellular amylase of the fused enzyme mutant of asparaginase is maximally improved by 2.25 times, the stability is maximally improved by 3.56 times, and the specific enzyme activity is maximally improved by 1.34 times.

Description

technical field [0001] The invention relates to a method for obtaining an enzyme mutant with high expression, high activity and high stability, and belongs to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Based on the important influence of expression amount and thermostability on the application performance of enzymes, obtaining enzymes with high expression amount and high thermostability has always been a research hotspot in the field of enzyme engineering. [0003] There is a certain mutual promotion relationship between expression level, thermal stability and enzyme catalytic activity. In terms of expression level, the prior art usually improves the expression level of protease by optimizing the expression element or fusing the expression tag at the end, but the optimization of the expression element has Certain uncertainties, and the most commonly used expression tags MBP and GST and other macromolecules have a certain...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/65C12N15/62C12N1/21C12N9/88C12N9/02C12N9/82C07K19/00C12R1/19
CPCC12N9/82C12N15/63C12N15/65C12Y305/01001C07K2319/35C12Y402/02002C12N9/88C12Y113/11012C12N9/0069
Inventor 刘松陈坚堵国成赵伟欣
Owner JIANGNAN UNIV
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