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Sucrose hydrolase mutant as well as preparation method and application of sucrose hydrolase mutant

A technology of sucrose hydrolase and mutants, applied in the field of genetic engineering and enzyme engineering, to achieve the effect of improving the activity of transglycosides

Active Publication Date: 2019-03-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been many reports on hydrolysis and transglycoside transfer, but most of them focus on the donor and acceptor sites, and methods that can significantly change the balance of hydrolysis and transglycoside transfer have been reported so far.

Method used

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  • Sucrose hydrolase mutant as well as preparation method and application of sucrose hydrolase mutant
  • Sucrose hydrolase mutant as well as preparation method and application of sucrose hydrolase mutant
  • Sucrose hydrolase mutant as well as preparation method and application of sucrose hydrolase mutant

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Experimental program
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Embodiment 1

[0033] Embodiment 1: Recombinant bacteria construction

[0034] According to the sucrose hydrolase gene sequence whose accession number is YP_002516566.2 on NCBI, the CcSH gene containing sucrose hydrolase was synthesized by chemical synthesis, and the sucrose hydrolase gene sequence whose accession number was CDG80999.1 was synthesized by chemical synthesis. JaSH gene, accession number is the sucrose hydrolase gene sequence of AAQ93678.1 The XaSH gene containing sucrose hydrolase is synthesized by chemical synthesis. CcSH gene, JaSH gene and XaSH gene were digested with pET-24a(+) plasmid with NdeI and HindⅢ, respectively, and the digested products were recovered by cutting gel, then ligated with T4 ligase, and the ligated products were transformed into E.coli JM109 competent cells , to obtain recombinant cells. The recombinant cells were cultured at 37°C for 8 hours, and the transformants were picked and cultured with shaking in LB liquid medium (containing 30mg / L kanamycin...

Embodiment 2

[0037] Embodiment 2: the preparation of sucrose hydrolase mutant

[0038] (1) Preparation of single mutation of sucrose hydrolase

[0039] According to the CcSH gene sequence of sucrose hydrolase, primers for introducing the S271A mutation were designed and synthesized, and the plasmid CcSH / pET-24a(+) carrying the gene encoding wild-type sucrose hydrolase was used as a template by rapid PCR technology to detect sucrose hydrolase Site-directed mutation was performed on the CcSH gene sequence, and the DNA coding sequence was determined to identify the gene in which the 271st Ser codon was changed to Ala codon, and the sucrose hydrolase single mutation S271A was obtained.

[0040] According to the JaSH gene sequence of sucrose hydrolase, primers for introducing the S279A mutation were designed and synthesized. Using rapid PCR technology, the plasmid JaSH / pET-24a(+) carrying the gene encoding wild-type sucrose hydrolase was used as a template to target sucrose hydrolase. The JaSH...

Embodiment 3

[0063] Embodiment 3: the concentration of crude enzyme liquid

[0064] Slowly add ammonium sulfate with a concentration of 20% relative to the mass fraction of the enzyme liquid to the enzyme liquid obtained in Examples 1 and 2 while stirring, stir until the ammonium sulfate is dissolved, and stand at 4°C for 8-10 hours to precipitate protein. The mixture was centrifuged (8000rpm, 10min) to collect the precipitate, and the minimum volume of 50mM KH 2 PO 4 -Na 2 HPO 4 The buffer (pH 7.0) was redissolved, and after reconstitution, the solid matter was removed by centrifugation again, and the supernatant was collected and dialyzed to obtain a concentrated enzyme solution.

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Abstract

The invention discloses a sucrose hydrolase mutant and belongs to the fields of gene engineering and enzyme engineering. The glucoside conversion activity of a monomutant enzyme obtained by modifyingsucrose hydrolase derived from Xanthomonas axonopodis pv.glycines, Janthinobacterium agaricidamnosum NBRC 102515 and Caulobacter crescentus NA1000 CB15 and respectively carrying out site-directed mutagenesis on serine residues on the 271st or 279th or 281st positions of the sucrose hydrolase is improved as comparison with that of a mild-type sucrose hydrolase. The sucrose hydrolase mutant is beneficial to the research on the glucoside conversion and hydrolysis mechanism of a glucoside hydrolase and can also be applied to industrial production of polysaccharide from the glucoside hydrolase.

Description

technical field [0001] The invention relates to a sucrose hydrolase mutant and its preparation method and application, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Sucrose hydrolase (SH, EC 3.2.1.–), belonging to the GH13 glycoside hydrolase family, is a strong hydrolase. It has almost no ability to transglycoside, and can hydrolyze sucrose molecules into glucose and fructose molecules in almost equal proportions. Sucrose hydrolase contains five domains (A, B, B', C and N), wherein the A, B and B'-domains constitute the catalytic core of sucrose hydrolase. [0003] Most of the existing sucrose hydrolases have strong hydrolysis ability and relatively weak ability to transglycoside. Studying the determining mechanism of hydrolysis and transglycoside has been a hot topic. At present, there have been many reports on hydrolysis and transglycoside transfer, but most of them focus on the donor and acceptor sites, and method...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12P19/14C12P19/04
CPCC12N9/2402C12N15/70C12P19/04
Inventor 吴敬宿玲恰郭志勇李玲玲姚锴琳
Owner JIANGNAN UNIV
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