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Amylosucrase mutant and preparation method and application thereof

A technology of amylosucrase and mutants, applied in the fields of genetic engineering and enzyme engineering

Active Publication Date: 2019-03-01
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 there are few reports on methods that can significantly change the balance of hydrolysis and transglycoside transfer.

Method used

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  • Amylosucrase mutant and preparation method and application thereof
  • Amylosucrase mutant and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Recombinant bacteria construction

[0035] According to the amylosucrase gene sequence with accession number ABF44874.1 on NCBI, the DgAS gene containing amylosucrase was synthesized by chemical synthesis, and the amylosucrase gene sequence with accession number Q9ZEU2.1 was synthesized by chemical synthesis. NpAS gene, accession number BAG82876.1 Amylosucrase gene sequence Synthesize AmAS gene containing amylosucrase by chemical synthesis. The DgAS gene, NpAS gene, AmAS gene and the pET-24a(+) plasmid were double-digested with NdeI and HindIII respectively, and the digested products were recovered by tapping the rubber, and then ligated with T4 ligase, and the ligated products were transformed into E.coliJM109 competent cells. 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 30 mg / L kanamycin), the plasmids were extracted, and the...

Embodiment 2

[0038] Embodiment 2: the preparation of amylosucrase mutant

[0039] (1) Preparation of amylosucrase single mutation

[0040] According to the DgAS gene sequence of amylosucrase, the primers for introducing the A285S mutation were designed and synthesized. Using rapid PCR technology, the plasmid DgAS / pET-24a(+) carrying the gene encoding wild-type amylosucrase was used as a template to target amylosucrase. The DgAS gene sequence was subjected to site-directed mutation, and the DNA coding sequence was determined to identify the gene in which the 285th Ala codon was changed to a Ser codon, and amylosucrase single mutation A285S was obtained.

[0041] According to the AmAS gene sequence of amylosucrase, the primers for introducing the A287S mutation were designed and synthesized, and the plasmid AmAS / pET-24a(+) carrying the gene encoding wild-type amylosucrase was used as a template by using rapid PCR technology to target amylosucrase. The AmAS gene sequence was subjected to sit...

Embodiment 3

[0064] Embodiment 3: the concentration of crude enzyme liquid

[0065] Slowly add ammonium sulfate with a concentration of 20% relative to the mass fraction of the enzyme liquid while stirring the crude enzyme liquid obtained in Examples 1 and 2, stir until the ammonium sulfate is dissolved, and stand at 4°C for 8 to 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 amylosucrase and belongs to the field of genetic engineering and enzyme engineering. Site-directed mutation is performed on alanine residue 285 of amylosucrase from Deinococcusgeothermalis, site-directed mutation is performed on alanine residue 287 of amylosucrase from Alteromonas macleodii, and site-directed mutation is performed on alanine residue 295 of amylosucrase from Neisseria polysaccharea; a single mutant enzyme acquired has higher hydrolytic activity than wild amylosucrase. The amylosucrase herein is helpful for the research on glucoside hydrolase glucoside conversion and hydrolytic mechanisms, and is also applicable to industrial production of polysaccharides from glucoside hydrolase.

Description

technical field [0001] The invention relates to a starch sucrase mutant and its preparation method and application, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Amylosucrase (AS) is a glucosyltransferase (glucosyltransferase, E.C.2.4.1.4), belonging to the glycoside hydrolase (glycoside hydrolase, GH) 13 family. Its main function is to catalyze the synthesis of insoluble polysaccharides. In the presence of dextran, amylosucrase can catalyze the synthesis of amylose with ordinary sucrose as the only energy source and substrate. It is an industrial polysaccharide Enzymes with high application value. Amylosucrase can use cheap sucrose as a substrate to produce polysaccharides without the need for expensive precursors such as UDP, making it widely applicable in industry. Amylosucrase contains five domains (A, B, B', C and N), wherein the A, B and B'-domains constitute the catalytic core of amylosucrase. [0003] Most of ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/56C12P19/04
CPCC12N9/1051C12P19/04C12Y204/01004
Inventor 吴敬宿玲恰郭志勇祝晓蕾徐星豪
Owner JIANGNAN UNIV
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