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Starch branching enzyme mutant with improved catalytic activity and application thereof

A starch branching enzyme and mutant technology, applied in the field of enzyme engineering, can solve the problems of large amount of enzyme added, long reaction time, limited effective modification, etc., and achieve the effects of improving stability and product stability.

Active Publication Date: 2021-02-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large amount of enzyme added and the long reaction time of this method, the effective modification of maltodextrin by starch branching enzymes is limited.

Method used

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  • Starch branching enzyme mutant with improved catalytic activity and application thereof
  • Starch branching enzyme mutant with improved catalytic activity and application thereof
  • Starch branching enzyme mutant with improved catalytic activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Contain the construction of the genetic engineering bacterium of starch branching enzyme mutant

[0047] 1. Construction of recombinant plasmids containing starch branching enzyme mutants

[0048]Using Rhodothermus obamensis genomic DNA as a template, the gbe gene (nucleotide sequence shown in SEQ ID NO.2) containing Nde I and Xho I restriction sites at both ends was amplified by PCR method, and inserted into into the pMD 18-T simple plasmid to obtain the recombinant plasmid pMD 18-T simple-gbe. After the recombinant plasmid was digested with double enzymes, the target gene fragment containing cohesive ends was recovered and inserted into the pET- 20b plasmid, the recombinant vector pET-20b-gbe was obtained. Using the expression vector pET-20b-gbe as a template, design the complementary primer strands required for the experiment (see Table 1). The primers were synthesized by Jinweizhi Biotechnology Co., Ltd., and the site-directed mutagenesis was performe...

Embodiment 2

[0056] Example 2: Expression of starch branching enzyme mutants

[0057] Specific steps are as follows:

[0058] (1) Activation culture of genetically engineered bacteria:

[0059] The genetically engineered bacteria E.coli BL21(DE 3) / pET-20b-gbe, E.coliBL21(DE 3) / pET-20b-G160R, E.coli BL21(DE 3) / pET- 20b-G160T, E.coli BL21(DE 3) / pET-20b-G160A, E.coli BL21(DE 3) / pET-20b-G160W were streaked and isolated on LB solid medium, and placed at a constant temperature of 37°C After culturing in the incubator for 12 h, pick positive single colonies and inoculate them into sterilized 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium (adding ampicillin with a final concentration of 100 μmg / mL before use), and place the Erlenmeyer flasks at 200 r / min rotary shaker, cultivated at 37°C for 8-10h to obtain seed liquid.

[0060] (2) Fermentation culture:

[0061] The seed liquid that step (1) obtains is inoculated respectively in the 250mL Erlenmeyer flask that contains 50mL TB...

Embodiment 3

[0064] Embodiment 3: Separation and purification of starch branching enzyme mutant

[0065] The crude enzyme solution containing wild-type starch branching enzyme WT obtained in Example 2, the crude enzyme solution containing mutant G160R, the crude enzyme solution containing mutant G160T, the crude enzyme solution containing mutant G160T, and the crude enzyme solution containing mutant G160A The crude enzyme solution was filtered through a 0.45μm aqueous membrane, and then used HisTrap TM HP column (specification: 5mL) for nickel column affinity chromatography purification. Equilibrate the nickel column (about 5 column volumes) with buffer A at a flow rate of 2 mL / min, then configure the sample into the same system as liquid A, with the same flow rate, load 60 mL of sample, and then use 60% (v / v) buffer solution B is eluted, and the eluate is collected to obtain pure enzymes, which are wild-type starch branching enzyme WT pure enzymes, mutant G160R pure enzymes, mutant G160...

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Abstract

The invention discloses a starch branching enzyme mutant with improved catalytic activity and an application thereof, and belongs to the technical field of enzyme engineering. The starch branching enzyme mutant with improved catalytic activity is obtained by adopting the technical scheme provided by the invention, and compared with wild type starch branching enzyme, starch branching enzyme mutantdisclosed by the invention has the advantages that the specific enzyme activities of the obtained starch branching enzyme mutants G160R and G160T are 12482 U / mg and 10497 U / mg respectively, and are improved by 88% and 58% respectively compared with those of the wild type starch branching enzyme; The starch branching enzyme provided by the invention is added into a reaction system taking DE11 maltodextrin as a substrate, compared with the addition of the wild type starch branching enzyme, the stability of a product is remarkably improved, after the starch branching enzyme is placed at 4 DEG C for 30 days, the clarity degrees of products obtained by adding the starch branching enzyme mutants G160T and G160R provided by the invention are respectively 10.7 times and 12 times of those of the product obtained by adding the wild type starch branching enzyme.

Description

technical field [0001] The invention relates to a starch branching enzyme mutant with improved catalytic activity and application thereof, belonging to the technical field of enzyme engineering. Background technique [0002] Starch branching enzyme (1,4-α-glucan branching enzyme; EC 2.4.1.18) is a type of glycosyltransferase belonging to the α-amylase family, which can catalyze the hydrolysis of α-1,4-glycosidic bonds in starch molecules to produce The free short chain at the non-reducing end is then connected to the acceptor chain in the form of α-1,6-glycosidic bonds by transglycosidation, thereby forming a new α-1,6-branch point. Through this transglycosylation reaction, the starch branching enzyme can increase the branching degree of starch and its derivatives, and improve the performance. Therefore, starch branching enzymes have become important amylases in the field of bioenzymatic modification of starch. [0003] Maltodextrin has the advantages of strong thickening ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/70C12N15/75C12N1/21C12P19/18C12P19/04C12R1/19C12R1/125
CPCC12N9/107C12N15/70C12N15/75C12P19/18C12P19/04C12Y204/01018
Inventor 李兆丰江海旻顾正彪李才明班宵逢程力洪雁
Owner JIANGNAN UNIV
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