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Starch branching enzyme mutants with improved thermal stability

A technology of starch branching enzyme and thermal stability, which is applied in the field of enzyme engineering and can solve the problems of low yield and poor stability of physical methods

Inactive Publication Date: 2018-10-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among these methods, the physical method has problems such as low yield and poor stability, and the application of the chemical method in the food field is limited to a certain extent.

Method used

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  • Starch branching enzyme mutants with improved thermal stability
  • Starch branching enzyme mutants with improved thermal stability
  • Starch branching enzyme mutants with improved thermal stability

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: express the preparation of starch branching enzyme mutant gene sequence of the present invention

[0035] Using the expression vector gbe / pET-20(+) as a template, design the complementary primer strands required for the experiment (see Table 1). The primers were synthesized by Jinweizhi Biotechnology Co., Ltd., according to the method shown in the instruction manual of the STAR Primer GXL kit from TaKaRa Company Perform site-directed mutagenesis. The PCR reaction system follows the conditions set in the STAR Primer kit instructions: 5×PrimeSTARBuffer (Mg 2+ Plus) 10 μL, template DNA 1 μL, both forward and reverse primers (10 μM) 1 μL, PrimeSTAR HS DNA Polymerase (2.5U / μL) 0.5 μL, dNTPs (each 2.5 mM) 4 μL, and finally add ultrapure water 32.5 μL. PCR amplification conditions were as follows: pre-denaturation at 98°C for 5 min; followed by 98°C for 10 s, 55°C for 10 s, and 72°C for 7 min as a cycle, and 35 cycles under the above conditions; finally, incubat...

Embodiment 2

[0039] Embodiment 2: Contain the construction of the genetically engineered bacteria expressing the starch branching enzyme mutant gene of the present invention

[0040] The construction of the engineering bacterium that contains the starch branching enzyme mutant gene is carried out according to the following method:

[0041] (1) Transfer the PCR product into E.coli DH5α according to the E.coli DH5α competent transformation method, and spread the recipient bacteria on the LB solid medium containing 100 μg / mL ampicillin;

[0042] (2) Place the coated LB solid medium upside down in a constant temperature incubator at 37°C for 12 hours;

[0043] (3) Pick positive single clones and inoculate them in LB liquid medium containing 100 μg / mL ampicillin and culture at 37°C for 10-12 hours;

[0044] (4) Plasmids were extracted from the collected bacteria and identified by double-enzyme electrophoresis and sequencing;

[0045] (5) At 37°C, treat the PCR product with DpnI for 2 hours, t...

Embodiment 3

[0046] Embodiment 3: the expression of starch branching enzyme mutant of the present invention

[0047] Activation culture of host bacteria: E.coli BL21(DE 3) containing the expression vector plasmid pET-20b(+) / gbe was streaked and isolated on LB solid medium, cultured overnight in a constant temperature incubator at 37°C, and picked Inoculate a positive single colony into a sterile 50mL centrifuge tube containing 15mL of LB liquid medium. The centrifuge tube was placed in a rotary shaker at 200 r / min, and incubated at 37° C. for 12 h.

[0048] Fermentation culture: Inoculate 200 μL of the activated strain culture solution into a 250 mL Erlenmeyer flask containing 50 mL of TB liquid medium, and place it in a rotary shaker at a rate of 200 r / min, and cultivate it to OD at 37 °C 600 Reach between 1.0-1.5. Add IPTG with a final concentration of 0.01mM / L and lower the culture temperature to 25°C, and continue to induce for 16h under this condition.

[0049] Ampicillin was added t...

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Abstract

The invention discloses starch branching enzyme mutants with improved thermal stability, and belongs to the technical field of enzyme engineering. According to the invention, amino acid residues in astarch branching enzyme are mutated into the amino acid residues with opposite charges to endogenous amino acid residues, an electrostatic interaction is formed between the mutant starch branching enzyme and the endogenous amino acid residues with opposite charges nearby, and the starch branching enzyme mutants with improved thermal stability are obtained; compared with a wild-type starch branching enzyme, half-life periods t1 / 2 (min, 60 DEG C) of the thermal stability of the starch branching enzyme mutants Q231R, Q231K, T339E, T339D, V37E, V37D and I571D obtained by the invention are respectively extended by 40%, 38%, 21%, 26%, 16% and 21% at 60 DEG C, and the half-life periods t1 / 2 (min, 65 DEG C) are extended by 1.5-2.0 times at 65 DEG C.

Description

technical field [0001] The invention relates to a starch branching enzyme mutant with improved thermostability, which belongs 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 glycosyltransferase belonging to glycoside hydrolase family 13 (GH 13), which can catalyze the α-1,4-glycosidic bond of starch molecules The cleavage of starch molecules forms free short chains, and through transglycosidation, the cleaved short chains are connected to the acceptor chains in the form of α-1,6-glycosidic bonds, forming new α-1,6 on the original main chain of starch molecules. - branch points. [0003] Through this transglycosylation reaction, starch branching enzyme can increase the branching degree of starch, improve the digestion resistance and slow digestibility of starch, delay the retrogradation process of starch, enhance the stability of starch and improve the performance of starch,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12P19/18C12P19/04
CPCC12N9/107C12P19/04C12P19/18C12Y204/01018
Inventor 李兆丰班宵逢顾正彪李才明程力洪雁
Owner JIANGNAN UNIV
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