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Amylosucrase mutant as well as preparation method and application thereof

A technology of amylosucrase and mutants, which is applied in the field of genetic engineering and enzyme engineering, can solve problems affecting product yield, etc., and achieve the effect of improving conversion rate

Active Publication Date: 2019-04-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, in the preparation process of turanose, although the polymerization reaction can be inhibited by high concentration of sucrose to a certain extent, when the sucrose concentration is reduced, the polymerization reaction will still affect the yield of the final product.

Method used

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  • Amylosucrase mutant as well as preparation method and application thereof

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Embodiment 1

[0032] Embodiment 1: Recombinant bacteria construction

[0033] According to the amino acid sequence of amylosucrase dgas on NCBI (PDB ID: 3UER), the dgas gene of amylosucrase was synthesized by chemical synthesis. The plasmid used for the construction of E. coli was pET24a(+) with T7 promoter. The pET24a(+) plasmid and dgas gene were double-digested with Nde I and Hind III, respectively, and the digested products were recovered by tapping the rubber, 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), the plasmid was extracted, and the expression plasmid dgas / pET24a(+) was obtained after enzyme digestion verification.

[0034] Transform the plasmid dgas / pET24a(+) into E.coli BL21(DE3) host bacteria, spread on LB plates (containin...

Embodiment 2

[0035] Embodiment 2: Preparation of single mutant

[0036] According to the dgas gene sequence of the amylosucrase synthesized by the chemical synthesis method in Example 1, design and synthesize primers for introducing the G399S mutation, carry out site-directed mutation to the amylosucrase dgas gene, determine the DNA coding sequence, and identify the 399th Gly code The codon becomes a Ser codon. The vector carrying the mutant gene is introduced into Bacillus subtilis, Escherichia coli or Bacillus pumilus for expression to obtain a single mutant amylosucrase.

[0037] Site-directed mutation of G399S: using rapid PCR technology, the expression vector dgas / pET24a(+) carrying the wild-type amylosucrase gene was used as a template.

[0038] The site-directed mutagenesis primers for introducing the G399S mutation are:

[0039] The nucleotide sequence is the forward primer of SEQ ID NO.3:

[0040] 5'-GTCATGATGATATT AGC TGGGCAATTAGCG-3' (the underline is the mutated base)

[...

Embodiment 3

[0047] Example 3: Fermentation of amylosucrase mutants

[0048] Pick the recombinant strain E.coli J BL21(DE3) / dgas / pET24a(+)(G399S) and grow it in LB liquid medium (containing 30μg / mL kanamycin) for 8-10h, and ferment the seeds according to the inoculum size of 5% Inoculated into TB medium (containing 30 μg / mL kanamycin), cultured on a shaker at 37°C for OD 600 After reaching 0.2, add 0.4mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce, after fermenting at 25°C for 24h, centrifuge the fermentation broth at 4°C, 8000rpm for 10min and discard it. The cells were collected to 30OD, and reconstituted with PBS buffer solution of pH 7.4, then subjected to high-pressure homogenization to break the wall, and centrifuged at 8000rpm for 10min to collect the supernatant to obtain mutant crude enzyme solution.

[0049] Using the same method, the broken enzyme solution obtained by fermenting the recombinant strain E.coli J BL21(DE3) / dgas / pET24a(+) in Example 1 was used as the wil...

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Abstract

The invention discloses an amylosucrase mutant as well as a preparation method and application thereof, and belongs to the field of gene engineering and enzyme engineering. According to the amylosucrase mutant as well as the preparation method and application thereof, glycine at the 399th site of amylosucrase derived from Deinococcus Geothermals is mutated into serine, and the obtained amylosucrase mutant is applied to the production of turanose; the yield of the turanose obtained through the amylosucrase mutant under the conditions that the temperature is 35 DEG C, and the initial pH value is7.0 reaches up to 32% and is 2 times of the conversion rate of the turanose produced through wild enzymes; and therefore, the amylosucrase mutant can be applied to preparation of the turanose, so that the conversion rate of the turanose is further increased.

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] Turanose is formed by linking a molecule of glucose and a molecule of fructose with α-1,3 glycosidic bonds. As an isomer of sucrose, its sweetness is only half of that of sucrose. It has the characteristics of easy crystallization, high solubility, slow hydrolysis rate, easy reconciliation with other substances, and no fermentation by cariogenic microorganisms. It is suitable for people suffering from obesity, hyperlipidemia, high blood pressure, and diabetes. Therefore, turanose has the potential to become a new generation of low-calorie functional sweetener in the food field. In addition, turanose is also widely used in the field of medicine, and can be used as an inhibitor of acid α-glucosidase for the medical diagnosis of Pompe disease; it also has anti-inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P19/14C12P19/12
CPCC12N9/1051C12N15/70C12P19/12C12P19/14C12Y204/01004
Inventor 吴敬吴丹宿玲恰赵雅琪
Owner JIANGNAN UNIV
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