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N-terminal truncated mutant enzyme of glucan sucrase and preparation method of N-terminal truncated mutant enzyme

A technology of glucan sucrase and mutant enzymes, which is applied in the field of N-terminal truncation mutant enzymes of dextran sucrase and its preparation, can solve the problems of substrate specific stability or catalytic efficiency limitation, and achieve stability Good results

Active Publication Date: 2022-07-08
GUANGXI ACAD OF SCI +1
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] When natural dextran sucrase is used in large-scale industrial synthesis to produce various oligosaccharides or glycosylated products, it is often limited by factors such as the enzyme's substrate-specific stability or catalytic efficiency.

Method used

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  • N-terminal truncated mutant enzyme of glucan sucrase and preparation method of N-terminal truncated mutant enzyme
  • N-terminal truncated mutant enzyme of glucan sucrase and preparation method of N-terminal truncated mutant enzyme
  • N-terminal truncated mutant enzyme of glucan sucrase and preparation method of N-terminal truncated mutant enzyme

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preparation example Construction

[0026] The present invention also provides a preparation method of the N-terminal truncation mutant enzyme of glucansucrase, comprising the following steps:

[0027] (1) After culturing the recombinant bacteria carrying the gene encoding the N-terminal truncation mutant enzyme of glucansucrase under the condition of 35-40°C for 10-14 hours, continue to culture until the OD600 of the bacterial solution reaches 0.5-0.7 Add IPTG to a final concentration of 0.8-1.2 mM, then culture at 24-26°C, 200-250 r / min for 3-5 h, collect the cells by centrifugation, resuspend the cells with sodium acetate buffer, and centrifuge to obtain the supernatant. The liquid is the crude enzyme liquid;

[0028] (2) Passing the obtained crude enzyme liquid through a nickel column for separation and purification to obtain the N-terminal truncation mutant enzyme of glucansucrase.

[0029] In the present invention, as described in step (1), the recombinant bacteria carrying the gene encoding the N-termina...

experiment example 1

[0037](1) Glucansucrase gene cloning: The dextransucrase gene dsrD of the present invention is derived from Leuconostoc mesenteroides, and primers P1 and P2 are designed with the full sequence of the bacteria (AY017384.1). Amplification. The nucleotide sequence of the P1 is shown in SEQ ID NO.10, the nucleotide sequence of the P2 is shown in SEQ ID NO.11, and PagI (PagI-qNcoI is a homozygous enzyme) and SacI are introduced into the primers The restriction site is convenient for ligation to the pET-30(a) vector. The amplified fragment is cloned to obtain the glucansucrase gene of the present invention. Wherein, the amino acid sequence of glucansucrase is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding glucansucrase is shown in SEQ ID NO.2.

[0038] (2) Inducible expression of N-terminal truncation mutant enzyme and determination of its activity:

[0039] Five mutant enzymes were obtained by truncation of the glucansucrase gene obtained in step (1) at d...

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Abstract

The invention belongs to the technical field of enzyme engineering, and provides an N-terminal truncated mutant enzyme of glucan sucrase and a preparation method of the N-terminal truncated mutant enzyme. The N-terminal of the known glucan sucrase gene sequence is truncated to obtain a truncated mutant enzyme, and the enzyme activity of the obtained mutant enzyme is about 20 times higher than that of the original gene enzyme through induced expression and activity screening. The enzymatic property analysis of the mutant enzyme finds that the mutant enzyme has better stability under an acidic condition, and the optimal induction expression temperature and reaction temperature are close to room temperature. The invention lays a foundation for rational design, practical application and enzyme engineering production of dextran.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to an N-terminal truncation mutant enzyme of glucansucrase and a preparation method thereof. Background technique [0002] Glucansuerased is a kind of α-transglycosidase, which uses sucrose as a substrate to polymerize the glucose group in sucrose into high molecular weight glucan and release fructose at the same time. In the presence of receptors, low molecular weight oligosaccharides are formed. Due to the specificity of the glucansucrase reaction, the diversity of products and the operability for product synthesis, it has become an important tool enzyme in biocatalytic synthesis, and has wide application prospects in industrial fields such as feed, food and medicine. . [0003] Generally, sucrose-utlizing transglucosidases can be divided into two families of glycoside hydrolase 70 and 13. Except for amylosucrases, which belong to the GH13 family, most glucanasucrases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12R1/19
CPCC12N9/1051C12N15/70C12Y204/01005
Inventor 左晓琼王青艳冼亮秦艳李亿梁戈徐秀颖李晓明陆迪
Owner GUANGXI ACAD OF SCI
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