A xylanase mutant with improved catalytic efficiency

A technology of xylanase mutation and carrier, which is applied in the fields of genetic engineering and protein expression, can solve the problems that need to be carried out and fail to reach industrial application, achieve great application potential, solve low catalytic activity, enzyme activity and kinetic parameters Improved effect

Active Publication Date: 2020-12-29
JIANGNAN UNIV
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Although many researchers have carried out molecular modification of xylanase through gene mutation technology and achieved certain results, it has not yet reached the level of industrial application, and research on molecular modification to improve the catalytic properties of xylanase remains to be done.

Method used

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  • A xylanase mutant with improved catalytic efficiency
  • A xylanase mutant with improved catalytic efficiency
  • A xylanase mutant with improved catalytic efficiency

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Experimental program
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Effect test

Embodiment 1

[0023] Construction of embodiment 1 mutant library

[0024] By searching the protein PDB database, a xylanase crystal structure (PDB: 2VGD) with a protein sequence identity of 76% to xylanase AEx11A was obtained, and the xylanase AEx11A was analyzed using SWISS-MODEL with 2VGD as a template. Three-dimensional structure simulation, and molecular docking of the simulated AEx11A. Through the conservative analysis of the three-dimensional structure of AEx11A and the analysis of amino acid positions and physical and chemical properties, the 98th threonine was selected to be mutated into aspartic acid at the same time, and the 124th valine was mutated on the above basis. Saturation mutations were performed to screen out the optimal transformants. Based on the mutation sites designed above, design and synthesize specific site-directed mutagenesis primers T98D-F, T7-R (as shown in SEQ ID NO.5, SEQ ID NO.6) and saturation site-directed mutagenesis primer V124X-F (as shown in shown in...

Embodiment 2

[0026] Example 2: Screening of mutant libraries

[0027] The saturation mutation library pET-28a-AEx11A constructed in Example 1 T98D / V124X To transform E.coli BL21 competent cells, the specific method is as follows:

[0028] 1) Inoculate activated E.coli BL21 on LB plate in 2 mL LB medium, culture overnight at 37°C, 220r / min; inoculate 2% of the above culture solution in 5mL LB medium, culture at 37°C, 220r / min for 4h;

[0029] 2) Take 1.4mL of the above bacterial solution and put it into a 1.5mLEP tube, bathe in ice for 10min, centrifuge at 4000r / min for 2min, and collect the bacteria;

[0030] 3) Add 1mL of pre-cooled 0.1M CaCl 2 The above-mentioned cells were resuspended in the solution, placed in an ice bath for 10 minutes, centrifuged at 4000 r / min for 2 minutes, and the bacteria were collected;

[0031] 4) Add 100 μL of pre-cooled 0.1M CaCl 2 Suspend the above cells in the solution and store at 4°C for 30 minutes to transform;

[0032] 5) Take 100 μL of competent c...

Embodiment 3

[0039] The purification of embodiment 3 recombinant xylanase and the mensuration of enzymatic property

[0040] E.coli / AEx11A and E.coli / AEx11A T98D / V124Q The expression was induced by 0.4mmol / L IPTG at 20°C for 8h and the cells were disrupted by sonication, and the supernatant was purified by Ni-NAT column to purify the target enzyme protein. SDS-PAGE detection of purified AEx11A and AEx11A T98D / V124Q All present a single band at a relative molecular mass of 26.2kDa. For purified AEx11A and AEx11A T98D / V124Q Enzymatic property analysis, as shown in Table 1, AEx11A T98D / V124Q The specific activity of the mutant enzyme is 3.04 of that of AEx11A, and the catalytic efficiency is 2.74 times of that of AEx11A. Increased mutant AEx11A due to improved substrate affinity and catalytic efficiency T98D / V124Q specific enzyme activity. This shows that the invention improves the enzymatic properties of xylanase through the innovative way of mutation.

[0041] The relative activity...

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Abstract

The invention discloses a xylanase mutant with improved catalytic efficiency, and belongs to the technical field of genetic engineering and protein expression. A molecular structure of xylanase is modified by site-specific mutagenesis and saturation mutation biological technologies to obtain a xylanase mutant AEx11AT98D / V124Q with improved catalytic activity. The specific activity and catalytic efficiency of mutate enzyme are separately 3.04 times and 3.74 times that of AEx11A, and moreover, the high temperature resistance and high thermal stability of the enzyme are still similar to those ofproenzyme. The limited problem of low catalytic activity of the xylanase is solved, the xylanase mutant with the improved catalytic efficiency has high application potential, and a theoretical basis is laid for research of the xylanase.

Description

technical field [0001] The invention relates to a xylanase mutant with improved catalytic efficiency, belonging to the technical fields of genetic engineering and protein expression. Background technique [0002] Xylan is an important component of hemicellulose and an important renewable biological resource. Endo-β-1,4-xylanases, referred to as xylanases (EC 3.2.1.8) for short, can hydrolyze the β-1,4-glycosidic bonds inside the main chain of xylan molecules, and are widely used in food , paper and feed and other fields. [0003] The enzymatic properties of xylanases from different sources are different. Studies have found that naturally occurring xylanases rarely have both high activity and high heat resistance. Therefore, more and more researchers carry out molecular modification on xylanase in order to obtain industrialized xylanase with excellent enzymatic properties. At present, the research on the molecular modification of xylanase is mainly focused on the modificat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/248C12N15/70
Inventor 邬敏辰张婷胡博淳苏永君文正徐雄峰刘艳胡蝶李剑芳
Owner JIANGNAN UNIV
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