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Novel beta-xylosidase and preparation thereof

A technology of xylosidase and mutant, applied in the field of genetic engineering of enzymes, can solve the problems of limited modification of enzyme functions, and achieve the effect of high-efficiency expression

Active Publication Date: 2020-12-22
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because it can only replace, delete or insert a few amino acids in natural enzyme proteins, the modification of enzyme functions is limited

Method used

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  • Novel beta-xylosidase and preparation thereof
  • Novel beta-xylosidase and preparation thereof
  • Novel beta-xylosidase and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Obtaining of wild-type XYL-encoding gene xyl

[0066] 1. The wild-type XYL-encoding gene xyl comes from the TCCC 11573 strain of Bacillus pumilus preserved in the laboratory, and the genome is extracted using the Bacterial DNA Kit of OMEGA Corporation of the United States.

[0067] (1) Strain activation: Dip the Bacillus pumilus bacteria liquid from the glycerol tube with an inoculation loop, inoculate it on an LB solid medium plate, draw three lines, and incubate at a constant temperature of 37°C for 12 hours;

[0068] (2) Transplantation: Pick a single colony with neat edges and smooth surface from the culture plate and inoculate it in 5 mL of liquid LB medium, and culture at 220 r / min and 37°C for 12 hours;

[0069] (3) Collect bacterial cells: Take an appropriate amount of cultured bacterial liquid and put it in 1.5mL EP tubes, centrifuge at 12000r / min for 2min, and discard the supernatant;

[0070] (4) Add 250μL ddH 2 O resuspend the bacteria, add 50 μ...

Embodiment 2

[0100] Embodiment 2: Obtaining of XYL mutant E455G

[0101] 1. Error-prone PCR: Error-prone PCR is performed using the wild-type coding gene xyl as a template, and the reaction system is as follows:

[0102] wxya 2 o

10 μL Recombinant plasmid pET22b-xyl (5ng / μL) 2μL Upstream primer P1 (10μmol / L) 2μL Downstream primer P2 (10μmol / L) 2μL Taq DNA polymerase 0.5μL 10×Taq buffer 5μL dATP (10mmol / L) 1μL dGTP (10mmol / L) 1μL dTTP (10mmol / L) 5μL dCTP (10mmol / L) 5μL MgCl 2 (25mmol / L)

14μL MnCl 2 (10mmol / L)

2.5 μL

[0103] Note: The reagents required above are from Takara, Takara Bioengineering Co., Ltd.

[0104] After the system is completed, the error-prone PCR reaction is performed, and the program is set as follows:

[0105] a. Pre-denaturation: 95°C for 5 minutes;

[0106] b. Denaturation: 95°C for 30s;

[0107] c. Annealing: 56°C 45s;

[0108] d. Extension: 72°C for 90s;

[...

Embodiment 3

[0118] Embodiment 3: Construction of β-xylosidase Bacillus subtilis recombinant bacteria

[0119] 1. Construction of expression plasmid pBSA43-xylm

[0120] Both xylm and Bacillus subtilis expression vector pBSA43 were digested with BamHI and NotI, and then ligated to construct the recombinant plasmid pBSA43-xylm, which was transformed into Escherichia coli DH5α competent cells, and positive transformants were selected, and the plasmid was extracted for enzyme digestion verification And sequenced, it was confirmed that the construction was successful, that is, the recombinant expression plasmid pBSA43-xylm was obtained.

[0121] 2. Transformation of Bacillus subtilis WB600 with the expression plasmid pBSA43-xylm

[0122] (1) Activation of Bacillus subtilis WB600 strain, three-section line was drawn on the LB plate without anti-antibody, and cultured for 12 hours;

[0123] (2) Pick a single colony, inoculate it into a test tube containing 5 mL of LB medium, and incubate at 37...

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Abstract

The invention belongs to the technical field of genetic engineering of enzymes, and particularly relates to a beta-xylosidase mutant with improved enzyme activity and preparation thereof. According tothe invention, a wild type beta-xylosidase gene of Bacillus pumilus is obtained through a molecular biological technology means, random mutation is carried out on the wild type beta-xylosidase gene by utilizing an error-prone PCR technology to obtain a beta-xylosidase mutant E455G and a coding gene xylm thereof, recombinant plasmids are reconstructed, efficient expression of the recombinant plasmids in Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens is realized, and high-activity beta-xylosidase is obtained through technologies such as fermentation and extraction.

Description

Technical field: [0001] The invention belongs to the technical field of gene engineering of enzymes, and in particular relates to a beta-xylosidase mutant with improved enzyme activity obtained through in vitro directed evolution of an error-prone PCR technique and its preparation. Background technique: [0002] β-xylosidase (β-xylosidase, XYL, EC 3.2.1.37) is an important part of the xylan degrading enzyme system, which hydrolyzes the non-reducing terminal residues of oligosaccharides and xyloside substrates in an exo-cutting manner , to generate xylose, which has been widely used in many fields by synergistically acting with xylanase. In terms of food application, the addition of xylanase and β-xylosidase can decompose long-chain arabinoxylan into short chains and reduce the viscosity of beer. Appropriate addition of some β-xylosidase in the bread production process can greatly improve the mechanical properties and texture of bread; in terms of energy applications, xylan ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/75C12N1/21C12Q1/34C12R1/125C12R1/10C12R1/07
CPCC12N9/2402C12Y302/01037C12N15/75C12Q1/34
Inventor 刘逸寒王凤华葛秀琪路福平
Owner TIANJIN UNIV OF SCI & TECH
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