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Thermophilic alkali-resistant xylanase recombinant engineering bacterium BL21-XA and application thereof

A technology of BL21-XA and recombinant engineering bacteria, which is applied in the fields of enzyme genetic engineering and enzyme engineering, can solve the problems of lack of recombinant engineering bacteria, and achieve the effects of strong thermal stability, strong thermal stability, and good heat resistance

Inactive Publication Date: 2010-09-29
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to solve the problem of lack of recombinant engineering bacteria for preparing heat-resistant and alkali-resistant xylanase in the prior art, and provide a heat-resistant and alkali-resistant xylanase recombinant engineering bacteria BL21-XA and its application , the thermostable xylanase prepared by the engineering bacteria BL21-XA has good activity, good tolerance, wide adaptability to pH value, heat and alkali resistance, etc.

Method used

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  • Thermophilic alkali-resistant xylanase recombinant engineering bacterium BL21-XA and application thereof
  • Thermophilic alkali-resistant xylanase recombinant engineering bacterium BL21-XA and application thereof
  • Thermophilic alkali-resistant xylanase recombinant engineering bacterium BL21-XA and application thereof

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1. Isolation and identification of thermophile TC-W7

[0027] The samples collected from Yongtai Hot Spring in Fujian were evenly spread on the screening plate with xylan as the carbon source, single colonies were picked for streaking and purification, and then the purified strains were planted on the transparent circle observation plate, 60 After culturing at ℃ for two days, stain with 0.1% Congo red for 30 min, and decolorize with 1mol / L NaCl solution. Select the colonies with transparent circles around them for re-screening in shake flask fermentation, and select the strain TC-W7 with larger enzyme activity.

[0028] The colonies on the plate of this strain are white, flat, viscous, translucent, with irregular edges and small "V"-shaped notches. Gram staining was positive, and the shape under the microscope was long rod. 16S rDNA identification: DNA was extracted using the instructions of the Tiangen Bacteria Genome Extraction Kit, PCR amplification was ...

Embodiment 2

[0029] Embodiment 2. Cloning of thermophile Gebacillus sp.TC-W7 xylanase gene

[0030]Using the extracted total DNA of thermophilic bacteria Gebacillus sp.TC-W7 as a template, the following degenerate primers were used: sense primerXA: [5'-CAT(C)ACA(G)C(T)TGGTTTGGCA-3'], antisense primer XA: [5'-A(T)CCCCAG(A)AAC(T)GTG(A)AC-3'] or a pair of specific primers derived from degenerate primers. The PCR reaction system is as follows: 95°C, 4 minutes, one cycle; 95°C, 1 minute, 55°C, 1 minute, 72°C, 1 minute, 30 cycles; 72°C, 10 minutes, one cycle. The product was verified by 1% agarose electrophoresis to obtain a DNA fragment of about 1200bp, which was verified by sequencing.

Embodiment 3

[0031] Example 3. Construction of Xylanase Recombinant Engineering Bacteria, Expression and Purification of Xylan Recombinase

[0032] The amplified DNA fragments were ligated into the plasmid vector pGEX using genetic engineering tool enzymes. Then, the ligation product was transferred into E. coli Escherichia coli BL21 (DE3) competent cells, and spread evenly on LB plates (containing Amp100 μg / ml). Pick the colony of positive recombinant engineered bacteria and extract the plasmid. Subsequently, the method of double enzyme digestion and sequencing was used to identify that the DNA fragment of the xylanase had been correctly integrated into the expression vector.

[0033] Pick a single colony containing the recombinant plasmid and put it in 3ml LB liquid medium (containing Amp 100μg / ml), shake it overnight at 37°C. Pipette 1ml of the bacterial liquid into a new 100ml LB liquid medium (containing Amp 100μg / ml), shake culture at 30°C until OD 600 is about 0.6, add IPTG to a ...

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Abstract

The invention a thermophilic alkali-resistant xylanase recombinant engineering bacterium BL21-XA and application thereof, belongs to the fields of enzyme gene engineering and enzyme engineering, and solves the problems that the thermophilic alkali-resistant xylanase recombinant engineering bacterium for preparing the xylanase is insufficient, has undesirable activity and thermophilic alkali resistance, and the like in the prior art. The invention discloses a recombinant plasmid pXA containing high-temperature xylanase gene of a Gebacillus sp. thermophilic bacterium TC-W7, which has a nucleotide sequence shown in SEQ No.1, the recombinant engineering bacterium BL21-XA containing the recombinant plasmid pXA, thermostable xylanase prepared from the engineering bacterium, and a preparation method and application of the xylanase. The engineering bacterium BL21-XA has the advantages of good heat resistance, wide pH value adaptability and good tolerance; and the thermostable xylanase prepared from the engineering bacterium has good activity, heat and alkali resistance and other performance.

Description

technical field [0001] The invention belongs to the fields of enzyme gene engineering and enzyme engineering, and more specifically relates to a heat-resistant and alkali-resistant xylanase recombinant engineering bacterium BL21-XA and application thereof. Background technique [0002] Xylanase is a class of hydrolytic enzymes that can catalyze the decomposition of hemicellulose, xylan, and branched xylooligosaccharides. They are different from general enzymes, it is an endonuclease system, which has good transglycosylation and degradation effects on substrates. In recent years, with the enhancement of people's understanding of the scope of application of this enzyme, this type of enzyme has been studied to a certain extent. However, because the use of xylanase in production often requires certain high-temperature conditions and higher alkali resistance, the breeding of thermostable alkaliphilic xylanase has great potential application value; but ideal in the prior art The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N1/21C12N9/42C12N15/56C12P19/14C12R1/19
Inventor 刘斌张宁宁吴祖建谢联辉
Owner FUJIAN AGRI & FORESTRY UNIV
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