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Alkaline xylanase with improved heat stability, and coding gene and application thereof

A thermal stability, xylanase technology, applied in the field of genetic engineering, can solve the problems of time-consuming and laborious, and achieve the effect of huge application potential and improved thermal stability.

Active Publication Date: 2014-01-22
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And so far, most of the xylanases used for pulp bleaching in industry are neutral and slightly acidic, and the optimum reaction temperature is mostly below 45°C, which requires the pH and temperature of the pulp to be adjusted before adding enzymes. Suitable for the range of xylanase action, which is a time-consuming and labor-intensive process

Method used

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  • Alkaline xylanase with improved heat stability, and coding gene and application thereof
  • Alkaline xylanase with improved heat stability, and coding gene and application thereof
  • Alkaline xylanase with improved heat stability, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, xylanase xyn-B230 gene synthesis, expression vector construction and mutant construction

[0037] (1) Structural optimization

[0038] Based on the crystal structure (PDB: 1IGO) of Bacillus subtillis B230 xylanase xyn-B230 (the amino acid sequence of which is shown in SEQ ID NO.3), the selected mutation sites are: S23H, N45I, D55P, H60Y, S61D, N65D, N75T, I129Y, T149D, Y185F,.

[0039] The amino acid sequence of Bacillus subtillis B230 xylanase xyn-B230 is shown in SEQ ID NO.3:

[0040] ATTITSNQTGTHDGYDYELWKDSGNTSMTLNSGGAFSAQWSNIGNALFRKGKKFDSTKTHSQ LGNISINYNATFNPGGNSYLCVYGWTKDPLTEYYIVDNWGTYRPTGTPKGTFTVDGGTYDIYE TTRINQPSIIGIATFKQYWSVRQTKRTSGTVSVSVSEHFKKWESLGMPMGKMYSNALTVEGY

[0041] (2) Gene optimization synthesis

[0042] The xyn-B230 gene xyn-B230 from Bacillus subtillis B230 was modified according to the preference of Pichia pastoris without changing its amino acid sequence. Avoid GT...AG site during transformation, and pay attention not to affect th...

Embodiment 2

[0050] Example 2 Expression of xylanase xyn-B230 and mutants thereof in Escherichia coli

[0051] The two expression vectors pET-22b(+)-xyn-B230 and its mutants S23H, N45I, D55P, H60Y, S61D, N65D, N75T, I129Y, T149D, and Y185F described in Example 1, a total of 11 expression plasmids Transform the bacillus E.coli BL21(DE3) by heat shock, and obtain recombinant strains containing the original gene and each mutant respectively.

[0052] Take the BL21(DE3) strain containing the recombinant plasmid and the BL21(DE3) strain containing the pET-22b(+) empty plasmid (as a control), inoculate them in 3 mL LB (containing 100 μg / mL Amp) culture solution, and rapidly Incubate overnight with shaking. Add 100 μL of overnight culture solution to 10 mL of LB culture solution (1% inoculum) containing Amp, and culture with rapid shaking for about 2-3 hours (OD 600 reach 0.6-0.8), then add the inducer IPTG with a final concentration of 1mmol / L, and continue shaking culture at 37°C for about 4h...

Embodiment 3

[0053] Embodiment 3, purification of xylanase xyn-B230 and mutants thereof

[0054] The specific process of purifying xylanase xyn-B230 and its mutants is basically the same, and the general process is as follows: the supernatant expressed in the shake flask is concentrated with a hollow fiber, and then ammonium sulfate is slowly added to the concentrated solution of every 100 mL, and Stir continuously for 3 hours for fractional precipitation, and then centrifuge at 12,000 rpm for 10 minutes to collect the precipitate. The pellet was dissolved with a small amount of 20 mM Tris-HCl buffer, pH 10.0, and dialyzed overnight against a large amount of this buffer. The dialyzed solution was purified by anion column (HiTrap Q Sepharose XL5mL). The anion column was equilibrated with 20mM Tris-HCl buffer at pH 10.0 in advance, and then eluted with a linear gradient of 0-1.0mol / L NaCl solution after loading, and the elution tubes with xylanase activity were collected and analyzed by SDS...

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Abstract

The invention relates to the field of genetic engineering, specifically to alkaline xylanase with improved heat stability and a coding gene and application thereof. According to the invention, the crystal structure of Bacillus subtillis B230 xylanase xyn-B230 is used as a starting structure, and selected mutation sites are S23H and I129Y. Compared with the xylanase xyn-B230, mutated xylanase has substantially improved heat stability and can retain 90% of enzymatic activity when maintained at a temperature of 70 DEG C for 120 min and more than 65% of enzymatic activity when treated at a temperature of 75 DEG C for 30 min. The xylanase xyn-B230-m has good properties and is applicable to the industry of papermaking.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an alkaline xylanase with improved thermostability, its encoding gene and application. Background technique [0002] Xylan is an important component of plant hemicellulose. It is the most common hemicellulose in the cell wall of terrestrial plants. Except for cellulose, xylan is the most abundant polysaccharide in nature. In broad-leaved wood and annual grass plants, xylan accounts for 20%-30% of the dry weight of plant raw materials, and the content of xylan in coniferous wood is less, generally 7%-10% of the dry weight of raw materials. [0003] Xylanase is a general term for a group of enzymes that degrade hemicellulose xylan, which mainly includes endo-1,4-β-xylanase, β-xylosidase and other coenzymes acting on the main chain. Since the discovery that xylanase can be used for pulp bleaching, the research and development of xylanase at home and abroad have paid great attenti...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19D21C5/00D21C5/02D21C9/10C12R1/84C12R1/125
CPCC12N9/248C12N9/2482C12N9/2485C12Y302/01008C12Y302/01032Y02W30/64
Inventor 姚斌黄火清罗会颖王亚茹石鹏君柏映国孟昆杨培龙
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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