Xylanase-m with high heat stability and its coding gene and application thereof

A technology of xylanase and xylan, which is applied in the field of genetic engineering, can solve the problems of large loss of activity and increased cost, and achieve the effects of improved thermal stability, improved practicability, and significant application value

Active Publication Date: 2019-04-19
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether it is the use of xylanase in papermaking or animal husbandry, there will be a process that requires high-temperature treatment. Wild-type xylanase loses a lot of activity after high-temperature treatment, which increases the cost of its industrial use, so There is an urgent need for a thermostable xylanase in industry

Method used

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  • Xylanase-m with high heat stability and its coding gene and application thereof
  • Xylanase-m with high heat stability and its coding gene and application thereof
  • Xylanase-m with high heat stability and its coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the construction of recombinant bacteria

[0037] The double-stranded DNA molecule represented by nucleotides 5-708 of sequence 4 in the sequence listing was inserted between the NcoI and EcoRI restriction sites of the vector pET-28a(+) to obtain the recombinant plasmid pET28a-xylanase-wt. After sequencing, the structure of the recombinant plasmid pET28a-xylanase-wt is described as follows: the exogenous DNA molecule is fused with part of the nucleotides on the vector backbone to form the fusion gene shown in Sequence 4 of the sequence listing, and the expression sequence 3 of the sequence listing. shown protein. In Sequence 3 of the Sequence Listing, the amino acids at positions 5-10 form his 6 Tag, amino acid residues 11-235 constitute wild-type xylanase. The recombinant plasmid pET28a-xylanase-wt was introduced into Escherichia coli BL21 (DE3) to obtain a recombinant bacteria, which was named as recombinant bacteria BL21 / xylanase-wt.

[0038] The dou...

Embodiment 2

[0039] Example 2. Preparation of xylanase

[0040] The test bacteria were: recombinant bacteria BL21 / xylanase-wt or recombinant bacteria BL21 / xylanase-m.

[0041] 1. Inoculate the test bacteria into the liquid LB medium, shake and cultivate to OD at 37°C and 250rpm. 600nm The value is 1.

[0042] 2. After completing step 1, IPTG was added to the system to make its concentration 0.1 mM, and cultured with shaking at 30°C and 200 rpm for 16 hours.

[0043] 3. After completing step 2, the cells were collected by centrifugation, resuspended with pH7.0, 50mM PBS buffer, and then sonicated (sonication parameters: power 30%, total time 20min, 5s for every 5s of work), and then 12000rpm Centrifuge for 10 min and collect the supernatant.

[0044] 4. Take the supernatant obtained in step 3 and perform nickel column affinity chromatography.

[0045] Nickel column (column volume is 2 mL): 6FF Ni Sepharose (Beijing Jiangchen Yuanyuan Biotechnology Co., Ltd.; product number: HA-0710-02). ...

Embodiment 3

[0053] Example 3. Activity analysis of xylanase (DNS method)

[0054] One unit of enzymatic activity (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions.

[0055] 1. Prepare the solution to be tested.

[0056] Take the xylanase-wt solution or xylanase-m solution prepared in Example 2, dilute it with a PBS buffer of pH 7.0 and 50 mM, and use it as the solution to be tested.

[0057] 2. Detect the protein concentration of the solution to be tested.

[0058] Detect the concentration of the target protein in the test solution prepared in step 1 (calculated as the total protein concentration).

[0059] 3. Detect the enzyme activity of the solution to be tested.

[0060] 1. Prepare the initial reaction system.

[0061] The initial reaction system (100 μL) consisted of the test solution, birch xylan and PBS buffer at pH 7.0 and 50 mM. In the initial reaction system, the protein concentration was 0.005 mg / mL, and the bir...

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Abstract

The invention discloses xylanase-m with high heat stability and its coding gene and an application thereof. The protein provided by the present invention is named as xylanase-m, also known as xylanase-m protein, which is as follows (a1), (a2) or (a3): (a1) a protein consisting of 11st to 235th amino acid residues in SEQ ID NO: 1 of a sequence table; (a2) a protein represented by SEQ ID NO: 1 of the sequence table; and (a3) a fusion protein obtained by connecting a label at the N-terminus or / and C-terminus of (a1). The invention also provides an application of xylanase-m protein as xylanase. The invention also provides an application of xylanase-m protein in degrading xylan. The invention also provides an application of the xylanase-m protein in reducing sugar production by taking xylan asa substrate. The xylanase-m can be used in the food industry, animal feed industry and paper bleaching industry, and has great application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a xylanase-m with high thermal stability and its encoding gene and application. Background technique [0002] Hemicellulose is the second most abundant component in plant cell walls and the second most abundant polysaccharide in nature. Xylan is the most important component of hemicellulose, accounting for about 15-30% of the dry weight of angiosperm cells. However, hemicellulose or xylan are difficult to degrade, and complete degradation of xylan requires multi-step enzymatic reactions and Synergistic action of multiple hydrolases, among which β-1,4-endo-xylanase (endo-1,4-β-xylanase, E.C.3.2.1.8) and β-xylosidase are the most critical hydrolases. Xylanase can open the xylosidic bond of xylan, and effectively degrade high-polymer xylan into xylose monomer or oligo-xylan. [0003] In papermaking, xylanase treatment of pulp can effectively release lignin, so that p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12P19/14
CPCC12N9/248C12P19/14
Inventor 胡美荣步依繁彭颖陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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