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A kind of xylanase xylanase-m with high thermostability and its coding gene and application

A xylan and gene technology, 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: 2020-10-09
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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  • A kind of xylanase xylanase-m with high thermostability and its coding gene and application
  • A kind of xylanase xylanase-m with high thermostability and its coding gene and application
  • A kind of xylanase xylanase-m with high thermostability and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction of recombinant bacteria

[0037] Insert the double-stranded DNA molecule represented by nucleotide 5-708 of sequence 4 in the sequence list between the NcoI and EcoRI restriction sites of 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 foreign 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 list, and the sequence shown in sequence 3 of the sequence list is expressed Protein shown. In sequence 3 of the sequence listing, the amino group of positions 5-10 constitutes his 6 Tag, amino acid residues 11-235 constitute wild-type xylanase. The recombinant plasmid pET28a-xylanase-wt was introduced into E. coli BL21 (DE3) to obtain a recombinant strain, which was named as recombinant strain BL21 / xylanase-wt.

[0038] Insert the double-stranded DN...

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 liquid LB medium, and cultivate to OD at 37℃ and 250rpm with shaking 600nm The value is 1.

[0042] 2. After completing step 1, add IPTG to the system and make the concentration of 0.1mM, 30℃, 200rpm shaking culture for 16h.

[0043] 3. After completing step 2, collect the bacteria by centrifugation, resuspend in pH7.0, 50mM PBS buffer, and ultrasonically disrupt (parameters of ultrasonic disruption: power 30%, total time 20min, stop for 5s every 5s of work), then 12000rpm Centrifuge for 10 min and collect the supernatant.

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

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

[0046] Washing so...

Embodiment 3

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

[0054] One unit (U) of enzyme activity 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, and dilute it with a pH 7.0, 50 mM PBS buffer as the test solution.

[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 (100μL) consists of the test solution, birchwood xylan and pH7.0, 50mM PBS buffer. In the initial reaction system, the concentration of protein is 0.005mg / mL, and the concentration of birchwood xylan is 1g / 100mL.

[006...

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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 specifically relates to a xylanase xylanase-m with high thermostability and its coding 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 is difficult to degrade. The complete degradation of xylan requires multiple enzymatic reactions and The synergistic effect of multiple hydrolases, of which β-1,4-endo-xylanase (endo-1,4-β-xylanase, EC3.2.1.8) and β-xylanase are the most critical hydrolases. Xylanase can open the xylan bonds of xylan and effectively degrade high xylan into xylose monomer or oligoxylan. [0003] In papermaking, xylanase treatment of pulp can effectively release lignin, so that pigments can be separat...

Claims

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Application Information

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Patent Type & Authority Patents(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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