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Alkaline xylanase as well as coding gene and application thereof

A xylanase and alkaline technology, applied in the field of alkaline xylanase and its coding gene and application, can solve the problems of high cost, poor direct utilization effect, poor stability, etc.

Inactive Publication Date: 2018-06-22
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are still some problems in the direct use of xylanase in industry: poor stability, low substrate specificity, short service life and high cost.
In addition, the specificity of xylanase is low, and the effect of direct utilization is poor. Therefore, the relationship between the type and mass concentration of the substrate and the dosage of xylanase should be studied in depth, and the compatibility effect between different enzyme preparations should be explored to improve the quality of xylanase. substrate specificity

Method used

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  • Alkaline xylanase as well as coding gene and application thereof
  • Alkaline xylanase as well as coding gene and application thereof
  • Alkaline xylanase as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] (1) Materials

[0054] 1. Bacterial species: Streptomyces alkalophilus, which is an alkaline xylanase-producing strain Streptomyces sp.WMN-3 screened from a soil sample on a certain coast in Shenzhen. CCTCC), deposit time: September 7, 2017, deposit address: China. Wuhan. Wuhan University, deposit number: CCTCC NO: M 2017477. E.coli TOP10F' was purchased from Invitrogen;

[0055] 2. Vector: The vector used for DNA connection is pMD-19T Simple Vector produced by TaKaRa Company.

[0056] 3. Medium

[0057] (1) Enrichment medium: xylan 8.0g, peptone 10g, NaCl 15g, KH 2 PO 4 1.5g, Na 2 HPO 4 12H 2 O9.0g, MgSO 4 ·7H 2 O 2.0g, dissolved in 1L ddH 2 O, pH 9.0.

[0058] (2) Selection medium: xylan 8.0g, KNO 3 1.0g, MgSO 4 ·7H 2 O 0.5g, NaCl 15g, KH 2 PO 4 1.5g, 20g agar, dissolved in 1L ddH 2 O, pH 9.0.

[0059] (3) Slant medium (preserved strain): 1.0% glucose, 0.5% peptone, 0.5% yeast extract, 0.1% KH 2 PO 4 , 0.02% MgCl 2 , 5.0% NaCl, 1.0% Na 2 CO 3 ...

Embodiment 2

[0151] (1) Materials

[0152] 1. Strain: Streptomyces sp. WMN-3 strain (CCTCC NO: M 2017477). The host bacteria E.coli BL21 Star(DE3) and E.coli TOP10F' were purchased from Invitrogen;

[0153] 2. Vector: Escherichia coli expression vector pET-28a(+) (Novagen, Kan R ) was purchased from Novagen; vector map see Image 6 , its multiple cloning site map is shown in Figure 7 .

[0154] 3. Medium and buffer

[0155] (1) Selection medium: xylan 8.0g / L, KNO 3 1.0g / L, MgSO 4 ·7H 2 O 0.5g / L, NaCl 15g / L, KH 2 PO 4 1.5g / L, solid medium plus agar 15-20g / L, pH 9.0;

[0156] (2) LB medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, solid medium plus agar 15-20g / L, pH 9.0, autoclaved at 121°C for 20min.

[0157] (3) TE buffer: 10mmol / L Tris-Hcl, pH8.0, 1mmol / L EDTA, pH8.0.

[0158] (4) Alkaline lysis solution I, II, III (plasmid extraction): Alkaline lysis solution I: glucose 50mmol / L, Tris-HCl (pH8.0) 25mmol / L, EDTA 10mmol / L. About 100mL per bottle, store at 4°C after s...

Embodiment 3

[0230] The enzymatic property research of embodiment 3 alkaline xylanase

[0231] (1) Experimental method

[0232] 1. Optimum reaction pH and pH stability

[0233] Take an appropriate amount of the pure enzyme solution of alkaline xylanase obtained in Example 2, add 1% xylan solutions with different pH values ​​respectively, and measure the enzyme activity according to a conventional method. At the same time, the pure enzyme solution was stored at 37° C. for 60 min under different predetermined pH conditions, and then the remaining enzyme activity was measured by conventional methods.

[0234] 2. Optimum reaction temperature and temperature stability

[0235] Take an appropriate amount of the pure enzyme solution of alkaline xylanase obtained in Example 2, respectively place it under different predetermined temperature conditions and react for 20 minutes, and measure its enzyme activity. At the same time, the pure enzyme solution was placed under different predetermined tem...

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PUM

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Abstract

The invention discloses alkaline xylanase as well as a coding gene and an application thereof and belongs to the technical field of biology. The gene sequence of WMN-3 is a complete ORF (open readingframe), wherein the ORF starts with start codon ATG, stops with stop codon TAA and totally contains 1476 nucleotides, and the nucleotide sequence is shown as SEQ ID NO:1. An amino acid sequence of a protein coded by a gene of alkaline xylanase WMN-3 is shown as SEQ ID NO:2. The invention further discloses the application of the alkaline xylanase WMN-3 to industries such as papermaking and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an alkaline xylanase, its coding gene and application. Background technique [0002] Xylan is a hemicellulose linked by β-1,4 linkages with the unit of xylanose, which is abundant in broad-leaved trees and most annual plants. The content of hemicellulose in nature accounts for 30% of biomass, second only to cellulose. [0003] Xylanase is a general term for enzymes that catalyze the hydrolysis of xylan into xylooligosaccharides or xylooligosaccharides, including a variety of endo-xylanases and exo-xylanases, widely distributed in bacteria, fungi, yeast, actin Bacteria, rumen of ruminants, snails, crustaceans, terrestrial plant tissues and various invertebrates, among which neutral or alkaline xylanases derived from bacteria have high alkali resistance and thermal stability. Xylanase has a wide range of applications. In the paper industry, it can be used in pulp bleachin...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/70C12N1/21D21C5/00
CPCC12N9/2482C12N15/70C12Y302/01008D21C5/005
Inventor 刘森林周鹏陈伟钊
Owner SHENZHEN UNIV
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