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A kind of alkaliphilic bacillus and the alkaline xylanase produced by it and its application

A technology of alkaliphilic bacillus and xylanase, which is applied in the field of alkaline xylanase and application, can solve the problem of not many excellent strains with high enzyme activity, achieve good application value, excellent enzyme properties, and stable enzyme activity Effect

Inactive Publication Date: 2021-10-01
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although there is a certain foundation for the research on xylanase at home and abroad, there are still not many excellent strains with high enzyme activity that can be produced on a large scale so far.

Method used

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  • A kind of alkaliphilic bacillus and the alkaline xylanase produced by it and its application
  • A kind of alkaliphilic bacillus and the alkaline xylanase produced by it and its application
  • A kind of alkaliphilic bacillus and the alkaline xylanase produced by it and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The screening of embodiment 1 target bacterial strain

[0041] (1) Enrichment culture: Weigh 1g of the collected soil sample, mix it thoroughly in 100mL of sterile water to make a bacterial suspension, take 1mL of the bacterial suspension and add it to the sterilized 5mL enrichment medium, 37 ℃ enrichment culture for 48h.

[0042] 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 O2.0g, dilute to 1L ddH 2 O, pH9.0.

[0043] (2) Plate initial screening: the bacterial solution after enrichment culture was diluted 10 times step by step, and the dilution factor was taken as 10 -3 、10 -5 、10 -7 0.5mL of the bacterial suspension was spread on the alkaline selective medium plate containing xylan, cultured at 37°C for 48h, and a single colony with a large transparent circle was screened out and picked into the alkaline slant medium, and obtained after multiple screenings A single colony, and preserved as the ...

Embodiment 2

[0052] Example 2 Separation and Purification of Alkaline Xylanase Produced by Bacillus sp.WMN1

[0053] (1) Materials

[0054] 1. Bacterial species: the alkalophilic Bacillus sp. WMN1 (CCTCC NO: M 2020007) obtained in Example 1.

[0055] 2. Medium and buffer

[0056] (1) Fermentation medium: bran 6g, peptone 1g, K 2 HPO 4 0.7g, dissolved in 100mL Gly-NaOH buffer, pH9.0.

[0057] (2) 1% xylan solution: 1 g of xylan was dissolved in 0.05M Gly-NaOH buffer solution with pH 9.0, the volume was adjusted to 100 mL, and shaken well before use.

[0058] (3) Gly-NaOH buffer (0.05M): glycine 3.8g, sodium hydroxide 0.35g, dissolved in 1L ddH 2 O, pH9.0.

[0059] (4) 3,5-dinitrosalicylic acid (DNS) solution: Accurately weigh DNS 7.5g, sodium hydroxide 14.0g (slowly added), potassium sodium tartrate 216g, sodium metabisulfite 6.0g, stir well, add 5.6 mL of phenol (poisonous, with a mask) melted in a 60°C water bath in advance, fully dissolved, added deionized water to make up to 1L,...

Embodiment 3

[0106] The enzymatic property research of embodiment 3 alkaline xylanase

[0107] (1) Experimental method

[0108] 1. Optimum reaction temperature and temperature stability

[0109] Take an appropriate amount of diluted pure enzyme solution, react at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 20 minutes, and measure its enzyme activity by DNS method. In order to determine the optimum reaction temperature. When studying temperature stability, an appropriate amount of diluted pure enzyme solution was placed at different temperatures for 1 hour, and then reacted at 45°C for 20 minutes to measure its enzyme activity.

[0110] 2. Optimum reaction pH and pH stability

[0111] Take an appropriate amount of diluted pure enzyme solution, react at pH2, pH3, pH4, pH5, pH6, pH7, pH8, pH8.5, pH9, pH9.5, pH10, pH11, pH12 respectively for 20 minutes, and measure its enzyme activity by DNS method. In order to determine the optimum reaction pH. When studying the st...

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Abstract

The invention discloses an alkalophilic bacillus and the alkaline xylanase produced by it and its application, belonging to the field of biotechnology. The alkalophilic Bacillus was screened from the soil, named Bacillus sp.WMN1, and was deposited in the Chinese Center for Type Culture Collection of Wuhan University, Wuhan, China on January 3, 2020, with the preservation number CCTCC NO: M 2020007. The invention also discloses the alkaline xylanase produced by the above-mentioned alkalophilic bacillus. The enzyme has a wide range of pH adaptation, heat resistance, alkali, metal ions and surfactants, etc., and is suitable for papermaking, washing and other industries. Excellent enzymology characteristic. The alkalophilic bacillus of the invention belongs to alkali-resistant extremophile microorganisms, can be cultivated under high alkaline (pH9.0) conditions, can more effectively prevent contamination by miscellaneous bacteria, and is more conducive to large-scale fermentation production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an alkaliphilic bacillus and the alkaline xylanase produced by it and its application. Background technique [0002] Foreign research on xylanase began in the 1950s, and it is relatively complete from strain construction to product development and production, and industrialized mass production of xylanase can be used. The current research hotspots mainly focus on the cloning and expression of microbial xylanase genes, the regulation mechanism and induction, purification and identification of xylanase, and the preparation of immobilized enzymes by nanotechnology. The research on xylanase in our country is relatively late, and the current research is mainly on the screening and domestication of excellent xylanase-producing strains, the optimization of enzyme production conditions, the purification of enzymes and the study of physical and chemical properties, xylanase gene c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/24C12R1/07
CPCC12N9/248C12N1/205C12R2001/07
Inventor 刘森林钟雅芳陈伟钊
Owner SHENZHEN UNIV
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