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Bacillus sphaericus with high yield and high-temperature xylanase resistance and application thereof

A technology of Bacillus sphaericus and xylanase, applied in the field of applied microorganisms, can solve the problems that hinder the development of xylanase application and energy consumption

Active Publication Date: 2014-09-10
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that the optimum reaction temperature of xylanase is generally lower than 45°C, so xylanase needs to be cooled in advance before it is put into use in the above process to improve the catalytic activity of the enzyme. These operations not only consume energy but also greatly hinder the development of xylanase. Application and Development of Glycanase

Method used

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  • Bacillus sphaericus with high yield and high-temperature xylanase resistance and application thereof
  • Bacillus sphaericus with high yield and high-temperature xylanase resistance and application thereof
  • Bacillus sphaericus with high yield and high-temperature xylanase resistance and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Screening and cultivation of high-yield and thermostable xylanase strains

[0022] Enrichment medium: NaNO 3 0.5g, K 2 HPO 4 l g, MgSO 4 ·7H 2 O0.5g, KCl0.5g, FeSO 4 ·7H 2 O0.005g, distilled water 1000mL, pH natural, sterilized at 121°C for 20min.

[0023] Screening medium: peptone 10g, yeast extract 3g, NaCl 5g, xylan 4g, agar powder 15g,

[0024] Distilled water 1000mL, pH8.0, sterilized at 121°C for 20min.

[0025] Fermentation medium: peptone 10g, yeast extract 3g, NaCl 5g, xylan 4g, distilled water 1000mL, pH natural, sterilized at 121°C for 20min.

[0026] LB medium: 10g of peptone, 5g of yeast extract, 10g of NaCl, 20g of agar, dissolved in 1000mL of distilled water, pH 7.0-7.2, sterilized at 121°C for 20min.

[0027] Collect several 5g samples from different positions of straw compost located around the biomethane production workshop of Nanjing University of Technology, put them in 250mL Erlenmeyer flasks, add 50mL sterile water, and incubate ...

Embodiment 2

[0028] The biological identification of embodiment 2 screening bacterial strains

[0029] 1. Morphological identification of strains

[0030] The isolated and purified strains were inoculated on solid LB medium and cultured at 37°C for 24 hours to observe the plate colony morphology of the strains. Such as figure 1 , strain Xyn-1, after growing on solid LB medium, the colony diameter is 0.6cm, the shape is a regular protrusion, which can form endophytic spores, the shape is round, the medium is white opaque colony, the surface is smooth, and the edge of the colony is regular .

[0031] 2. Molecular identification of strains

[0032]The strains of the screened bacteria were identified based on the bacterial 16S rDNA sequence library of the NCBI database. The DNA of the strain was extracted using a bacterial genome extraction kit, and PCR amplification was carried out using bacterial 16S rDNA universal primers. The PCR reaction system is (25 μL): template DNA 10ng, 10×PCR b...

Embodiment 3

[0034] The optimization of embodiment 3 fermentation culture conditions

[0035] The culture conditions of strain Xyn-1 were optimized by single factor analysis method, and the best culture conditions were determined: the amount of culture medium was 25mL / 250mL conical flask, the culture temperature was 45°C, the rotation speed of shaker flask was 200r / min, and the concentration of bacterial suspension was 5 ×10 6 Individual / mL, the inoculum volume per bottle is 1.0mL, the initial pH is 7.0, and the fermentation time is 72 hours. Optimization of medium composition:

[0036] The 9 factors related to the medium of Xyn-1 enzyme production were determined by single factor analysis. The Plackett-Burman design was used to screen the important influencing factors of enzyme production, and the design with the number of trials N=12 was selected to control the lactose, maltose, xylan, yeast powder, peptone, sodium chloride, magnesium sulfate, ammonium sulfate and phosphoric acid in th...

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Abstract

The invention discloses a bacillus sphaericus with high yield and high-temperature xylanase resistance and an application thereof. The bacillus sphaericus is classified and named as Lysinibacillus sphaericus Xyn-1, and is collected in China Center for Type Culture Collection (CCTCC) on May, 5th, 2014 with the collection number CCTCCM2014183. The optimal fermentation enzyme production condition of a strain screening shake flask is determined by adopting a Plackett-Burman design and a response surface analysis method, and the fermentation level under the condition can reach 5678.5IU / mL. When the enzyme produced by fermentation of the strain is applied to degradation of plant hemicellulose, the xylobiose yield of the hemicellulose can reach over 60% through enzymolysis.

Description

technical field [0001] The invention relates to a high-yielding high-temperature resistant xylanase bacillus sphaericus and an application thereof, belonging to the technical field of applied microorganisms. Background technique [0002] Xylanase (Xylanase E.C3.2.1.8) is a major xylan degrading enzyme, which degrades the β-1,4 xylosidic bonds in xylan by endocutting, and the hydrolyzed products are mostly Xylooligosaccharides, accompanied by a small amount of xylose and arabinose. Xylanase has a wide range of application prospects. In the paper industry: xylanase can be used as a biological bleaching agent to replace traditional chemical bleaching, thereby greatly reducing the amount of chlorine and reducing the environmental pollution of papermaking. Xylanase is used as " "Pulping enzyme" has been widely used in western developed countries, bringing good economic and social benefits; xylanase is widely used in the food industry for the production of flour products, fruit j...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/42C12P19/14C12R1/01
Inventor 贾红华钟超王春明王凤学韦萍
Owner NANJING UNIV OF TECH
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