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High-yield strain of high temperature resistant 1,4-beta-D-xylanase, method for producing high temperature resistant 1,4-beta-D-xylanase through fermentation of high-yield strain, and high temperature resistant 1,4-beta-D-xylanase

A technology of xylanase and high-yield strains, which is applied in the field of biotechnology and engineering, can solve problems such as application limitations, and achieve good stability and broad application prospects

Active Publication Date: 2012-01-18
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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AI Technical Summary

Problems solved by technology

A number of enzyme preparation manufacturers such as Novozymes of Denmark and Sandaus Chemical Company of the United States have launched new products of xylanase and cellulase specially used for pulp treatment, but so far, the industrially used for pulp bleaching Most xylanases are neutral and slightly acidic, and the optimum reaction temperature is mostly around 50°C. As we all know, pulp cooking and bleaching are basically carried out under the conditions of high temperature and strong alkali, so that the existing low-temperature acidic xylanase The application of carbohydrase products in this field has been greatly limited. In addition, in the field of feed and food, xylanase is used in the granulation of feed and the baking process of food, and it is still required that the xylanase used in high temperature High enzyme activity can be maintained under certain conditions, therefore, the research and development of high temperature resistant xylanase products will bring good economic and social benefits

Method used

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  • High-yield strain of high temperature resistant 1,4-beta-D-xylanase, method for producing high temperature resistant 1,4-beta-D-xylanase through fermentation of high-yield strain, and high temperature resistant 1,4-beta-D-xylanase
  • High-yield strain of high temperature resistant 1,4-beta-D-xylanase, method for producing high temperature resistant 1,4-beta-D-xylanase through fermentation of high-yield strain, and high temperature resistant 1,4-beta-D-xylanase
  • High-yield strain of high temperature resistant 1,4-beta-D-xylanase, method for producing high temperature resistant 1,4-beta-D-xylanase through fermentation of high-yield strain, and high temperature resistant 1,4-beta-D-xylanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Screening Example 1: Screening and identification of xylanase-producing strains

[0060] (1) Enrichment culture

[0061] Take 0.1g of soil sample, add it into 10mL of sterile water, shake and mix well, then pipette 1mL of bacterial suspension into 5mL of enrichment medium, and incubate in a 37°C water bath for 48h with shaking.

[0062] (2) Primary screening by transparent circle method

[0063] Dilute the enriched culture 10-fold step by step, and take the dilution factor as 10 -3 , 10 -5 and 10 -7 0.5 mL of the bacterial suspension was spread on the selective medium plate, cultured at 37°C for 24 hours, and single colonies with relatively large transparent circles were picked for isolation and purification ( figure 1 ), access to the slant medium to preserve the original strains.

[0064] (3) Shake flask fermentation re-screening

[0065] Inoculate the primary screened strains in 50mL seed medium, cultivate overnight at 37°C, 180rpm, take 1mL of the overnight cu...

Embodiment 2

[0068] Enzyme Activity Example 2: Study on Fermentation Conditions and Enzyme Production Activity of P. campinasensis G1-1

[0069] (1) The assay method for xylanase activity is (DNS method):

[0070] Draw 0.1mL of appropriately diluted enzyme solution into a 5mL stoppered scale test tube, then add 0.1mL of 10g / L xylan substrate solution, cover the test tube tightly, react in a water bath at 50°C for 10min, and immediately add 0.6 Mix mL DNS reagent and mix to terminate the reaction, then boil in boiling water for 10 min, add water to make up to 5 mL after cooling, shake well, and calculate the sugar content of the reaction system according to the regression equation of the xylose standard curve.

[0071] The definition of xylanase activity unit in the test is: 1mL enzyme solution produces 1 μ moL of reducing sugar (calculated as xylose) per minute as an activity unit, expressed in IU:

[0072] IU=N×R / 10min×0.1mL

[0073] In the formula: N is the dilution factor of the enzym...

Embodiment 3

[0077] Enzyme separation example 3: separation and purification of high temperature resistant xylanase

[0078] (1) Centrifuge the fermentation broth at 8000rpm for 10min at 4°C, and take the supernatant;

[0079] (2) Slowly add ammonium sulfate to the fermentation supernatant to make the saturation of ammonium sulfate reach 70%, and salt out overnight at 4°C;

[0080] (3) The fermentation supernatant that was salted out overnight was centrifuged at 4° C., 8000 rpm, for 10 minutes, and the precipitate was taken, and an appropriate volume of PBS buffer solution of pH 7.0 was used to dissolve the precipitate to obtain a crude enzyme solution of high-temperature-resistant xylanase for later use;

[0081] (4) Octyl Sepharose Fast Flow hydrophobic interaction chromatography: adjust the ammonium sulfate saturation of the crude enzyme solution to 40%, and perform hydrophobic interaction chromatography, column type: Equilibrium solution: 0.02mol / L PBS buffer solution (pH7.0) contain...

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Abstract

The present invention discloses a high-yield strain of high temperature resistant 1,4-beta-D-xylanase, and a method for producing the high temperature resistant 1,4-beta-D-xylanase through fermentation of the high-yield strain, and provides partial enzymatic properties of the high temperature resistant 1,4-beta-D-xylanase. After the strain identification, the high-yield strain is named Paenibacillus campinasensis G1-1, and is preserved in the China general microbiological culture collection center. The preservation number of the strain is CGMCC No. 5023. According to the present invention, the high temperature resistant 1,4-beta-D-xylanase produced through the fermentation of the high-yield strain is subjected to separation and purification to obtain a single component, wherein the single component has a relative molecular weight of 41.3 KDa, an optimal operation temperature of 60 DEG C and an optimal operation pH of 7.0; the relatively stable enzyme activity of the high temperature resistant 1,4-beta-D-xylanase is remained in the temperature range of 40-70 DEG C and the pH range of 5.0-9.0; the Paenibacillus campinasensis G1-1 provided by the present invention is applicable for a plurality of fields such as paper making, food, feedstuff and the like, and has a broad application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology and engineering, in particular to a high-yielding strain of high-temperature-resistant xylanase, a method for producing high-temperature-resistant xylanase by fermentation of the bacteria, and the obtained enzyme. Background technique [0002] Xylanase (1,4-β-D-xylanase; EC3.2.1.8) is an important industrial enzyme, which has shown broad application prospects in the fields of paper industry, food, energy, feed and environment , especially the huge application potential in pulp biobleaching has already attracted great attention from all walks of life. The role of xylanase treatment of various pulps is to degrade the xylan in the pulp, reduce the content of hemicellulose in the pulp, and make the cellulose cell wall structure loose, and at the same time make the remaining lignin in the pulp The attached hemicellulose degrades, forming delignification or a state favoring delignification. The pretreatme...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/42C12R1/01
Inventor 路福平刘逸寒郑宏臣王春霞王建玲
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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