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Method for immobilizing hemicellulase by using yeast spores

A technology of hemicellulase and spores, which is applied in the field of enzyme immobilization, can solve the problems of decreased enzyme activity, unsatisfactory use effect, and spores are easily broken and germinated.

Inactive Publication Date: 2019-01-11
BIOLOGY INST OF SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the defective yeast can display enzymes that need to be immobilized on the surface during the process of producing ascospores, the effect of using hemicellulase is not ideal in the process of actually displaying hemicellulase, mainly because the spores are easy to break during actual use. Wall germination, resulting in a sharp drop in enzyme activity; currently there is no better solution

Method used

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  • Method for immobilizing hemicellulase by using yeast spores
  • Method for immobilizing hemicellulase by using yeast spores
  • Method for immobilizing hemicellulase by using yeast spores

Examples

Experimental program
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Effect test

Embodiment 1

[0086] A method for immobilizing hemicellulase using yeast spores, comprising the steps of:

[0087] (1) Genomic DNA of Bacillus subtilis is extracted, respectively amplified by PCR to obtain xylanase xynA gene and signal peptide Epr gene; the upstream and downstream primer nucleotide sequences of the PCR amplification are as follows:

[0088] xynA gene amplification primers

[0089] Upstream primer: 5'-CA GGATCC TTCTCAAGGAACGATCAG-3' ("_" is the recognition site for BamH I digestion)

[0090] Downstream primer: 5'-TT GAGCTC ACGAGTGCTACCTCAAAG-3' ("_" is the recognition site for Sac I digestion)

[0091] Epr gene amplification primer

[0092] Primer on: 5'-CG GAATTC ATGAAAAACATGTCTTG-3' ("_" is the recognition site of EcoR I enzyme digestion)

[0093] Downstream primer: 5'-TT GGATCC ATAACCTCTTTCTCGC-3' ("_" is the recognition site for BamH I digestion)

[0094] The PCR amplification systems of the xylanase xynA gene and the signal peptide Epr gene are as follows, wit...

Embodiment 2

[0129] For the different recombinant bacteria constructed in Example 1, 0.5 g of 9 immobilized enzyme granules were taken in parallel for each strain, and the enzyme activity was measured at different temperatures (45 ° C ~ 85 ° C) to determine the optimum temperature of the immobilized enzyme ; At the optimum temperature, each bacterial strain took 8 parts of immobilized enzyme granules in parallel, each 0.5g, measured the enzyme activity at different pH values ​​(1.0~6.0) respectively, and determined the optimal reaction pH value of the immobilized enzyme; Under the appropriate pH value and temperature, take 6 immobilized enzyme granules for each strain in parallel, 0.5g each, and add phosphate to the reaction system of the same volume of the enzyme solution to be tested, so that the final concentration is between 5 and 30mmol / L , to determine the optimum phosphate concentration; take 5 copies of immobilized enzyme particles in parallel for each strain, 0.5g each, and add MgS...

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Abstract

The invention relates to a method for immobilizing hemicellulase by yeast spores. As the hemicellulase express gene and the Saccharomyces cerevisiae strain are screened for the first time, Hemicellulase immobilized between the dityrosine layer and the chitosan layer of spore is successfully obtained; by detecting the immobilized hemicellulase, it is found that the optimum temperature of immobilized enzyme is increased to 75 DEG C, the optimum pH value is decreased to 3.2, and the enzyme activity is the highest when the concentration of phosphate is less than 20 mmol / L and Mg 2+ is less than 15mmol / L, which increased the difference between the optimum temperature for spore germination and the optimum pH value at 30 DEG C, which is beneficial to the stability of the immobilized enzyme.

Description

technical field [0001] The invention relates to a method for immobilizing hemicellulase by using yeast spores, belonging to the technical field of enzyme immobilization. Background technique [0002] Immobilization of enzymes (Immobilization of enzymes), generally refers to the assembly of enzymes and certain materials through certain methods and principles, so as to achieve the purpose of immobilizing enzymes in a limited range. The final result is to make Enzymes are a new technology that can not only carry out normal catalytic reactions, but also realize the recycling of enzymes. Therefore, the immobilized enzyme not only retains the basic characteristics, high efficiency and specificity, but also has more advantages. Although enzymes are more efficient and specific than chemical catalysts, most enzymes are proteins, and proteins are very unstable in the environment. Therefore, free enzymes are also very unstable, and they are easily inactivated if they are not properly ...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N11/10C12N15/81C12N1/19C12R1/865
CPCC12N9/248C12N11/10C12N15/81
Inventor 刘可春何秋霞李宁楚杰韩利文
Owner BIOLOGY INST OF SHANDONG ACAD OF SCI