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Method for improving beta glucanase activity

A glucanase and activity technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problem of low catalytic efficiency of β-glucanase and achieve the goal of improving the activity of β-glucanase Effect

Inactive Publication Date: 2011-11-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the problem of low catalytic efficiency of the existing β-glucanase, the present invention provides a technology for exposing and expressing the β-glucanase on the outer surface of the cell wall of Saccharomyces cerevisiae, so as to realize the complete contact between the enzyme and the extracellular substrate, thereby significantly Increases beta glucanase activity

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  • Method for improving beta glucanase activity

Examples

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Effect test

Embodiment 1

[0015] Example 1 β-glucanase expression

[0016] The β-glucanase gene (from Bacillus subtilis, Genbank number: U60830) was connected to the N-terminal of the cell wall component of Saccharomyces cerevisiae α-lectin (from Saccharomyces cerevisiae, Genbank number: M28164), and then inserted into the Saccharomyces cerevisiae expression vector GAL1 Promoter (from Saccharomyces cerevisiae expression vector pYES263, Genbank number: AY428072) downstream of the C-terminal MFα1 signal peptide (from Saccharomyces cerevisiae, Genbank number: M17301) sequence, constructed into an expression cassette (from N-terminal to C-terminal): GAL1 Promoter + MFα1 signal peptide sequence + β-glucanase gene + α-lectin. The yeast plasmid containing the expression cassette is transformed into Saccharomyces cerevisiae cells (Saccharomyces cerevisiae, purchased from Angel Yeast Co., Ltd.), then the MFα1 signal peptide will guide the secretion of β-glucanase to Saccharomyces cerevisiae cells, and β-glucana...

Embodiment 2

[0017] Example 2 Induction of expression of beta glucanase exposure

[0018] In the YPD medium for culturing Saccharomyces cerevisiae, add 6% galactose (purchased from Shanghai Sangon Biological Engineering Co., Ltd., Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.) and 4.5-6% lactose (purchased from Shanghai Sangon Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.), the GAL1 promoter in the expression cassette "GAL1 promoter + MFα1 signal peptide sequence + β glucanase gene + α lectin" will be activated , to induce the expression of β-glucanase on the outer surface of the cell wall of Saccharomyces cerevisiae.

Embodiment 3

[0019] Example 3 The effect of galactose and lactose on the expression of β-glucanase

[0020] In the YPD medium for culturing Saccharomyces cerevisiae, if galactose and lactose are not included, no β-glucanase activity is detected because the expression box "GAL1 promoter + MFα1 signal peptide sequence + β-glucanase gene The GAL1 promoter in the +FLO sequence" fails to remain active.

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Abstract

The invention relates to a method for improving beta glucanase activity. The method comprises the following steps that: beta glucanase gene is linked to a N-terminal of alpha agglutinin sequence of saccharomycescerevisiae cell wall composition; the resulting sequence is inserted into a C-terminal of MF alpha 1 signal peptide sequence positioned on the downstream of a GAL1 promoter of a galactosidase expression vector to construct a expression cassette, wherein the expression cassette sequentially comprises the GAL1 promoter, the MF alpha 1 signal peptide sequence, the beta glucanase gene and the alpha agglutinin sequence from the N-terminal to the C-terminal; yeast plasmids containing the expression cassette are transformed into the saccharomycescerevisiae cells. According to the present invention, the beta glucanase is exposed and expressed on the outer surface of the saccharomycescerevisiae cell wall, such that complete contacts of the beta glucanase and the extracellular sunstrates are realized so as to substantially improve the beta glucanase activity.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for exposing and expressing beta glucanase on the outer surface of the cell wall of Saccharomycescerevisiae to improve the activity of the enzyme. Background technique [0002] Grain and its processing by-products are commonly used feed ingredients, but these feeds contain high content of β-glucan, which will increase the viscosity of the digestive tract of monogastric animals and hinder the digestion and utilization of feed by animal digestive enzymes. Adding exogenous β-glucanase has the effect of improving the digestibility, remuneration rate and production performance of monogastric animals. [0003] Exogenous β-glucanases have been widely used in feedstuffs, but the currently used β-glucanases mainly have the following problems: the recombinant expression of the enzyme is usually intracellular expression and secreted expression. During intracellular expression, the en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/81C12R1/865
Inventor 阮晖陈美龄马风兰陈赟廖文艳徐娟王睿之周陈伟何国庆
Owner ZHEJIANG UNIV
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