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Method for raising alpha galactosidase activity

A technology of α-galactosidase and galactose, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problem of low catalytic efficiency of α-galactosidase

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

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem of low catalytic efficiency of the existing α-galactosidase, the present invention provides a technology for exposing and expressing the α-galactosidase 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 improving Alpha-galactosidase activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Example 1 α-galactosidase expression

[0011] The α-galactosidase gene (from Saccharomyces cerevisiae, Genbank No.: X03102) was connected to the N-terminal of the Saccharomyces cerevisiae cell wall component α-lectin sequence (from Saccharomyces cerevisiae, Genbank No.: M28164), and then inserted into Saccharomyces cerevisiae for expression The C-terminal of the MFα1 signal peptide sequence (from Saccharomyces cerevisiae, Genbank No.: M17301) downstream of the vector GAL1 promoter (from Saccharomyces cerevisiae expression vector pYES263, Genbank No.: AY428072) was constructed into an expression cassette (from N-terminal to C-terminal) : GAL1 promoter + MFα1 signal peptide sequence + α-galactosidase 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 α-galactosidase to Saccharomyces...

Embodiment 2

[0012] Example 2 Induction of α-Galactosidase Exposure Expression

[0013] In the YPD medium for culturing Saccharomyces cerevisiae, add 7% galactose (purchased from Shanghai Sangon Biological Engineering Co., Ltd., Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.) and 2.5-5% 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 + α-galactosidase gene + α-lectin" will be activated , inducing the expression of α-galactosidase on the outer surface of the cell wall of Saccharomyces cerevisiae.

Embodiment 3

[0014] Example 3 The effect of galactose and lactose on the expression of α-galactosidase

[0015] In the YPD medium for culturing Saccharomyces cerevisiae, if galactose and lactose are not included, no α-galactosidase activity is detected because the expression box "GAL1 promoter + MFα1 signal peptide sequence + α-galactosidase gene The GAL1 promoter in the +FLO sequence" fails to remain active.

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Abstract

The invention provides a method for raising alpha galactosidase activity, which comprises the following steps: connecting an alpha galactosidase gene (Genbank number: X03102) with a N terminus of a saccharomyces cerevisiae cell wall constituents alpha lectin sequence (Genbank number:M28164), then inserting a C terminus of downstream MFalphal1 signal peptide sequence (Genbank number:M17301) of a saccharomyces cerevisiae expression vector GAL1 promotor (Genbank number:AY428072), constructing an expression cassette, wherein the expression cassette is from the N terminus to the C terminus: the GAL1 promoter, a MF alpha1 signal peptide sequence, the alpha galactosidase gene, the alpha lectin sequence; converting a cerevisiae plasmid containing the expression cassette to the saccharomyces cerevisiae cell. The invention has the advantages that the alpha galactosidase is expressed out of an external surface of the saccharomyces cerevisiae cell wall so that a total contact of enzyme and an extracellular substrate can be realized, therefore the alpha galactosidase activity can be obviously improved.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for exposing and expressing α-galactosidase on the outer surface of the cell wall of Saccharomycescerevisiae to improve the activity of the enzyme. Background technique [0002] The feed contains stachyose and other sugars that are difficult for animal intestinal digestive enzymes containing α-galactoside to digest, which not only affects the digestion and absorption of carbohydrates by animals, but also increases the viscosity of the digestive tract of monogastric animals and hinders the intestinal digestive enzymes of animals. Digestion and absorption of other nutrients in the feed. [0003] The addition of α-galactosidase in the feed is expected to effectively remove the adverse effects of stachyose on the digestive function of animals. However, the α-galactosidases that have been successfully developed so far mainly have the following problems: the recombinant expressi...

Claims

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Application Information

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IPC IPC(8): C12N9/40C12N15/81C12R1/865
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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