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Method for improving continuity of alpha galactosidase activity

A galactosidase, sustainable technology, applied in the field of bioengineering, can solve the problems of insufficient alpha galactosidase activity

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

[0005] Aiming at the problem of insufficient activity persistence of the existing α-galactosidase, the present invention provides a technology for making the α-galactosidase an integral part of S. Can maintain activity, thereby significantly improving the activity persistence of α-galactosidase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1 α-galactosidase expression

[0012] The α-galactosidase gene (from Saccharomyces cerevisiae, Genbank No.: X03102) was connected to the N-terminal of the Saccharomyces cerevisiae cell wall component FLO sequence (from Saccharomyces cerevisiae, Genbank No.: S73336), and then inserted into the Saccharomyces cerevisiae expression vector GAL1 The C-terminal of the MF α1 signal peptide sequence (from Saccharomyces cerevisiae, Genbank No.: M17301) downstream of the promoter (from Saccharomyces cerevisiae expression vector pYES263, Genbank number: AY428072) was constructed into an expression cassette (from N-terminal to C-terminal): GAL1 promoter + MF α1 signal peptide sequence + α-galactosidase gene + FLO sequence. The Saccharomyces cerevisiae plasmid containing the expression cassette is transformed into Saccharomyces cerevisiae cells (Saccharomyces cerevisiae, purchased from Angel Yeast Co., Ltd.), then the MF α1 signal peptide will guide the secretion of α-galacto...

Embodiment 2

[0013] Example 2 Induction of α-Galactosidase Exposure Expression

[0014] In the YPD medium for cultivating Saccharomyces cerevisiae, add 3.5-7% galactose (purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.), the expression box "GAL1 promoter + MF α1 signal The GAL1 promoter in the peptide sequence + α-galactosidase gene + FLO sequence" will be activated to induce the expression of α-galactosidase on the outer surface of the cell wall of Saccharomyces cerevisiae.

Embodiment 3

[0015] Example 3 The effect of galactose on the expression of α-galactosidase

[0016] In the YPD medium for culturing Saccharomyces cerevisiae, if there is no galactose, no α-galactosidase activity can be detected because the expression box "GAL1 promoter + MF α1 signal peptide sequence + α-galactosidase gene + The GAL1 promoter in the FLO sequence" cannot be activated.

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PUM

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Abstract

The invention relates to a method for improving continuity of alpha galactosidase activity. The method comprises the following steps that: alpha galactosidase gene is linked to a N-terminal of FLO 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 alpha galactosidase gene and the FLO 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 alpha galactosidase is linked to the FLO sequence of the saccharomycescerevisiae cell wall composition, such that if the saccharomycescerevisiae cells are in survival states, the alpha galactosidase can remain the activity.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for exposing and expressing alpha galactosidase on the outer surface of the cell wall of Saccharomyces cerevisiae to improve the activity persistence 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, insufficient enzyme activity persistence is a problem in the currently used α-galactosidase. There are two w...

Claims

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Application Information

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IPC IPC(8): C12N9/40C12N15/81C12R1/865
Inventor 阮晖陈美龄马风兰廖文艳陈赟徐娟王睿之周陈伟何国庆
Owner ZHEJIANG UNIV
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