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Method for raising glutamate decarboxylase activity

A glutamate decarboxylase and activity technology, applied in the field of bioengineering, can solve the problems of low catalytic efficiency of glutamate decarboxylase, and achieve the effect of improving the activity of glutamate decarboxylase

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

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Problems solved by technology

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

Method used

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  • Method for raising glutamate decarboxylase activity

Examples

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Effect test

Embodiment 1

[0013] Example 1 Expression of Glutamate Decarboxylase

[0014] Link the glutamate decarboxylase gene (from Drosophila melanogaster, Genbank number: NM_168056) to the N end of the sequence of the cell wall component α lectin (from Saccharomyces cerevisiae, Genbank number: M28164) of Saccharomyces cerevisiae, and then insert it into the expression vector GAL1 of Saccharomyces cerevisiae Promoter (from Saccharomyces cerevisiae expression vector pYES263, Genbank number: AY428072) downstream MF α1 signal peptide sequence (from Saccharomyces cerevisiae, Genbank number: M17301) C-terminus, constructed into an expression box (from N-terminus to C-terminus): GAL1 promoter + MF α1 signal peptide sequence + glutamate decarboxylase 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 MF α1 signal peptide will guide the secretion of glutamate decarbo...

Embodiment 2

[0015] Example 2 Induction of revealing expression of glutamate decarboxylase

[0016] In the YPD medium for cultivating Saccharomyces cerevisiae, 8.5% galactose (purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.) and 5-8.5% lactose (purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.) Co., Ltd., Shanghai Sangon Biological Engineering Technology&Services Co., Ltd.), the GAL1 promoter in the expression box "GAL1 promoter + MF α1 signal peptide sequence + glutamate decarboxylase gene + α lectin" will activate and induce Glutamate decarboxylase is expressed on the outer surface of the cell wall of Saccharomyces cerevisiae.

Embodiment 3

[0017] Example 3 The effect of galactose and lactose on the expression of glutamate decarboxylase

[0018] In the YPD medium for culturing Saccharomyces cerevisiae, if galactose and lactose are not present, glutamate decarboxylase activity is not detected, this is because the expression box "GAL1 promoter + MF α1 signal peptide sequence + glutamate decarboxylase The GAL1 promoter in the "gene + FLO sequence" cannot remain activated.

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Abstract

A method for raising glutamate decarboxylase activity of the present invention comprises steps of: connecting a glutamate decarboxylase gene (Genbank No: NM_168056) to an N end of an alpha agglutinin sequence (Genbank No: M28164) of a saccharomyces cerevisiae cell wall component; inserting into a C end of a downstream MFalpha1 signal peptide sequence (Genbank No: M17301) of a saccharomyces cerevisiae expression vector GAL1 promoter (Genbank No: AY 428072) to construct an expression frame of, from an N end to a C end, GAL1 promoter + MFalpha1 signal peptide sequence + glutamate decarboxylase gene + alpha agglutinin sequence; and conversing a yeast plasmid containing the expression frame to a saccharomyces cerevisiae cell. The invention has the advantage that the glutamate decarboxylase is expressed in exposure on an external surface of the saccharomyces cerevisiae cell wall to realize complete contact between the enzyme and an extracellular substrate, so as to substantially raise glutamate decarboxylase activity.

Description

Technical field [0001] The invention relates to the field of bioengineering, in particular to a method for expressing glutamate decarboxylase on the outer surface of the cell wall of Saccharomyces cerevisiae to improve the activity of the enzyme. Background technique [0002] Gamma-aminobutyric acid is a natural health factor with a significant effect on lowering blood pressure. Glutamate can be transformed into glutamate decarboxylase-producing microbial cells to produce γ-aminobutyric acid in one step. However, the currently used glutamate decarboxylase mainly has the following problems: the recombinant expression mode of the enzyme is generally intracellular expression and secreted expression. During intracellular expression, the enzyme cannot contact the extracellular substrate, which greatly affects the catalytic efficiency of the enzyme, and the accumulation of enzyme molecules in the cell forms feedback inhibition, which also affects the expression efficiency. The main d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/81C12R1/865
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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