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Method for raising linoleate isomerase activity

A linoleic acid isomerase and activity technology, applied in the field of bioengineering, can solve the problems of low catalytic efficiency of linoleic acid isomerase and the like

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 linoleic acid isomerase, the present invention provides a technology for exposing and expressing the linoleic acid isomerase 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 increased linoleic acid isomerase activity

Method used

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  • Method for raising linoleate isomerase activity

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Effect test

Embodiment 1

[0015] Example 1 The expression of linoleic acid isomerase

[0016] The linoleic acid isomerase gene (from Lactobacillus plantarum, Genbank number: DQ227322) was connected to the N-terminal of the Saccharomyces cerevisiae cell wall component α-lectin sequence (from Saccharomyces cerevisiae, Genbank number: M28164), and then inserted into the Saccharomyces cerevisiae expression vector The C-terminal of the MFα1 signal peptide sequence (from Saccharomyces cerevisiae, Genbank No.: M17301) downstream of the 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+linoleic acid isomerase 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 l...

Embodiment 2

[0017] Example 2 Induction of expression of linoleic acid isomerase exposure

[0018] In the YPD medium for culturing Saccharomyces cerevisiae, add 3.5% galactose (purchased from Shanghai Sangon Biological Engineering Co., Ltd., Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.) and 4-7.5% lactose (purchased from Shanghai Sangon Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.), the GAL1 promoter in the expression frame "GAL1 promoter+MFα1 signal peptide sequence+linoleic acid isomerase gene+α lectin" will be Activated to induce the expression of linoleic acid isomerase 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 linoleic acid isomerase

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

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Abstract

A method for raising linoleate isomerase activity of the present invention comprises steps of: connecting a linoleate isomerase gene (Genbank No: DQ22732) 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: AY428072) to construct an expression frame of, from an N end to a C end, GAL1 promoter + MFalpha1 signal peptide sequence + linoleate isomerase 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 linoleate isomerase 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 linoleate isomerase activity.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for exposing and expressing linoleic acid isomerase on the outer surface of the cell wall of Saccharomycescerevisiae to improve the activity of the enzyme. Background technique [0002] Conjugated linoleic acid is a general term for geometric isomers of 18-carbon conjugated dienoic acid derived from essential fatty acid linoleic acid with conjugated unsaturated double bonds at different positions. Many microorganisms can use their own linoleic acid isomerase to convert linoleic acid into conjugated linoleic acid. However, the currently used linoleic acid isomerase mainly has the following problems: the recombinant expression of the enzyme is generally intracellular expression and secretory expression. During intracellular expression, the enzyme cannot contact the extracellular substrate, which greatly affects the catalytic efficiency of the enzyme, and the accumulation of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/81C12R1/865
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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