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Method for improving activity of lipase

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

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

[0005] Aiming at the problem of low catalytic efficiency of existing lipase, the present invention provides a technology for exposing and expressing lipase 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 the activity of lipase

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  • Method for improving activity of lipase

Examples

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Effect test

Embodiment 1

[0016] Example 1 Lipase expression

[0017] The lipase gene (from Rhizopus oryzae, Genbank number: AF229435) 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 GAL1 promoter ( From the C-terminal of the downstream MFα1 signal peptide (from Saccharomyces cerevisiae, Genbank No.: M17301) sequence of Saccharomyces cerevisiae expression vector pYES263, Genbank No.: AY428072), construct an expression cassette (from N-terminal to C-terminal): GAL1 promoter + MFα1 signal peptide sequence + lipase gene + α lectin. Transform the yeast plasmid containing the expression cassette into Saccharomyces cerevisiae cells (Saccharomycescerevisiae, purchased from Angel Yeast Co., Ltd.), then the MFα1 signal peptide will guide the lipase to secrete outside the Saccharomyces cerevisiae cells, and the α-lectin downstream of ...

Embodiment 2

[0018] Example 2 Induction of Lipase Exposure Expression

[0019] In the YPD medium for culturing Saccharomyces cerevisiae, add 6.5% galactose (purchased from Shanghai Sangon Biological Engineering Co., Ltd., Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.) and 5-8% 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 + lipase gene + α lectin" will be activated to induce fat The enzyme is expressed on the outer surface of the cell wall of Saccharomyces cerevisiae.

Embodiment 3

[0020] Example 3 The effect of galactose and lactose on the expression of lipase

[0021] In the YPD medium for cultivating Saccharomyces cerevisiae, if galactose and lactose are not included, no lipase activity is detected, because the expression box "GAL1 promoter + MFα1 signal peptide sequence + lipase gene + FLO sequence" The GAL1 promoter fails to remain active.

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Abstract

The invention provides a method for improving activity of lipase. The method comprises the following steps: connecting a lipase gene (Genebank number: AF229435) to the N terminal of a Saccharomyces cerevisiae cell wall component alpha lectin sequence (Genebank number: M28164); then inserting into the C terminal of a downstream MFalpha 1 signal peptide sequence of a Saccharomyces cerevisiae expression vector GAL1 promoter (Genebank number: AY428072) so as to construct an expression frame, wherein from the N end to the C end, the expression frame successively comprises: a GAL1 promoter, a MFalpha signal peptide sequence, a lipase gene and an alpha lectin sequence; and transforming yeast plasmid containing the expression frame into a Saccharomyces cerevisiae cell. The method has the advantages that linoleate isomerase is revealed and expressed on the outer surface of a Saccharomyces cerevisiae cell wall, and the complete contact of the linoleate isomerase and an extracellular substrate is achieved, thereby obviously improving the activity of the linoleate isomerase.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for exposing and expressing lipase on the outer surface of the cell wall of Saccharomycescerevisiae to improve the enzyme activity. Background technique [0002] Esters are a large class of important products or intermediates widely used in chemical, food, pharmaceutical and other fields. There are hundreds of esters used only as food flavors. The synthesis routes of various esters are the same, and they are prepared by esterification reaction using carboxyl donors (such as acids, acid anhydrides) and hydroxyl donors (such as alcohols, starches, etc.) as raw materials. [0003] The biosynthesis of many esters has been performed using lipase-producing microbial cells. Compared with the chemical synthesis of esters, the biosynthesis of esters has the advantages of higher efficiency, mild reaction conditions, and low energy consumption. However, the lipase currently used mai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/81C12R1/865
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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