CN102242089A - Method for improving activity of lipase - Google Patents
Method for improving activity of lipase Download PDFInfo
- Publication number
- CN102242089A CN102242089A CN2010101728431A CN201010172843A CN102242089A CN 102242089 A CN102242089 A CN 102242089A CN 2010101728431 A CN2010101728431 A CN 2010101728431A CN 201010172843 A CN201010172843 A CN 201010172843A CN 102242089 A CN102242089 A CN 102242089A
- Authority
- CN
- China
- Prior art keywords
- lipase
- saccharomyces cerevisiae
- sequence
- signal peptide
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
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
The present invention relates to a kind of bioengineering field, particularly a kind of the demonstration at yeast saccharomyces cerevisiae (Saccharomycescerevisiae) cell walls outside surface expressed lipase to improve the method for this enzymic activity.
Background technology
Ester is staple product or the intermediate of a big class in chemical industry, food, medicine and other fields widespread use, and only the ester as flavouring agent promptly reaches hundreds of.The synthetic route of various esters is identical, is raw material with carboxyl donor (as acid, acid anhydrides) and hydroxyl donor (as alcohol, starch etc.) all, prepares by esterification.
Microorganism cells with yielding lipase can carry out the biosynthesizing of many kinds of esters.Compare with the chemosynthesis of ester, the biosynthesizing of ester has that efficient is higher, reaction conditions is gentle, low power consumption and other advantages.But mainly there is following problem in the lipase of using at present: the recombinant expressed mode of enzyme generally is cell inner expression and secreting, expressing.During cell inner expression since enzyme can not contact with the extracellular substrate, thereby greatly influence the catalytic efficiency of enzyme, and the enzyme molecule is collected at formation feedback inhibition in the cell, also influences expression efficiency.The main drawback of secreting, expressing is that expression efficiency is not high, nor can realize complete secreting, expressing, always has quite a few expression product to be stranded in the cell, thereby can not realize contacting fully of enzyme and extracellular substrate.These two kinds of phraseologies all cause enzyme catalysis efficient not high.
The present invention develops and a kind of lipase demonstration is expressed in the technology of brewing yeast cell wall outside surface, realizes contacting fully of enzyme and extracellular substrate, thereby significantly improves lipase activity.
Summary of the invention
At existing lipase-catalyzed inefficient problem, the invention provides and a kind of lipase demonstration is expressed in the technology of brewing yeast cell wall outside surface, realize contacting fully of enzyme and extracellular substrate, thereby significantly improve lipase activity.
A kind of method that improves lipase activity of the present invention, may further comprise the steps: (Genbank number: (Genbank number: N M28164) holds AF229435) to be connected brewing yeast cell wall composition α lectin sequence with lipase gene, (Genbank number: AY428072) downstream MF α 1 signal peptide sequence is (Genbank number: C end M17301) to insert yeast saccharomyces cerevisiae expression vector GAL1 promotor then, be built into expression cassette, expression cassette is held the C end from N: GAL1 promotor+MF α 1 signal peptide sequence+lipase gene+α lectin sequence; The yeast plasmid that will comprise this expression cassette is transformed into brewing yeast cell.
The brewing yeast cell that will comprise above-mentioned expression cassette is incubated in the YPD substratum that is added with 6.5% (weight percent) semi-lactosi and 5-8% lactose (weight percent).
Advantage of the present invention:
The linoleate isomerase demonstration is expressed in brewing yeast cell wall outside surface, and the realization enzyme contacts fully with the extracellular substrate, thereby significantly improves the linoleate isomerase activity.
Embodiment
The present invention is further described by the following examples:
The present invention develops and a kind of lipase demonstration is expressed in the technology of brewing yeast cell wall outside surface, realizes contacting fully of enzyme and extracellular substrate, thereby significantly improves lipase activity.
Three kinds of enzyme developed by molecule modes are as follows:
-: the enzyme molecule
The demonstration of cell inner expression secreting, expressing is expressed
The demonstration of embodiment 1 lipase is expressed
With lipase gene (from Rhizopus oryzae, Genbank number: AF229435) be connected brewing yeast cell wall composition α lectin sequence (from Saccharomyces Cerevisiae in S accharomyces cerevisiae, Genbank number: N end M28164), insert yeast saccharomyces cerevisiae expression vector GAL1 promotor then (from yeast saccharomyces cerevisiae expression vector pYES263, Genbank number: AY428072) downstream MF α 1 signal peptide is (from Saccharomyces Cerevisiae in S accharomyces cerevisiae, Genbank number: the M17301) C of sequence end is built into expression cassette (holding the C end from N): GAL1 promotor+MF α 1 signal peptide sequence+lipase gene+α lectin.The yeast plasmid that will comprise this expression cassette is transformed into brewing yeast cell (Saccharomycescerevisiae, available from Angel Yeast Co.,Ltd), then MF α 1 signal peptide will guide lipase to the brewing yeast cell external secretion, the α lectin in lipase downstream then is anchored in the brewing yeast cell wall, thereby makes the lipase demonstration be expressed in brewing yeast cell wall outside surface.
What the demonstration of embodiment 2 lipase was expressed induces
In the YPD substratum of cultivating yeast saccharomyces cerevisiae, add 6.5% semi-lactosi and (give birth to worker's biotechnology company limited, Shanghai Sangon Biological Engineering Technology ﹠amp available from Shanghai; ServicesCo., Ltd.) (give birth to worker's biotechnology company limited, Shanghai SangonBiological Engineering Technology ﹠amp available from Shanghai with the 5-8% lactose; Services Co., Ltd.), then the GAL1 promotor in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+lipase gene+α lectin " will activate, and the induced lipolysis enzyme is expressed in the demonstration of brewing yeast cell wall outside surface.
The effect that demonstration is expressed to lipase of embodiment 3 semi-lactosis and lactose
In the YPD substratum of cultivating yeast saccharomyces cerevisiae, if do not contain semi-lactosi and lactose, then do not detect lipase activity, this is because the GAL1 promotor in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+lipase gene+FLO sequence " can't keep active state.
The active raising in back is expressed in the demonstration of embodiment 4 lipase
The lipase activity that demonstrates expression according to step shown in embodiment 1 and the embodiment 2 is 733U/mL (6.5% semi-lactosi and 5% lactose) and 790U/mL (6.5% semi-lactosi and 8% lactose), if and express removal α lectin sequence in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+lipase gene+α lectin " in the demonstration shown in embodiment 1 and the embodiment 2, then lipase carries out secreting, expressing, and activity is 175U/mL (6.5% semi-lactosi and 5% lactose) and 170U/mL (6.5% semi-lactosi and 8% lactose).Therefore, under the expression system situation the same, increase this demonstration Expression element of α lectin and can make lipase activity improve several times with the primary expression element.
Adopt lipid acid to discharge assay method to lipase activity determination, activity unit is defined as: 25% sweet oil at pH 7.0 (is given birth to worker's biotechnology company limited, Shanghai Sangon BiologicalEngineering Technology ﹠amp available from Shanghai; Services Co., Ltd.) in the solution, per minute discharges 1 μ mol lipid acid, and (standard substance are given birth to worker's biotechnology company limited, Shanghai Sangon BiologicalEngineering Technology ﹠amp available from Shanghai; Services Co., enzyme amount Ltd.) is defined as 1 lipase activity unit of force (U).
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (2)
1. method that improves lipase activity, it is characterized in that may further comprise the steps: lipase gene is connected brewing yeast cell wall composition α lectin sequence of N end, insert the C end of yeast saccharomyces cerevisiae expression vector GAL1 promotor downstream MF α 1 signal peptide sequence then, be built into expression cassette, expression cassette is held the C end from N: GAL1 promotor+MF α 1 signal peptide sequence+lipase gene+α lectin sequence; The yeast plasmid that will comprise this expression cassette is transformed into brewing yeast cell.
2. method according to claim 1 is characterized in that: the brewing yeast cell that will comprise " claim 1 " described expression cassette is incubated in the YPD substratum that is added with weight percent 6.5% semi-lactosi and weight percent 5-8% lactose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101728431A CN102242089A (en) | 2010-05-14 | 2010-05-14 | Method for improving activity of lipase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101728431A CN102242089A (en) | 2010-05-14 | 2010-05-14 | Method for improving activity of lipase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102242089A true CN102242089A (en) | 2011-11-16 |
Family
ID=44960337
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101728431A Pending CN102242089A (en) | 2010-05-14 | 2010-05-14 | Method for improving activity of lipase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102242089A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154347A (en) * | 2015-06-26 | 2015-12-16 | 中国环境科学研究院 | Genetic recombination brewer's yeast for degrading fat, construction method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1293571A2 (en) * | 1992-07-08 | 2003-03-19 | Unilever N.V. | Process for immobilizing enzymes to the cell wall of a microbial cell by producing a fusion protein |
-
2010
- 2010-05-14 CN CN2010101728431A patent/CN102242089A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1293571A2 (en) * | 1992-07-08 | 2003-03-19 | Unilever N.V. | Process for immobilizing enzymes to the cell wall of a microbial cell by producing a fusion protein |
Non-Patent Citations (8)
| Title |
|---|
| 《Appl Microbiol Biotechnol》 20040110 A. Kondo & M. Ueda Yeast cell-surface display-applications of molecular display 全文 1-2 第64卷, * |
| A. KONDO & M. UEDA: "Yeast cell-surface display—applications of molecular display", 《APPL MICROBIOL BIOTECHNOL》 * |
| M. WASHIDA ET AL.: "Spacer-mediated display of active lipase on the yeast cell surface", 《APPL MICROBIOL BIOTECHNOL》 * |
| MAARTEN P. SCHREUDER ET AL.: "Immobilizing proteins on the surface of yeast cells", 《TIBTECH APRIL》 * |
| SANG YUP LEE ET AL.: "Microbial cell-surface display", 《TRENDS IN BIOTECHNOLOGY》 * |
| SEIZABURO SHIRAGA ET AL.: "Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| WEI-GUO ZHANG ET AL.: "Functional display of Rhizomucor miehei lipase on surface of Saccharomyces cerevisiae with higher activity and its practical properties", 《JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY》 * |
| YOSHIDA ET AL.: "Cell Surface Engineering of Yeast: Construction of Arming Yeast with Biocatalyst", 《JOURNAL OP BIOSCIENCE AND BIOENGINEERING》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154347A (en) * | 2015-06-26 | 2015-12-16 | 中国环境科学研究院 | Genetic recombination brewer's yeast for degrading fat, construction method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Anasontzis et al. | Homologous overexpression of xylanase in Fusarium oxysporum increases ethanol productivity during consolidated bioprocessing (CBP) of lignocellulosics | |
| WO2006060062A2 (en) | Trichoderma reesei glucoamylase and homologs thereof | |
| US20190276905A1 (en) | Yeast with improved alcohol production | |
| CN110741014B (en) | Yeast with improved alcohol production | |
| Aydemir et al. | Genetic modifications of Saccharomyces cerevisiae for ethanol production from starch fermentation: a review | |
| US9751917B2 (en) | Polynucleotide for cell surface layer expression | |
| US20230063426A1 (en) | Heterologous protease expression for improving alcoholic fermentation | |
| US10323260B2 (en) | Reducing batch time in fermentation processes by subjecting corn-based fermentation medium to a xylanase and a pectinase during fermentation | |
| EP2271765A1 (en) | Use of rhizopus amylases in granular starch hydrolysis | |
| CN102242088A (en) | Method for improving lipase activity persistency | |
| CN102242089A (en) | Method for improving activity of lipase | |
| US20220251608A1 (en) | Disruption of cdc42 effectors in yeast for increased alcohol and lysine production | |
| US20220251582A1 (en) | Modified yeast and method for increasing lysine content in fermentation co-products | |
| US10392624B2 (en) | Gene engineering yeast having saccharification function, method of preparing same, and application of same | |
| Yang et al. | Heterologous expression and efficient ethanol production of a Rhizopus glucoamylase gene in Saccharomyces cerevisiae | |
| US20210179674A1 (en) | Yeast with improved alcohol production | |
| CN102242095A (en) | Method for raising alpha galactosidase activity | |
| Sakdaronnarong et al. | Enzyme matching design approach on very high gravity liquefaction and saccharification of cassava root and cassava starch for ethanol fermentation | |
| CN102242087A (en) | Method for improving phytase activity persistency | |
| Stewart et al. | Biochemical and genetic control of sugar and carbohydrate metabolism in yeasts | |
| US11208670B2 (en) | Method for producing a fermentation product | |
| CN102242094A (en) | Method for improving continuity of alpha galactosidase activity | |
| CN102242086A (en) | Method for improving phytase activity | |
| US20210040474A1 (en) | Yeast with improved alcohol production under high dissolved solids conditions | |
| CN102242108A (en) | Method for raising linoleate isomerase activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20111116 |