CN102242106A - Method for improving persistency of glutamic acid decarboxylase activity - Google Patents
Method for improving persistency of glutamic acid decarboxylase activity Download PDFInfo
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- CN102242106A CN102242106A CN2010101718177A CN201010171817A CN102242106A CN 102242106 A CN102242106 A CN 102242106A CN 2010101718177 A CN2010101718177 A CN 2010101718177A CN 201010171817 A CN201010171817 A CN 201010171817A CN 102242106 A CN102242106 A CN 102242106A
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- glutamic acid
- acid decarboxylase
- saccharomyces cerevisiae
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- expression cassette
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Abstract
The invention provides a method for improving the persistency of glutamic acid decarboxylase activity. The method comprises the steps of: connecting a glutamic acid decarboxylase gene (Genbank No.: NM_168056) (with a conventional method) to the N end of a saccharomyces cerevisiae cell wall component FLO sequence (Genbank No.: S73336), then inserting the glutamic acid decarboxylase gene (in a conventional approach) to the C end of a downstream MF alpha1 signal peptide sequence (Genbank No.: M17301) of the GAL1 promoter (Genbank No.: AY428072) of a saccharomyces cerevisiae expression vector so as to construct an expression cassette, which is, from the N end to the C end, as follows: GAL1 promoter + MF alpha1 signal peptide sequence + glutamic acid decarboxylase gene + FLO sequence; shifting a yeast plasmid containing the expression cassette into the saccharomyces cerevisiae cell. According to the invention, the glutamic acid decarboxylase is connected to the saccharomyces cerevisiae cell wall component FLO, so that as long as the saccharomyces cerevisiae cell maintains alive, the glutamic acid decarboxylase can keep active.
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 L-Glutamic decarboxylase to improve the method for this enzyme activity persistence.
Background technology
γ-An Jidingsuan is a kind of natural health factor, has obvious hypotensive activity.L-glutamic acid transforms and can a step generate γ-An Jidingsuan through L-Glutamic decarboxylase.But there is not enough this problem of enzyme activity persistence in the L-Glutamic decarboxylase of using at present.The recombinant expressed mode that comprises the various enzymes of L-Glutamic decarboxylase has two kinds of cell inner expression and secreting, expressings, the recombinant expressed mode of these two kinds of enzymes make do not have between enzyme molecule and the host cell related, be that the enzyme molecule is not the integral part of host cell, thereby be easy to inactivation.If make the enzyme molecule become the organic component of cell, then molecule also can maintain vigour as long as cell keeps the existing state enzyme, thereby can significantly improve the vigor persistence of enzyme.
The present invention develops a kind of technology that makes L-Glutamic decarboxylase become the brewing yeast cell organic composition, soon L-Glutamic decarboxylase and brewing yeast cell wall composition FLO are formed by connecting and are the integral part of brewing yeast cell, L-Glutamic decarboxylase remains active condition as long as brewing yeast cell keeps surviving then, thereby has significantly improved L-Glutamic decarboxylase vigor persistence.
Summary of the invention
At the insufficient problem of existing L-Glutamic decarboxylase vigor persistence, the invention provides a kind of technology that makes L-Glutamic decarboxylase become the brewing yeast cell organic component, make L-Glutamic decarboxylase as long as brewing yeast cell keeps survival to maintain vigour, thereby significantly improve L-Glutamic decarboxylase vigor persistence.
A kind of method that improves L-Glutamic decarboxylase vigor persistence of the present invention may further comprise the steps:
(Genbank number: (Genbank number: N S73336) holds in brewing yeast cell wall composition FLO sequence NM_168056) to connect (ordinary method) with glutamic acid decarboxylase gene, (Genbank number: AY428072) downstream MF α 1 signal peptide sequence is (Genbank number: C end M17301) to insert (ordinary method) 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+glutamic acid decarboxylase gene+FLO 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 6.5-8.5% (weight percent) semi-lactosi (common semi-lactosi).
Advantage of the present invention:
L-Glutamic decarboxylase is connected with brewing yeast cell wall composition FLO, and so then as long as brewing yeast cell keeps existing state, L-Glutamic decarboxylase can maintain vigour.
Embodiment
The present invention is further described by the following examples:
The demonstration of embodiment 1 L-Glutamic decarboxylase is expressed
With glutamic acid decarboxylase gene (from Drosophila melanogaster, Genbank number: NM_168056) be connected brewing yeast cell wall composition FLO sequence (from Saccharomyces Cerevisiae in S accharomyces cerevisiae, Genbank number: N end S73336), 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+glutamic acid decarboxylase gene+FLO sequence.The yeast saccharomyces cerevisiae plasmid that will comprise this expression cassette is transformed into brewing yeast cell (Saccharomyces cerevisiae, available from Angel Yeast Co.,Ltd), then MF α 1 signal peptide will guide L-Glutamic decarboxylase to the brewing yeast cell external secretion, the FLO sequence in L-Glutamic decarboxylase downstream then is anchored in the brewing yeast cell wall, thereby makes the L-Glutamic decarboxylase demonstration be expressed in brewing yeast cell wall outside surface.
What the demonstration of embodiment 2 L-Glutamic decarboxylases was expressed induces
In the YPD substratum of cultivating yeast saccharomyces cerevisiae, add the 6.5-8.5% semi-lactosi and (give birth to worker's biotechnology company limited, Shanghai Sangon Biological Engineering Technology﹠amp available from Shanghai; ServicesCo., Ltd.), then the GAL1 promotor in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+glutamic acid decarboxylase gene+FLO sequence " will activate, and induces L-Glutamic decarboxylase to express in the demonstration of brewing yeast cell wall outside surface.
The effect that demonstration is expressed to L-Glutamic decarboxylase of embodiment 3 semi-lactosis
In the YPD substratum of cultivating yeast saccharomyces cerevisiae, if do not contain semi-lactosi, then do not detect the L-Glutamic decarboxylase activity, this is because the GAL1 promotor in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+glutamic acid decarboxylase gene+FLO sequence " can't activate.
The demonstration of embodiment 4 L-Glutamic decarboxylases is expressed its vigor persistence of back and is significantly improved
The L-Glutamic decarboxylase activity that demonstrates expression according to step shown in embodiment 1 and the embodiment 2 is 2005U/mL (6.5% semi-lactosi) and 2014U/mL (8.5% semi-lactosi), and the transformation period is 75 days, can keep half of initial activity in the time of promptly the 75th day.And if express removal FLO sequence nucleotide sequence in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+glutamic acid decarboxylase gene+FLO sequence " in the demonstration shown in embodiment 1 and the embodiment 2, then L-Glutamic decarboxylase carries out secreting, expressing, activity is 1156U/mL (6.5% semi-lactosi) and 1130U/mL (8.5% semi-lactosi), transformation period all only is 8 days, can keep half of initial activity in the time of promptly the 8th day.Therefore, L-Glutamic decarboxylase and brewing yeast cell wall composition FLO be formed by connecting be the integral part of brewing yeast cell, L-Glutamic decarboxylase remains active condition as long as brewing yeast cell keeps surviving then, thereby has significantly improved L-Glutamic decarboxylase vigor persistence.
The U of L-Glutamic decarboxylase activity unit is defined as: (give birth to worker's biotechnology company limited, Shanghai Sangon Biological Engineering Technology﹠amp available from Shanghai at 0.05mol/L L-glutamic acid; ServicesCo., Ltd.) in, per minute catalysis generates 1 μ mol γ-An Jidingsuan, and (standard substance are given birth to worker's biotechnology company limited, Shanghai Sangon Biological Engineering Technology﹠amp available from Shanghai; ServicesCo., Ltd.) required enzyme amount is a unit of activity (U).
At last, it should be noted that above what 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 L-Glutamic decarboxylase vigor persistence is characterized in that may further comprise the steps:
Glutamic acid decarboxylase gene is connected brewing yeast cell wall composition FLO 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+glutamic acid decarboxylase gene+FLO 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 6.5-8.5% semi-lactosi.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1681942A (en) * | 2002-09-13 | 2005-10-12 | 韩国生命工学研究院 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
| CN1746302A (en) * | 2005-07-18 | 2006-03-15 | 山东大学 | Production of Non-N glycosylated protein from yeast |
| CN101643941A (en) * | 2009-09-07 | 2010-02-10 | 湖北大学 | High-efficiency directed evolution method of lipase gene |
-
2010
- 2010-05-14 CN CN2010101718177A patent/CN102242106A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1681942A (en) * | 2002-09-13 | 2005-10-12 | 韩国生命工学研究院 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
| CN1746302A (en) * | 2005-07-18 | 2006-03-15 | 山东大学 | Production of Non-N glycosylated protein from yeast |
| CN101643941A (en) * | 2009-09-07 | 2010-02-10 | 湖北大学 | High-efficiency directed evolution method of lipase gene |
Non-Patent Citations (4)
| Title |
|---|
| 《Applied and Environmental Microbiology》 19970228 J. Marcel Van Der Vaart et al. "Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins" 第63卷, 第2期 * |
| J. MARCEL VAN DER VAART ET AL.: ""Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins"", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| SATO N, ET AL.: "Long anchor using Flo1 protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates", 《APPL MICROBIOL BIOTECHNOL》 * |
| 郭钦 等: "酿酒酵母表面展示表达***及应用", 《中国生物工程杂志》 * |
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Application publication date: 20111116 |