CN102242107A - Method for improving vitality persistence of linoleic acid isomerase - Google Patents
Method for improving vitality persistence of linoleic acid isomerase Download PDFInfo
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- CN102242107A CN102242107A CN201010171804XA CN201010171804A CN102242107A CN 102242107 A CN102242107 A CN 102242107A CN 201010171804X A CN201010171804X A CN 201010171804XA CN 201010171804 A CN201010171804 A CN 201010171804A CN 102242107 A CN102242107 A CN 102242107A
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- linoleic acid
- acid isomerase
- persistence
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- linoleate isomerase
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Abstract
The invention relates to a method for improving the vitality persistence of a linoleic acid isomerase. The method comprises the following steps: connecting linoleic acid isomerase gene (Genbank number: DQ227322) to an N terminus of a sequence (Genbank number: M28164) of a Saccharomycescerevisiae cell wall component alpha agglutinin; inserting a C terminus of an MFalpha1 signal peptide sequence (Genbank number: M17301) of a downstream GAL1 promoter of a Saccharomycescerevisiae expression vector (Genbank number: AY428072) to construct an expression cassette which sequentially comprises the GAL1 promoter, the MFalpha1 signal peptide sequence, the linoleic acid isomerase gene, and the alpha agglutinin sequence from the N terminus to the C terminus; and transforming yeast plasmid containing the expression cassette to Saccharomyces cerevisiaes. The method of the present invention has the advantage that the vitality persistence of the linoleic acid isomerase is substantially improved because a complete contact of the linoleic acid isomerase and extracellular substrates is realized through revealedly expressing the linoleic acid isomerase on external surfaces of Saccharomyces cerevisiae walls.
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 linoleate isomerase to improve the method for this enzyme activity persistence.
Background technology
Conjugated linolic acid is the general name that is positioned at the 18 carbon conjugated dieness acid geometrical isomer of different positions by essential fatty acids linoleic deutero-conjugation unsaturated double-bond.Linoleate isomerase can change into conjugated linolic acid with linolic acid.But there is not enough this problem of enzyme activity persistence in the linoleate isomerase of using at present.The recombinant expressed mode that comprises the various enzymes of linoleate isomerase 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 linoleate isomerase become the brewing yeast cell organic composition, soon linoleate isomerase and brewing yeast cell wall composition FLO are formed by connecting and are the integral part of brewing yeast cell, linoleate isomerase remains active condition as long as brewing yeast cell keeps surviving then, thereby has significantly improved linoleate isomerase vigor persistence.
Summary of the invention
At the insufficient problem of existing linoleate isomerase vigor persistence, the invention provides a kind of technology that makes linoleate isomerase become the brewing yeast cell organic component, make linoleate isomerase as long as brewing yeast cell keeps survival to maintain vigour, thereby significantly improve linoleate isomerase vigor persistence.
A kind of method that improves linoleate isomerase 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 DQ227322) to connect (ordinary method) with linoleate isomerase 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+linoleate isomerase gene+FLO sequence; The yeast plasmid that will comprise this expression cassette is transformed into brewing yeast cell.
The brewing yeast cell of described expression cassette is incubated in the YPD substratum that is added with 4-5% (weight percent) semi-lactosi (common semi-lactosi).
Advantage of the present invention:
Linoleate isomerase is connected with brewing yeast cell wall composition FLO, and so then as long as brewing yeast cell keeps existing state, linoleate isomerase can maintain vigour.
Embodiment
The present invention is further described by the following examples:
The demonstration of embodiment 1 linoleate isomerase is expressed
With linoleate isomerase gene (from Lactobacillus plantarum, Genbank number: DQ227322) 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 GAL 1 promotor then (from yeast saccharomyces cerevisiae expression vector pYES263, Genbank number: AY428072) downstream MF α 1 signal peptide sequence is (from Saccharomyces Cerevisiae in S accharomyces cerevisiae, Genbank number: C end M17301) is built into expression cassette (holding the C end from N): GAL1 promotor+MF α 1 signal peptide sequence+linoleate isomerase 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 linoleate isomerase to the brewing yeast cell external secretion, the FLO sequence in linoleate isomerase downstream then is anchored in the brewing yeast cell wall, thereby makes the linoleate isomerase demonstration be expressed in brewing yeast cell wall outside surface.
What the demonstration of embodiment 2 linoleate isomerases was expressed induces
In the YPD substratum of cultivating yeast saccharomyces cerevisiae, add the 4-5% 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+linoleate isomerase gene+FLO sequence " will activate, and induces linoleate isomerase to express in the demonstration of brewing yeast cell wall outside surface.
The effect that demonstration is expressed to linoleate isomerase 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 linoleate isomerase activity, this is because the GAL1 promotor in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+linoleate isomerase gene+FLO sequence " can't activate.
The demonstration of embodiment 4 linoleate isomerases is expressed its vigor persistence of back and is significantly improved
The linoleate isomerase activity that demonstrates expression according to step shown in embodiment 1 and the embodiment 2 is 862U/mL (4% semi-lactosi) and 856U/mL (4-5% semi-lactosi), and the transformation period is 58 days, can keep half of initial activity in the time of promptly the 58th day.And if express removal FLO sequence nucleotide sequence in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+linoleate isomerase gene+FLO sequence " in the demonstration shown in embodiment 1 and the embodiment 2, then linoleate isomerase carries out secreting, expressing, activity is 325U/mL (4% semi-lactosi) and 330U/mL (5% semi-lactosi), transformation period all only is 5 days, can keep half of initial activity in the time of promptly the 5th day.Therefore, linoleate isomerase and brewing yeast cell wall composition FLO be formed by connecting be the integral part of brewing yeast cell, linoleate isomerase remains active condition as long as brewing yeast cell keeps surviving then, thereby has significantly improved linoleate isomerase vigor persistence.
The U of linoleate isomerase activity unit is defined as: under 30 ℃ of conditions, in the 5mL reaction system, per hour (give birth to worker's biotechnology company limited, Shanghai SangonBiological Engineering Technology﹠amp available from Shanghai from the 0.15g/mL linolic acid; Services Co., Ltd.) (standard substance are given birth to worker's biotechnology company limited, Shanghai Sangon BiologicalEngineering Technology﹠amp available from Shanghai to middle generation 1 μ g conjugated linolic acid; Services Co., Ltd.) required enzyme amount is 1 enzyme U of unit alive.
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 linoleate isomerase vigor persistence is characterized in that may further comprise the steps:
Linoleate isomerase 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+linoleate isomerase 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 4-5% semi-lactosi.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201010171804XA CN102242107A (en) | 2010-05-14 | 2010-05-14 | Method for improving vitality persistence of linoleic acid isomerase |
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| CN201010171804XA CN102242107A (en) | 2010-05-14 | 2010-05-14 | Method for improving vitality persistence of linoleic acid isomerase |
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| CN102242107A true CN102242107A (en) | 2011-11-16 |
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| CN201010171804XA Pending CN102242107A (en) | 2010-05-14 | 2010-05-14 | Method for improving vitality persistence of linoleic acid isomerase |
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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 |
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2010
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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 (3)
| Title |
|---|
| 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》 * |
| 侯进等: "木糖异构酶在酿酒酵母表面表达及对木糖代谢影响的初步研究", 《生物加工过程》 * |
| 刘文山等: "脂肪酶表面展示技术", 《中国生物工程杂志》 * |
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Application publication date: 20111116 |