CN102242086A - Method for improving phytase activity - Google Patents
Method for improving phytase activity Download PDFInfo
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- CN102242086A CN102242086A CN 201010171789 CN201010171789A CN102242086A CN 102242086 A CN102242086 A CN 102242086A CN 201010171789 CN201010171789 CN 201010171789 CN 201010171789 A CN201010171789 A CN 201010171789A CN 102242086 A CN102242086 A CN 102242086A
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- CN
- China
- Prior art keywords
- phytase
- expression cassette
- saccharomyces cerevisiae
- sequence
- signal peptide
- 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
- 108010011619 6-Phytase Proteins 0.000 title claims abstract description 44
- 229940085127 phytase Drugs 0.000 title claims abstract description 29
- 230000000694 effects Effects 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 19
- 101150094690 GAL1 gene Proteins 0.000 claims abstract description 13
- 102100028501 Galanin peptides Human genes 0.000 claims abstract description 13
- 101100121078 Homo sapiens GAL gene Proteins 0.000 claims abstract description 13
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 12
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 12
- 239000013604 expression vector Substances 0.000 claims abstract description 5
- 239000013612 plasmid Substances 0.000 claims abstract description 4
- 210000005253 yeast cell Anatomy 0.000 claims description 15
- 108090001090 Lectins Proteins 0.000 claims description 11
- 102000004856 Lectins Human genes 0.000 claims description 11
- 239000002523 lectin Substances 0.000 claims description 11
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 9
- 239000008101 lactose Substances 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 abstract description 6
- 239000000758 substrate Substances 0.000 abstract description 6
- 210000002421 cell wall Anatomy 0.000 abstract description 3
- 101710204899 Alpha-agglutinin Proteins 0.000 abstract 2
- 108090000790 Enzymes Proteins 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 6
- 230000003248 secreting effect Effects 0.000 description 5
- 229940081969 saccharomyces cerevisiae Drugs 0.000 description 4
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 239000002366 mineral element Substances 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 229940068041 phytic acid Drugs 0.000 description 3
- 235000002949 phytic acid Nutrition 0.000 description 3
- 239000000467 phytic acid Substances 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 240000000894 Lupinus albus Species 0.000 description 1
- 235000010649 Lupinus albus Nutrition 0.000 description 1
- FENRSEGZMITUEF-ATTCVCFYSA-E [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OP(=O)([O-])O[C@@H]1[C@@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H]1OP(=O)([O-])[O-] Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OP(=O)([O-])O[C@@H]1[C@@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H]1OP(=O)([O-])[O-] FENRSEGZMITUEF-ATTCVCFYSA-E 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 229940083982 sodium phytate Drugs 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a method for improving the phytase activity, comprising the steps of: connecting a phytase gene (Genbank No.: AB508806) to the N end of a saccharomyces cerevisiae cell wall component alpha agglutinin sequence (Genbank No.: M28164), then inserting the phytase gene 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 + phytase gene + alpha agglutinin sequence; shifting a yeast plasmid containing the expression cassette into the saccharomyces cerevisiae cell. According to the invention, the phytase, revealed and expressed outside the saccharomyces cerevisiae cell wall, is in complete connection with an extracellular substrate, so that the phytase activity is obviously improved.
Description
Technical field
The present invention relates to a kind of bioengineering field, particularly a kind of in yeast saccharomyces cerevisiae (Saccharomycescerevisiae) cell walls outside surface demonstration Expressing Recombinant Phytase to improve the method for this enzymic activity.
Background technology
The phytic acid that contains in the feed easily with mineral element formation infusible precipitates such as calcium, phosphorus, influence the absorption of animal to these mineral elements.The phytase phytic acid of can degrading adds phytase and can effectively eliminate phytic acid absorbs mineral elements such as calcium, phosphorus to animal disadvantageous effect in feed.
But mainly there is following problem in the phytase that adds in feed 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 the catalytic efficiency of enzyme not high.
Summary of the invention
At the low problem of existing phytase catalytic efficiency, the invention provides and a kind of phytase demonstration is expressed in the technology of brewing yeast cell wall outside surface, realize contacting fully of enzyme and extracellular substrate, thereby significantly improve phytase activity.
A kind of method that improves phytase activity of the present invention, may further comprise the steps: (Genbank number: (Genbank number: N M28164) holds AB508806) to be connected brewing yeast cell wall composition α lectin sequence with phytase 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+phytase 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% (weight percent) semi-lactosi and 6-9% lactose (weight percent).
Advantage of the present invention: the phytase demonstration is expressed in brewing yeast cell wall outside surface, and the realization enzyme contacts fully with the extracellular substrate, thereby significantly improves phytase activity.
Embodiment
The present invention is further described by the following examples:
The present invention develops and a kind of phytase demonstration is expressed in the technology of brewing yeast cell wall outside surface, realizes contacting fully of enzyme and extracellular substrate, thereby significantly improves phytase 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 phytase is expressed
With phytase gene (from Lupinus albus, Genbank number: AB508806) 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 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+phytase 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 phytase to the brewing yeast cell external secretion, the α lectin in phytase downstream then is anchored in the brewing yeast cell wall, thereby makes the phytase demonstration be expressed in brewing yeast cell wall outside surface.
What the demonstration of embodiment 2 phytases was expressed induces
In the YPD substratum of cultivating yeast saccharomyces cerevisiae, add 6% 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 6-9% lactose; Services Co., Ltd.), then the GAL1 promotor in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+phytase gene+α lectin " will activate, and induces phytase to express in the demonstration of brewing yeast cell wall outside surface.
The effect that demonstration is expressed to phytase 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 phytase activity, this is because the GAL1 promotor in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+phytase gene+FLO sequence " can't keep active state.
The active raising in back is expressed in the demonstration of embodiment 4 phytases
The phytase activity that demonstrates expression according to step shown in embodiment 1 and the embodiment 2 is 1186U/mL (6% semi-lactosi and 6% lactose) and 1145U/mL (6% semi-lactosi and 9% lactose), if and express removal α lectin sequence in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+phytase gene+α lectin " in the demonstration shown in embodiment 1 and the embodiment 2, then phytase carries out secreting, expressing, and activity is 353U/mL (6% semi-lactosi and 6% lactose) and 360U/mL (6% semi-lactosi and 9% lactose).Therefore, under the expression system situation the same, increase this demonstration Expression element of α lectin and can make phytase activity improve several times with the primary expression element.
Phytase activity unit U is defined as: under 37 ℃, the condition of pH 5.5, sodium phytate from 0.0051 mol in 1 minute (is given birth to worker's biotechnology company limited, Shanghai SangonBiological Engineering Technology﹠amp available from Shanghai; Services Co., Ltd.) discharging the needed phytase enzyme of 1 micromole's inorganic phosphorus amount in the solution is 1U.
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 phytase activity, it is characterized in that may further comprise the steps: phytase 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+phytase 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% semi-lactosi and weight percent 6-9% lactose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010171789 CN102242086A (en) | 2010-05-14 | 2010-05-14 | Method for improving phytase activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010171789 CN102242086A (en) | 2010-05-14 | 2010-05-14 | Method for improving phytase activity |
Publications (1)
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|---|---|
| CN102242086A true CN102242086A (en) | 2011-11-16 |
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Family Applications (1)
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| CN 201010171789 Pending CN102242086A (en) | 2010-05-14 | 2010-05-14 | Method for improving phytase activity |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805025A (en) * | 2015-04-20 | 2015-07-29 | 江南大学 | Engineering strain of saccharomyces cerevisiae for expressing phytase 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 CN 201010171789 patent/CN102242086A/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 (5)
| 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 全文 1-2 第63卷, 第2期 * |
| 《Biotechnology and Bioprocess Engineering》 20051231 Ae-Young Mo et al. Expression of Fungal phytase on the cell surface of saccharomyces cerevisiae 第577页右栏最后1段至第578页左栏第2段 1-2 第10卷, 第6期 * |
| 《FEMS RESEARCH LETTER》 20091005 Piyanun Harnpicharnchai et al. Cell-surfacephytaseonPichiapastoris cellwallo¡ersgreatpotential as a feed supplement 全文 1-2 第302卷, 第1期 * |
| 《GENETICS》 19891231 Monica C. Flessel et al. The MFal Gene of Saccharomyces cerevisiae: Genetic Mapping and Mutational Analysis of Promoter Elements 全文 1-2 第121卷, * |
| 《Proc. Nati. Acad. Sci.》 19831231 SCOTT D. EMR et al. An MFalphal-SUC2 (a-factor-invertase) gene fusion for study of protein localization and gene expression in yeast 1-2 全文 第80卷, * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805025A (en) * | 2015-04-20 | 2015-07-29 | 江南大学 | Engineering strain of saccharomyces cerevisiae for expressing phytase and application |
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Application publication date: 20111116 |