CN102242094A - Method for improving continuity of alpha galactosidase activity - Google Patents
Method for improving continuity of alpha galactosidase activity Download PDFInfo
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- CN102242094A CN102242094A CN 201010171805 CN201010171805A CN102242094A CN 102242094 A CN102242094 A CN 102242094A CN 201010171805 CN201010171805 CN 201010171805 CN 201010171805 A CN201010171805 A CN 201010171805A CN 102242094 A CN102242094 A CN 102242094A
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- Prior art keywords
- alpha
- sequence
- expression cassette
- flo
- yeast cell
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- 108010030291 alpha-Galactosidase Proteins 0.000 title claims abstract description 18
- 230000000694 effects Effects 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 8
- 102000005840 alpha-Galactosidase Human genes 0.000 title abstract description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 20
- 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 23
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 230000002688 persistence Effects 0.000 claims description 11
- 150000008195 galaktosides Chemical class 0.000 claims description 8
- 229940081969 saccharomyces cerevisiae Drugs 0.000 abstract description 9
- 210000004027 cell Anatomy 0.000 abstract description 7
- 210000002421 cell wall Anatomy 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 abstract description 2
- 210000004899 c-terminal region Anatomy 0.000 abstract 2
- 102000002464 Galactosidases Human genes 0.000 abstract 1
- 108010093031 Galactosidases Proteins 0.000 abstract 1
- 229960002203 tilactase Drugs 0.000 description 21
- 229940088598 enzyme Drugs 0.000 description 16
- 238000005516 engineering process Methods 0.000 description 6
- 230000001079 digestive effect Effects 0.000 description 3
- UQZIYBXSHAGNOE-USOSMYMVSA-N Stachyose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO[C@@H]2[C@@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O2)O1 UQZIYBXSHAGNOE-USOSMYMVSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- UQZIYBXSHAGNOE-XNSRJBNMSA-N stachyose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)O2)O)O1 UQZIYBXSHAGNOE-XNSRJBNMSA-N 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- IFBHRQDFSNCLOZ-IIRVCBMXSA-N 4-nitrophenyl-α-d-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-IIRVCBMXSA-N 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- -1 p-nitrophenyl p-nitrophenyl Chemical group 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for improving continuity of alpha galactosidase activity. The method comprises the following steps that: alpha galactosidase gene is linked to a N-terminal of FLO sequence of saccharomycescerevisiae cell wall composition; the resulting sequence is inserted into a C-terminal of MF alpha 1 signal peptide sequence positioned on the downstream of a GAL1 promoter of a galactosidase expression vector to construct a expression cassette, wherein the expression cassette sequentially comprises the GAL1 promoter, the MF alpha 1 signal peptide sequence, the alpha galactosidase gene and the FLO sequence from the N-terminal to the C-terminal; yeast plasmids containing the expression cassette are transformed into the saccharomycescerevisiae cells. According to the present invention, the alpha galactosidase is linked to the FLO sequence of the saccharomycescerevisiae cell wall composition, such that if the saccharomycescerevisiae cells are in survival states, the alpha galactosidase can remain the activity.
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 the alpha tilactase to improve the method for this enzyme activity persistence.
Background technology
Contain the carbohydrate that animal intestinal digestive ferment that stachyose etc. contains the α galactoside is difficult to digest in the feed, not only influence animal digesting and assimilating to carbohydrate, and can improve monogastric animal digestive tube viscosity, hinder animal intestinal digestive ferment digesting and assimilating to other nutritive ingredient in the feed.
In feed, add the α tilactase and be expected effectively to remove the disadvantageous effect to the animal digestion function such as stachyose.But enzyme activity persistence deficiency is the problem that the α tilactase of application at present exists.The recombinant expressed mode that comprises the various enzymes of α tilactase 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 the technology that a kind of α of making tilactase becomes the brewing yeast cell organic composition, soon α tilactase and brewing yeast cell wall composition FLO are formed by connecting and are the integral part of brewing yeast cell, the α tilactase remains active condition as long as brewing yeast cell keeps surviving then, thereby has significantly improved α galactoside enzyme activity persistence.
Summary of the invention
At the insufficient problem of existing α galactoside enzyme activity persistence, the invention provides a kind of α of making tilactase becomes the technology of brewing yeast cell organic component, make the α tilactase as long as brewing yeast cell keeps survival to maintain vigour, thereby significantly improve α galactoside enzyme activity persistence.
A kind of method that improves alpha galactoside enzyme activity persistence of the present invention may further comprise the steps:
The alpha galactosidase 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+alpha galactosidase 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 " claim 1 " described expression cassette is incubated in the YPD substratum that is added with weight percent 3.5-7% semi-lactosi.
Advantage of the present invention:
The α tilactase is connected with brewing yeast cell wall composition FLO, and so then as long as brewing yeast cell keeps existing state, the α tilactase can maintain vigour.
Embodiment
The demonstration of embodiment 1 α tilactase is expressed
With the α galactosidase gene (from Saccharomyces Cerevisiae in S accharomyces cerevisiae, Genbank number: X03102) 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 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+α galactosidase 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 the α tilactase to the brewing yeast cell external secretion, the FLO sequence in α tilactase downstream then is anchored in the brewing yeast cell wall, thereby makes the demonstration of α tilactase be expressed in brewing yeast cell wall outside surface.
What the demonstration of embodiment 2 α tilactases was expressed induces
In the YPD substratum of cultivating yeast saccharomyces cerevisiae, add the 3.5-7% 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+α galactosidase gene+FLO sequence " will activate, and induces the α tilactase to express in the demonstration of brewing yeast cell wall outside surface.
The effect that demonstration is expressed to the α tilactase 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 α galactosidase activity, this is because the GAL1 promotor in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+α galactosidase gene+FLO sequence " can't activate.
The demonstration of embodiment 4 α tilactases is expressed its vigor persistence of back and is significantly improved
The α galactosidase activity that demonstrates expression according to step shown in embodiment 1 and the embodiment 2 is 629U/mL (3.5% semi-lactosi) and 678U/mL (3.5-7% semi-lactosi), and the transformation period is 73 days, can keep half of initial activity in the time of promptly the 73rd day.And if express removal FLO sequence in the expression cassette " GAL1 promotor+MF α 1 signal peptide sequence+α galactosidase gene+FLO sequence " in the demonstration shown in embodiment 1 and the embodiment 2, then the α tilactase carries out secreting, expressing, activity is 228U/mL (3.5% semi-lactosi) and 216U/mL (3.5-7% semi-lactosi), transformation period all only is 5 days, can keep half of initial activity in the time of promptly the 5th day.Therefore, α tilactase and brewing yeast cell wall composition FLO be formed by connecting be the integral part of brewing yeast cell, the α tilactase remains active condition as long as brewing yeast cell keeps surviving then, thereby has significantly improved α galactoside enzyme activity persistence.
The U of α galactosidase activity unit is defined as: under 37 ℃, the condition of pH 5.0, in 1 minute from the pNPG (p-nitrophenyl-alpha-D-galactopyranoside of 0.01 mol, give birth to worker's biotechnology company limited, Shanghai Sangon Biological Engineering Technology﹠amp available from Shanghai; Services Co., (p-nitrophenyl p-nitrophenyl gives birth to worker's biotechnology company limited, Shanghai Sangon Biological EngineeringTechnology﹠amp available from Shanghai Ltd.) to discharge 1 micromole pNP in the solution; Services Co., Ltd.) needed α tilactase enzyme amount is 1U.
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 alpha galactoside enzyme activity persistence is characterized in that may further comprise the steps:
The alpha galactosidase 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+alpha galactosidase 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 3.5-7% semi-lactosi.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 201010171805 CN102242094A (en) | 2010-05-14 | 2010-05-14 | Method for improving continuity of alpha galactosidase activity |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201010171805 CN102242094A (en) | 2010-05-14 | 2010-05-14 | Method for improving continuity of alpha galactosidase activity |
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| CN 201010171805 Pending CN102242094A (en) | 2010-05-14 | 2010-05-14 | Method for improving continuity of alpha galactosidase activity |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164398A (en) * | 2017-06-30 | 2017-09-15 | 浙江工业大学 | A kind of recombinant alpha galactosidase gene, carrier, engineering bacteria and its application |
| WO2018207889A1 (en) * | 2017-05-11 | 2018-11-15 | 関西化学機械製作株式会社 | MICROORGANISM CAPABLE OF DISPLAYING α-GALACTOSIDASE ON SURFACE LAYER THEREOF, AND USE THEREOF |
Citations (3)
| 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 |
| US6838254B1 (en) * | 1993-04-29 | 2005-01-04 | Conopco, Inc. | Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae |
| CN101475914A (en) * | 2008-10-15 | 2009-07-08 | 山东大学 | Method for producing oligo-galactose by cyclic utilization of recombinant Saccharomyces cerevisiae |
-
2010
- 2010-05-14 CN CN 201010171805 patent/CN102242094A/en active Pending
Patent Citations (3)
| 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 |
| US6838254B1 (en) * | 1993-04-29 | 2005-01-04 | Conopco, Inc. | Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae |
| CN101475914A (en) * | 2008-10-15 | 2009-07-08 | 山东大学 | Method for producing oligo-galactose by cyclic utilization of recombinant Saccharomyces cerevisiae |
Non-Patent Citations (1)
| 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 615-620 1,2 第63卷, 第2期 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018207889A1 (en) * | 2017-05-11 | 2018-11-15 | 関西化学機械製作株式会社 | MICROORGANISM CAPABLE OF DISPLAYING α-GALACTOSIDASE ON SURFACE LAYER THEREOF, AND USE THEREOF |
| US11306300B2 (en) | 2017-05-11 | 2022-04-19 | Kansai Chemical Engineering Co., Ltd. | Microorganism capable of displaying α-galactosidase on surface layer thereof, and use thereof |
| CN107164398A (en) * | 2017-06-30 | 2017-09-15 | 浙江工业大学 | A kind of recombinant alpha galactosidase gene, carrier, engineering bacteria and its application |
| CN107164398B (en) * | 2017-06-30 | 2020-05-26 | 浙江工业大学 | Recombinant α -galactosidase gene, vector, engineering bacterium and application thereof |
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Application publication date: 20111116 |