CN102154231A - Mixture of enzymes, high-flux enzyme screen plate and applications thereof - Google Patents
Mixture of enzymes, high-flux enzyme screen plate and applications thereof Download PDFInfo
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- CN102154231A CN102154231A CN2011100379194A CN201110037919A CN102154231A CN 102154231 A CN102154231 A CN 102154231A CN 2011100379194 A CN2011100379194 A CN 2011100379194A CN 201110037919 A CN201110037919 A CN 201110037919A CN 102154231 A CN102154231 A CN 102154231A
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- OHLRLMWUFVDREV-UHFFFAOYSA-N CCOC(CC(CCl)=O)=O Chemical compound CCOC(CC(CCl)=O)=O OHLRLMWUFVDREV-UHFFFAOYSA-N 0.000 description 1
- ZAJNMXDBJKCCAT-YFKPBYRVSA-N CCOC(C[C@@H](CCl)O)=O Chemical compound CCOC(C[C@@H](CCl)O)=O ZAJNMXDBJKCCAT-YFKPBYRVSA-N 0.000 description 1
- GZPHSAQLYPIAIN-UHFFFAOYSA-N N#Cc1cccnc1 Chemical compound N#Cc1cccnc1 GZPHSAQLYPIAIN-UHFFFAOYSA-N 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N OC(c1cnccc1)=O Chemical compound OC(c1cnccc1)=O PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a mixture of enzymes. The mixture comprises enzymes and a buffer salt. The invention also discloses a high-flux enzyme screen plate, wherein the screen plate is taken as a pore plate; and the mixture of enzymes, comprising the enzymes and the buffer salt, is arranged in pores of the pore plate. The invention also discloses the applications of the mixture of enzymes and the high-flux enzyme screen plate. Due to the adoption of the mixture of enzymes, the operation of an enzymic catalytic reaction is more convenient and efficient. The high-flux enzyme screen plate disclosed by the invention can be used for efficiently and conveniently performing high-flux enzyme screening on a certain substrate, so that the efficiency is greatly increased and the cost is saved.
Description
Technical field
The present invention relates to technical field of enzyme engineering, relate in particular to a kind of mixture, high-throughput enzyme screen plate and application thereof of enzyme.
Background technology
The mankind that develop into of chemical industry have brought huge change and interests, but have also brought harm for human beings'health, community's safety and ecotope simultaneously.Along with global problem of environmental pollution increasingly sharpen and the energy, resource sharply reduce, Public Environmental Awareness progressively improves, Green Chemistry becomes the theme of 21 century gradually.
Based on the criterion of Green Chemistry, bio-transformation becomes one of current the most green internationally recognized chemical conversion technology.Bio-transformation has the unrivaled advantage of some chemical processes, and for example specificity is strong, and selectivity is good, reaction conditions gentleness, environmental friendliness.Bio-transformation is applied to large-scale commercial production, not only alleviates environmental problem, cost also can reduce, and produces remarkable economic efficiency.
The enzyme catalysis bio-transformation is a kind of highly selective reaction, and different types of enzyme can act on not isomorphism type and different types of specific substrates, thereby reaches the directed purpose that transforms.Just because of these characteristics of enzyme, will carry out the screening of enzyme at a certain substrate.The screening of enzyme is to determine a project key of success, but also is the work of relatively wasting time and energy simultaneously, if can carry out the screening of enzyme efficiently, will raise the efficiency greatly, also can correspondingly reduce cost simultaneously.Existing researchist pays close attention to this, for example, Yazbeck etc. are positioned over enzyme in advance and make precoated plate in 96 orifice plates, preserve then, add other auxiliary reagents and substrate (Adv.Synth.Catal.2003,345 (4): 524-532) such as damping fluid when to be used more voluntarily; Codeix company has also released the enzyme screen plate, respectively various auxiliary reagents such as damping fluid is prepared simultaneously, and the ratio that provides in the said firm is mixed in use.Though these effort have improved the efficient of enzyme screening to a certain extent, operate not easyly, still can not carry out enzyme efficiently and screen.
Add damping fluid in the enzyme-catalyzed reaction and be for the soda acid buffer system of an appropriate pH value being provided to enzyme, making the maximum effect of enzyme performance, prevent peracid or cross alkali and influence the activity of enzyme.In traditional technology, the storage stage of enzyme before being used for catalyzed reaction, active for the stable and maintenance of enzyme, the common individual packages of enzyme when catalyzed reaction, is prepared required damping fluid and other auxiliary reagent again and is mixed with enzyme.Yet, now preparing damping fluid and auxiliary reagent makes troubles for the operation of enzymic catalytic reaction, when especially carrying out the high-throughput enzyme screening, because the damping fluid that the enzyme of more or less a hundred different activities is required and the kind of auxiliary reagent and amount are all different, giving these enzymes interpolation damping fluids and auxiliary reagent respectively is a job very loaded down with trivial details and consuming time, makes the enzyme screening efficiency very low.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of mixture of enzyme, and the mixture of this enzyme makes the operation of enzymic catalytic reaction convenient more, efficient.
In addition, also will provide a kind of high-throughput enzyme screen plate, this enzyme screen plate can be efficiently, carry out the high-throughput enzyme screening at a certain substrate easily.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of mixture of enzyme, comprised enzyme and buffering salt.
The concentration of described enzyme is 0.1g/l～50g/l, and the pH value of described buffering salt is 5.0～9.0, and its concentration is 10mM～500mM.
Described enzyme comprises the various enzymes that are used for bio-transformation such as ketoreductase, lytic enzyme or transaminase; Described buffering salt comprises phosphoric acid, Tri-HCl or other commonly used buffer salt classes.
For oxydo-reductase, transferring enzyme or some lyase etc., when the catalysis respective reaction, need NAD (P)
+, coenzyme such as NAD (P) H or PLP participation.And after these coenzyme participation reactions, if there is not the regenerating coenzyme system to make it regeneration, in case coenzyme is consumed fully, enzyme reaction will stop, therefore, also need to add the regenerating coenzyme system, as Hexose phosphate dehydrogenase (GDH) and D-glucose or hydrogenlyase (FDH) and ammonium formiate etc.
Preferably, for oxydo-reductase, transferring enzyme or some lyase etc., the mixture of described enzyme also comprises coenzyme and regenerating coenzyme system.The concentration of coenzyme is 0.01g/l～2.0g/l, and the concentration of described regenerating coenzyme system is 0.5g/l～100g/1.
In another aspect of this invention, provide a kind of high-throughput enzyme screen plate, this screen plate is an orifice plate, is placed with the mixture of enzyme in the hole of described screen plate, and this mixture comprises enzyme and buffering salt.
The concentration of described enzyme is 0.1g/l～50g/l, and the pH value of described buffering salt is 5.0～9.0, and its concentration is 10mM～500mM.
Preferably, for screening oxydo-reductase, transferring enzyme or some lyase etc., the mixture of described enzyme also comprises coenzyme and regenerating coenzyme system.The concentration of coenzyme is 0.01g/l～2.0g/l, and the concentration of described regenerating coenzyme system is 0.5g/l～100g/l.
In another aspect of this invention, also provide a kind of preparation method of above-mentioned high-throughput enzyme screen plate, may further comprise the steps:
Damping fluid and each enzyme concentrated solution are tested in preparation respectively;
To test damping fluid and each concentrated solution is mixed in proportion;
Above-mentioned mixed solution branch is filled to each hole of screen plate;
After removing the moisture of mixed solution in the screening plate hole, cryopreservation.
For the enzyme screen plate that needs coenzyme and regenerating coenzyme system,, also comprise step: the concentrated solution of preparing coenzyme and regenerating coenzyme system respectively testing before damping fluid and each concentrated solution be mixed in proportion.
A kind of application of mixture in the enzyme product of preparation catalyzed reaction of above-mentioned enzyme also is provided in another aspect of this invention.
In another aspect of this invention, also provide the application of a kind of above-mentioned high-throughput enzyme screen plate in the product of preparation screening enzyme.
The mixture of enzyme of the present invention makes the operation of enzymic catalytic reaction convenient more, efficient.High-throughput enzyme screen plate of the present invention can be efficiently, carry out the high-throughput enzyme screening at a certain substrate easily, improved efficient greatly, saved cost.High-throughput enzyme screen plate of the present invention, it is wide to have suitability, good stability, using method is easy, the economic dispatch characteristics, can be widely used in a plurality of industries such as scientific research, medicine, fine chemistry industry.
Embodiment
Convenient more, efficient for the operation that makes enzymic catalytic reaction, the present invention develops a kind of mixture of enzyme, and this mixture comprises enzyme and buffering salt.Wherein, the concentration of enzyme is 0.1g/l～50g/l, and the pH value of buffering salt is 5.0～9.0, and the concentration of buffering salt is 10mM～500mM.For oxydo-reductase, transferring enzyme or some lyase etc., the mixture of enzyme also comprises coenzyme and regenerating coenzyme system.The concentration of coenzyme is 0.01g/l～2.0g/l, and the concentration of regenerating coenzyme system is 0.5g/l～100g/l.Coenzyme comprises NAD (P)
+, NAD (P) H or PLP etc., the regenerating coenzyme system comprises Hexose phosphate dehydrogenase (GDH) and D-glucose or hydrogenlyase (FDH) and ammonium formiate etc.
The present invention also provides a kind of high-throughput enzyme screen plate, and this screen plate is an orifice plate, is placed with the mixture of enzyme in the hole, and this mixture comprises enzyme and buffering salt.The concentration of enzyme is 0.1g/l～50g/l, and the pH value of buffering salt is 5.0～9.0, and the concentration of buffering salt is 10mM～500mM.For screening oxydo-reductase, transferring enzyme or some lyase etc., the mixture of enzyme also comprises coenzyme and regenerating coenzyme system.The concentration of coenzyme is 0.01g/l～2.0g/l, and the concentration of regenerating coenzyme system is 0.5g/l～100g/l.
The preparation method of high-throughput enzyme screen plate of the present invention may further comprise the steps:
Damping fluid and each enzyme concentrated solution are tested in preparation respectively;
To test damping fluid and each concentrated solution is mixed in proportion;
Above-mentioned mixed solution branch is filled to each hole of screen plate;
After under not damaging enzyme prerequisite alive, removing the moisture of mixed solution in the screen plate hole, cryopreservation.
For the enzyme screen plate that needs coenzyme and regenerating coenzyme system,, also comprise step: the concentrated solution of preparing coenzyme and regenerating coenzyme system respectively testing before damping fluid and each concentrated solution be mixed in proportion.
In actual production process, remove the moisture of mixed solution in the screening plate hole after, also to pack, the step of labelling, cryopreservation then.
The using method of high-throughput enzyme screen plate of the present invention following (with the reaction scale is 0.1ml, and adding concentration of substrate is that 1g/l is an example):
1. add 90 μ l deionized waters in each hole of enzyme screen plate, suggestion added before using, in order to avoid the coenzyme in the screen plate degenerates;
2. preparation substrate mother liquor is dissolved in the 10mg substrate in the 1ml deionized water, if substrate is water insoluble, substrate can be dissolved in (for example, DMSO, methyl alcohol, acetonitrile, Virahol etc.) in the organic solvent;
3. 10 μ l substrate mother liquors are joined respectively and begin the screening reaction in each hole of enzyme screen plate;
4. the enzyme screen plate is positioned over constant temperature and the incubator of certain stirring velocity is arranged or shaking table in reacted 24～72 hours;
5. according to the analytical procedure termination reaction of having set up, as be used for positive HPLC or gas phase GC analyzes, add 0.1ml ethyl acetate or MTBE; As be used for the reversed-phase HPLC analysis, then add the 0.1ml acetonitrile.The concussion back is centrifugal, and organic phase or acetonitrile solution are taken out, and analyzes transformation efficiency and selectivity.
The present invention is further detailed explanation below by embodiment.
The high-throughput enzyme screening of embodiment 1 ketoreductase
Use ketoreductase high-throughput enzyme screen plate, comprising 1mg ketoreductase (totally 85 kinds), 1.07mg K
2HPO
4, 0.52mg KH
2PO
4, 0.02mg NADP
+/ NAD
+, 0.4mg D-glucose, 0.2mg Hexose phosphate dehydrogenase (GDH).
In each hole of enzyme screen plate, add 90 μ l deionized waters; The 10mg substrate is dissolved in preparation substrate mother liquor among the 1ml DMSO; 10 μ l substrate mother liquors are joined respectively begin screening reaction in each hole of enzyme screen plate; The enzyme screen plate is positioned over 30 ℃ ﹠amp; Reaction is 24 hours in the 160rpm shaking table; Add the 0.1ml acetonitrile in each hole of enzyme screen plate, the concussion back is centrifugal, acetonitrile solution is taken out HPLC analyze, and 34 reactions is arranged better in 85 ketoreductases.
The high-throughput enzyme screening of embodiment 2 nitrilases
Use nitrilase high-throughput enzyme screen plate, comprising 1mg nitrilase (totally 40 kinds), 1.07mg K
2HPO
4, 0.52mg KH
2PO
4
In each hole of enzyme screen plate, add 90 μ l deionized waters; The 10mg substrate is dissolved in preparation substrate mother liquor among the 1ml DMSO; 10 μ l substrate mother liquors are joined respectively begin screening reaction in each hole of enzyme screen plate; The enzyme screen plate is positioned over 30 ℃ ﹠amp; Reaction is 24 hours in the 160rpm shaking table; Add the 0.1ml acetonitrile in each hole of enzyme screen plate, the concussion back is centrifugal, acetonitrile solution is taken out HPLC analyze, and 9 reactions is arranged better in 40 nitrilases.
The high-throughput enzyme screening of embodiment 3 transaminases
Use transaminase high-throughput enzyme screen plate, comprising 10mg transaminase (totally 20 kinds), 10.7mg K
2HPO
4, 5.2mgKH
2PO
4, 0.4mg PLP, 20mg L-α-Ben Yian.
In each hole of enzyme screen plate, add 180 μ l deionized waters; The 10mg substrate is dissolved in preparation substrate mother liquor among the 1ml DMSO; 20 μ l substrate mother liquors are joined respectively begin screening reaction in each hole of enzyme screen plate; The enzyme screen plate is positioned over 30 ℃ ﹠amp; Reaction is 24 hours in the 160rpm shaking table; Add the 0.2ml acetonitrile in each hole of enzyme screen plate, the concussion back is centrifugal, acetonitrile solution is taken out HPLC analyze, and 7 reactions is arranged better in 20 transaminases.
The high-throughput enzyme screening of embodiment 4 ketoreductases
Use ketoreductase high-throughput enzyme screen plate, comprising 0.02mg ketoreductase (totally 120 kinds), 0.21mg K
2HPO
4, 0.10mg KH
2PO
4, 0.002mg NADP
+/ NAD
+, 0.08mg D-glucose/ammonium formiate, 0.02mg Hexose phosphate dehydrogenase (GDH)/hydrogenlyase (FDH).
In each hole of enzyme screen plate, add 180 μ l deionized waters; The 30mg substrate is dissolved in preparation substrate mother liquor among the 3ml DMSO; 20 μ l substrate mother liquors are joined respectively begin screening reaction in each hole of enzyme screen plate; The enzyme screen plate is positioned over 30 ℃ ﹠amp; Reaction is 24 hours in the 160rpm shaking table; Add the 0.2ml ethyl acetate in each hole of enzyme screen plate, the concussion back is centrifugal, organic phase is taken out GC analyze, and 55 reactions is arranged better in 120 ketoreductases.
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (12)
1. the mixture of an enzyme is characterized in that, comprises enzyme and buffering salt.
2. the mixture of enzyme according to claim 1 is characterized in that, the concentration of described enzyme is 0.1g/l～50g/l, and the pH value of described buffering salt is 5.0～9.0, and its concentration is 10mM～500mM.
3. the mixture of enzyme according to claim 1 and 2 is characterized in that, this mixture also comprises coenzyme and regenerating coenzyme system.
4. the mixture of enzyme according to claim 3 is characterized in that, the concentration of described coenzyme is 0.01g/l～2.0g/l, and the concentration of described regenerating coenzyme system is 0.5g/l～100g/l.
5. high-throughput enzyme screen plate, this screen plate is an orifice plate, it is characterized in that, is placed with the mixture of enzyme in the hole of described screen plate, this mixture comprises enzyme and buffering salt.
6. high-throughput enzyme screen plate according to claim 5 is characterized in that, the concentration of described enzyme is 0.1g/l～50g/l, and the pH value of described buffering salt is 5.0～9.0, and its concentration is 10mM～500mM.
7. according to claim 5 or 6 described high-throughput enzyme screen plates, it is characterized in that the mixture of described enzyme also comprises coenzyme and regenerating coenzyme system.
8. high-throughput enzyme screen plate according to claim 7 is characterized in that, the concentration of described coenzyme is 0.01g/l～2.0g/l, and the concentration of described regenerating coenzyme system is 0.5g/l～100g/l.
9. the preparation method of the described high-throughput enzyme screen plate of claim 5 is characterized in that, may further comprise the steps:
Damping fluid and each enzyme concentrated solution are tested in preparation respectively;
To test damping fluid and each concentrated solution is mixed in proportion;
Above-mentioned mixed solution branch is filled to each hole of screen plate;
After removing the moisture of mixed solution in the screening plate hole, cryopreservation.
10. preparation method according to claim 9, it is characterized in that, for the enzyme screen plate that needs coenzyme and regenerating coenzyme system,, also comprise step: the concentrated solution of preparing coenzyme and regenerating coenzyme system respectively testing before damping fluid and each concentrated solution be mixed in proportion.
11. the application of the mixture of the described enzyme of claim 1 in the enzyme product of preparation catalyzed reaction.
12. the application of the described high-throughput enzyme screen plate of claim 5 in the product of preparation screening enzyme.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100379194A CN102154231A (en) | 2011-02-15 | 2011-02-15 | Mixture of enzymes, high-flux enzyme screen plate and applications thereof |
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|---|---|---|---|
| CN2011100379194A CN102154231A (en) | 2011-02-15 | 2011-02-15 | Mixture of enzymes, high-flux enzyme screen plate and applications thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012120086A1 (en) | 2011-03-10 | 2012-09-13 | Zach System S.P.A. | Asymmetric reduction process |
| CN107630052A (en) * | 2017-03-29 | 2018-01-26 | 武汉茵茂特生物技术有限公司 | The bioconversion method of L glufosinate-ammoniums |
-
2011
- 2011-02-15 CN CN2011100379194A patent/CN102154231A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| DANIEL R. YAZBECK ET AL: "Automated enzyme screening methods for the preparation of enantiopure pharmaceutical intermediates", 《ADV. SYNTH. CATAL》 * |
| 张翀 等: "辅酶再生***的研究进展", 《生物工程学报》 * |
| 王秋雨 等: "伴有辅酶再生的生物催化过程", 《化学通报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012120086A1 (en) | 2011-03-10 | 2012-09-13 | Zach System S.P.A. | Asymmetric reduction process |
| CN107630052A (en) * | 2017-03-29 | 2018-01-26 | 武汉茵茂特生物技术有限公司 | The bioconversion method of L glufosinate-ammoniums |
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Application publication date: 20110817 |