CN101430279A - Method for screening catalysis non-aqueous phase system transesterification enzyme by fluorospectrophotometry - Google Patents
Method for screening catalysis non-aqueous phase system transesterification enzyme by fluorospectrophotometry Download PDFInfo
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- CN101430279A CN101430279A CNA2008101949189A CN200810194918A CN101430279A CN 101430279 A CN101430279 A CN 101430279A CN A2008101949189 A CNA2008101949189 A CN A2008101949189A CN 200810194918 A CN200810194918 A CN 200810194918A CN 101430279 A CN101430279 A CN 101430279A
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Abstract
The invention relates to a method for filtering enzyme applied to catalyze nonaqueous phase system transesterification reaction and belongs to the technical field of enzyme reaction and fluorescence measuration. In the method, a. vinyl ester and primary alcohol substance are catalyzed by enzyme to take transesterification reaction, as one of the reaction products vinyl alcohol generates acetaldehyde by keto-enol tautomerization to ensure that the transesterification reaction is nonreversible; b. the acetaldehyde reacts with 4-diazanyl-7-nitryl-2, 1, 3-benzoxadiazole (NBD-H.NH2NH2) to generate fluorescent derivative, and the fluorescence intensity of the fluorescent derivative is mensurated to reflect the catalytic capability of the enzyme indirectly. The invention has simple operation and can intuitively and easily judge whether enzyme catalysis transesterification reaction in nonaqueous phase can occur or not, the invention can compare the catalytic activity of the enzyme catalyzing the transesterification reaction, and the method can be used for studying on the factor influencing enzymic catalytic reaction.
Description
Technical field
The present invention relates to a kind of screening technique of enzyme of catalysis non-aqueous system transesterification, be to utilize fluorescence spectrophotometry can in nonaqueous phase, judge by the catalysis transesterification specifically to enzyme, and determine enzymatic ability power according to fluorescence intensity, belong to enzyme reaction and fluorometric assay technical field.
Background technology
Some lipase, esterase and proteinase but can synthesize and transesterification in non-aqueous system at the aqueous phase cartalytic decomposition effect in catalysis.Enzymatic transesterification even can carry out modification to solid high molecular polymer is introduced the macromolecular compound side chain with functional group.Therefore, if can introduce different groups on the cotton fiber surface, then can under the condition of not damaging cotton fiber, give cotton fiber new function by modification by the enzymatic transesterification.
So far, the mensuration of enzymic catalytic reaction is used gas chromatography or high performance liquid chromatography usually, and enzyme, reaction substrate and reaction dissolvent consumption are more in these methods, and working sample pre-treatment requirement is high, and the single sample minute is longer.By contrast, fluorescence spectrophotometry needs sample size few, and reaction system can be measured a plurality of samples in microlitre at every turn simultaneously.Fluorescence spectrophotometry utilizes the effect of one of the fluorogenic substrate that self do not have fluorescence and product of enzymic catalytic reaction to generate fluorescent derivative, reflects the catalytic capability of enzyme indirectly by the fluorescence intensity of measuring fluorescent derivative.This method requires enzymic catalytic reaction and fluorescent derivative generation reaction to take place synchronously.Therefore, the conditions that these two reactions take place must be coordinated mutually, and the condition that a lot of fluorogenic substrate reaction generates fluorescent derivatives is inconsistent and cause mensuration to carry out with enzyme-catalyzed reaction condition, thereby have limited the application of fluorescence spectrophotometry in enzyme catalysis field.
4-diazanyl-7-nitro-2,1,3-benzo 4-oxadiazole hydrazine (NBD-H.NH
2NH
2) be a kind of commercialization fluorogenic substrate, can be used for the fluorometric assay of aldehyde material.
Summary of the invention
Technical matters to be solved by this invention mainly provides a kind of method of judging that non-aqueous system enzymatic transesterification takes place, can compare the activity of energy catalysis transesterification enzyme simultaneously.This method operation is succinct, simple and clear.
Technical scheme of the present invention: a kind of method of judging that non-aqueous system enzymatic transesterification takes place, the while can be compared the activity of energy catalysis transesterification enzyme.The method of fluorescence spectrophotometry screening catalysis non-aqueous system transesterification enzyme, a, enzymatic vinyl esters and primary alconol class material generation transesterification, the vinyl alcohol of one of reaction product generates acetaldehyde by keto-enol tautomerization, makes transesterification irreversible; B, acetaldehyde and 4-diazanyl-7-nitro-2,1,3-benzo 4-oxadiazole hydrazine (NBD-H.NH
2NH
2) react the generation fluorescent derivative, and reflect the catalytic capability of enzyme indirectly by the fluorescence intensity of measuring this fluorescent derivative; The aitiogenic reaction equation of enzymic catalytic reaction and fluorescent derivative is:
Now that several gordian technique divisions in the method are as follows:
(1) freeze drying of enzyme
Compound concentration is the enzyme solutions of 1.333g/L, and adding enzyme solutions weight percent 5% sucrose fully after the vibration dissolving, installs in the 0.5mL centrifuge tube with the liquid-transfering gun branch as freezing drying protective agent, and 300 μ L/ pipe carries out freeze drying, gets freeze drying enzyme powder, and is standby;
Selected enzyme, enzyme solutions are configured to:
The proteinase bacillolysin K of pH7.5,0.1mol/L
2HPO
4-KH
2PO
4Buffer preparation;
The proteinase subtilisin Carlsberg K of pH7.5,0.1mol/L
2HPO
4-KH
2PO
4Buffer preparation;
The lipase LPL-3 K of pH7.5,0.1mol/L
2HPO
4-KH
2PO
4Buffer preparation;
The protease A lcalase 3.0T K of pH8.0,0.1mol/L
2HPO
4-KH
2PO
4Buffer preparation.
(2) preparation NBD-H.NH
2NH
2Isooctane solution
Accurately take by weighing a certain amount of NBD-H.NH
2NH
2In the fixed measured isooctane, compound concentration is the NBD-H.NH of 200 μ mol/L extremely in advance
2NH
2Isooctane solution uses the vortex vortex mixer to shake up to NBD-H.NH continuously
2NH
2Dissolving fully, this solution uses in back 3 days of preparation; NBD-H.NH
2NH
2Also operation like this when being dissolved in toluene and the acetonitrile;
(3) enzyme reaction and fluorometric assay
1., the NBD-H.NH of 200 μ L, 200 μ mol/L in the 0.5mL centrifuge tube that freeze drying enzyme powder is housed, add successively:
2NH
2Isooctane solution, 2., the 0.2mmol n-propanol, be 15 μ L and 3., 0.02mmol vinyl propionate base ester is that 2 μ L or 0.02mmol lauric acid vinyl esters are 5 μ L;
Use vortex vortex mixer concussion mixing, in the isothermal vibration water-bath, reacting 1.5h under the temperature of reaction of enzyme, after finishing, reaction leaves standstill a moment, draw 200 μ L supernatants with liquid-transfering gun and to black non transparent 96 orifice plates, measure fluorescence intensity, each condition is made 3 parallel samples, measurement result is averaged, the fluorometric assay condition is excitation wavelength 435nm, wavelength of transmitted light 535nm, firing time 1s, take the top reading model, adopt relative intensity of fluorescence to represent during data analysis, being 100% with high fluorescent in batch mensuration.
The temperature of reaction of selected enzyme is: 37 ℃ of proteinase bacillolysin temperature; 37 ℃ of proteinase subtilisinCarlsberg temperature; 50 ℃ of lipase LPL-3 temperature; 50 ℃ of protease A lcalase 3.0T temperature.
Beneficial effect of the present invention: acetaldehyde is volatile matter, and can the acetaldehyde that the enzymatic transesterification produces fully and NBD-H.NH
2NH
2Effect generates fluorescent derivative also influences the mensuration of fluorescence spectrophotometry to the enzymatic ability, but and the working sample pre-treatment requires gas chromatography and high performance liquid chromatography high, that the single sample minute is long to compare, fluorescence spectrophotometry needs sample size few, reaction system can be in microlitre, can measure a plurality of samples simultaneously, be a kind of method of reaction enzymes catalytic activity directly perceived preferably at every turn.The present invention operates succinctly, and can the enzymatic transesterification take place in the simple and clear judgement nonaqueous phase of energy, can compare the catalytic activity of energy catalysis transesterification enzyme, and can study the factor that influences enzymic catalytic reaction by the inventive method.
Description of drawings
Fig. 1 difference is participated in the experiment with the ability of enzymatic transesterification.1, lipase LPL-3; 2, protease A lcalase 3.0T; 3, proteinase subtilisin Carlsberg; 4, proteinase bacillolysin.
The different transesterification ester of Fig. 2 substrate is to the influence of transesterification.1, proteinase bacillolysin; 2, lipase LPL-3; 3, proteinase subtilisin Carlsberg; 4, protease A lcalase 3.0T.
Fig. 3 different organic solvents is to the influence of transesterification.1, proteinase bacillolysin; 2, lipase LPL-3; 3, proteinase subtilisin Carlsberg; 4, protease A lcalase 3.0T.
Embodiment
Embodiment 1: difference is participated in the experiment with the ability of enzymatic transesterification
Experiment is the commercialization enzyme preparation with enzyme, proteinase bacillolysin (37 ℃ of optimum temperatures, optimal pH 7.5) changes into Industrial Co., Ltd available from the Tokyo, lipase LPL-3 (50 ℃ of optimum temperatures, optimal pH 7.0) available from Japanese amano enzyme goods Co., Ltd., proteinase subtilisin Carlbergs (37 ℃ of optimum temperatures, optimal pH 7.5) is available from Sigma company, protease A lcalase 3.0T (50 ℃ of optimum temperatures, optimal pH 8.5) is available from Novozymes company.NBD-H.NH
2NH
2Buy in the Tokyo and change into Co., Ltd., chromatographically pure.Lauric acid vinyl esters (chromatographically pure) is bought in the Tokyo and is changed into Industrial Co., Ltd.Vinyl propionate base ester (chromatographically pure) is bought in Sigma.The multi-functional resolution luminoscope of the used 1420 multilabel counter types of fluorometric assay is that U.S. PerkinElmer company produces.
Study the ability of the catalysis transesterification of above-mentioned 4 kinds of enzymes according to the method described above, as shown in Figure 1.The reaction system fluorescence intensity of lipase LPL-3 catalysis enters plateau behind 1.5h, the fluorescence intensity ratio 1.5h of reaction 2h has only increased by 2.3%, and the enzymatic system fluorescence intensity of its excess-three kind presents rising tendency always in experimental period.The used 4 kinds of enzymes of experimental result illustrative experiment can both be in isooctane catalysis vinyl propionate base ester and n-propanol generation transesterification, catalytic capability is: lipase LPL-3〉protease A lcalase3.0T proteinase subtilisin Carlsberg proteinase bacillolysin.
When reaction proceeds to 2h, the relative intensity of fluorescence of lipase LPL-3 catalyst system and catalyzing is 100%, the relative intensity of fluorescence of protease A lcalase3.0T catalyst system and catalyzing is 73.5%, the relative intensity of fluorescence of proteinase subtilisinCarlsberg catalyst system and catalyzing is 43.4%, and the relative intensity of fluorescence of proteinase bacillolysin catalyst system and catalyzing is 7.9%.
Embodiment 2: study the influence of different transesterification ester substrates to reaction
Vinyl propionate base ester identical with the alcohol moiety structure, that carboxylic moiety carbochain length is different and lauric acid vinyl esters are as the substrate of enzymatic transesterification, and research different molecular size ester substrate is to the influence of reaction.The ability of testing used 4 kinds of enzymes difference catalysis vinyl propionate base ester and n-propanol reaction illustrates that all greater than the ability of catalysis lauric acid vinyl esters and n-propanol reaction micromolecule ester substrate helps the enzyme effect.
Embodiment 3: the research different organic solvents is to the influence of reaction
With vinyl propionate base ester and n-propanol is substrate, more different hydrophobic organic solvents: isooctane (logP=4.5), toluene (log P=2.5), acetonitrile (logP=-0.33) are to the influence of enzymatic ability.The catalytic capability of these 4 kinds of enzymes presents consistent trend in isooctane and toluene, acetonitrile system, but along with the hydrophobic decline of organic solvent, the catalytic activity of these 4 kinds of enzymes descends rapidly: proteinase bacillolysin, lipase LPL-3, proteinase subtilisin Carlsberg and the catalytic capability of protease A lcalase 3.0T in toluene respectively only in isooctane 11.5%, 10.1%, 10.8% and 8.8%; Catalytic capability in acetonitrile respectively only in isooctane 5.7%, 4.1%, 5.3% and 4.7%.Compare with toluene, acetonitrile, the isooctane that hydrophobicity is stronger relatively is fit to the solvent as the enzymatic transesterification.
The gained result is:
1) the used 4 kinds of enzymes of experiment catalysis vinyl propionate base ester and n-propanol in isooctane, the ability that transesterification takes place is: lipase LPL-3〉protease A lcalase 3.0T〉proteinase subtilisin Carlsberg〉proteinase bacillolysin.
2) ability of experiment used 4 kinds of enzymatic vinyl propionate base esters and n-propanol reaction is all greater than the ability of catalysis lauric acid vinyl esters and n-propanol reaction.
3) compare with toluene, acetonitrile, the isooctane that hydrophobicity is stronger is more suitable for the solvent as the enzymatic transesterification.
Claims (1)
1, the method for fluorescence spectrophotometry screening catalysis non-aqueous system transesterification enzyme, it is characterized in that a, enzymatic vinyl esters and primary alconol class material generation transesterification, the vinyl alcohol of one of reaction product generates acetaldehyde by keto-enol tautomerization, makes transesterification irreversible; B, acetaldehyde and 4-diazanyl-7-nitro-2,1,3-benzo 4-oxadiazole hydrazine NBD-H.NH
2NH
2Reaction generates fluorescent derivative, and reflects the catalytic capability of enzyme indirectly by the fluorescence intensity of measuring this fluorescent derivative; Step is:
(1) freeze drying of enzyme
Compound concentration is the enzyme solutions of 1.333g/L, and adding enzyme solutions weight percent 5% sucrose fully after the vibration dissolving, installs in the 0.5mL centrifuge tube with the liquid-transfering gun branch as freezing drying protective agent, and 300 μ L/ pipe carries out freeze drying, and it is standby to get freeze drying enzyme powder;
Selected enzyme, enzyme solutions are formulated as:
The proteinase bacillolysin K of pH7.5,0.1mol/L
2HPO
4-KH
2PO
4Buffer preparation;
The proteinase subtilisin Carlsberg K of pH7.5,0.1mol/L
2HPO
4-KH
2PO
4Buffer preparation;
The lipase LPL-3 K of pH7.5,0.1mol/L
2HPO
4-KH
2PO
4Buffer preparation;
The protease A lcalase 3.0T K of pH8.0,0.1mol/L
2HPO
4-KH
2PO
4Buffer preparation;
(2) preparation NBD-H.NH
2NH
2Isooctane solution
Accurately take by weighing a certain amount of NBD-H.NH
2NH
2In the fixed measured isooctane, compound concentration is the NBD-H.NH of 200 μ mol/L extremely in advance
2NH
2Isooctane solution uses the vortex vortex mixer to shake up to NBD-H.NH continuously
2NH
2Dissolving fully, this solution uses in back 3 days of preparation; NBD-H.NH
2NH
2Also operation like this when being dissolved in toluene and the acetonitrile;
(3) enzyme reaction and fluorometric assay
1., the NBD-H.NH of 200 μ L, 200 μ mol/L in the 0.5mL centrifuge tube that freeze drying enzyme powder is housed, add successively:
2NH
2Isooctane solution, 2., the 0.2mmol n-propanol be 15 μ L and 3., 0.02mmol vinyl propionate base ester is that 2 μ L or 0.02mmol lauric acid vinyl esters are 5 μ L;
Use vortex vortex mixer concussion mixing, in the isothermal vibration water-bath, reacting 1.5h under the temperature of reaction of enzyme, after finishing, reaction leaves standstill a moment, draw 200 μ L supernatants with liquid-transfering gun and to black non transparent 96 orifice plates, measure fluorescence intensity, each condition is made 3 parallel samples, measurement result is averaged, the fluorometric assay condition is excitation wavelength 435nm, wavelength of transmitted light 535nm, firing time 1s, take the top reading model, adopt relative intensity of fluorescence to represent during data analysis, being 100% with high fluorescent in batch mensuration;
The temperature of reaction of selected enzyme is: 37 ℃ of proteinase bacillolysin temperature; 37 ℃ of proteinase subtilisinCarlsberg temperature; 50 ℃ of lipase LPL-3 temperature; 50 ℃ of protease A lcalase 3.0T temperature.
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Cited By (13)
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| US9259393B2 (en) | 2000-11-15 | 2016-02-16 | Aptalis Pharma S.R.L. | Microspheres of pancreatic enzymes with high stability and production method thereof |
| US9884025B2 (en) | 2000-11-15 | 2018-02-06 | Aptalis Pharma S.R.L. | Microspheres of pancreatic enzymes with high stability and production method thereof |
| US10206882B2 (en) | 2007-02-20 | 2019-02-19 | Allergan Pharmaceuticals International Limited | Stable digestive enzyme compositions |
| US10087493B2 (en) | 2008-03-07 | 2018-10-02 | Aptalis Pharma Canada Ulc | Method for detecting infectious parvovirus in pharmaceutical preparations |
| US11364205B2 (en) | 2010-10-01 | 2022-06-21 | Societe Des Produits Nestle S.A. | Stable low digestive enzyme content formulation |
| CN103732245B (en) * | 2011-08-08 | 2016-07-06 | 阿普塔利斯制药有限公司 | For the method that the dissolution of the compositions containing digestive enzyme is tested |
| US9976171B2 (en) | 2011-08-08 | 2018-05-22 | Allergan Pharmaceuticals International Limited | Method for dissolution testing of solid compositions containing digestive enzymes |
| CN103732245A (en) * | 2011-08-08 | 2014-04-16 | 阿普塔利斯制药有限公司 | Method for dissolution testing of solid compositions containing digestive enzymes |
| US10184121B2 (en) | 2013-06-28 | 2019-01-22 | Allergan Pharmaceuticals International Limited | Methods for removing viral contaminants from pancreatic extracts |
| US10993996B2 (en) | 2013-08-09 | 2021-05-04 | Allergan Pharmaceuticals International Limited | Digestive enzyme composition suitable for enteral administration |
| CN109752356A (en) * | 2018-11-27 | 2019-05-14 | 河南师范大学 | A kind of method of quick visualization screening ionic-liquid catalyst |
| CN109752356B (en) * | 2018-11-27 | 2021-02-26 | 河南师范大学 | Method for rapidly and visually screening ionic liquid catalyst |
| CN109734709A (en) * | 2019-02-12 | 2019-05-10 | 温州医科大学 | A kind of small-molecule fluorescent probe and the preparation method and application thereof |
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Open date: 20090513 |