CN1928116A - Rapid determination method of lipase synthetase vitality in non-aqueous phase - Google Patents
Rapid determination method of lipase synthetase vitality in non-aqueous phase Download PDFInfo
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Abstract
一种非水相中脂肪酶合成酶活力的快速测定方法，属于非水相脂肪酶催化技术领域。本发明是利用脂肪酶在非水相体系中催化对硝基苯酚酯与醇之间的转酯化反应，反应一定时间后，吸取一定体积的反应液用0.1M的NaOH溶液萃取，用比色法于410nm处测定吸光度以确定对硝基苯酚的生成量，一个脂肪酶酶活单位定义为生成1μmol的对硝基苯酚所需的酶量。本发明用于高酯合成活性脂肪酶生产菌的直接筛选，将会得到目的性更强，更适合于非水相催化的脂肪酶生产菌。本发明方法与国际上已有的有关脂肪酶合成酶活力测定与筛选方法相比，无需气相色谱、液相色谱，无需价格昂贵的荧光底物和荧光分光光度计。
The invention discloses a method for quickly measuring lipase synthetase activity in non-aqueous phase, which belongs to the technical field of non-aqueous phase lipase catalysis. The present invention utilizes lipase to catalyze the transesterification reaction between p-nitrophenol ester and alcohol in a non-aqueous phase system. After reacting for a certain period of time, draw a certain volume of reaction solution and extract it with 0.1M NaOH solution. Absorbance was measured at 410nm to determine the amount of p-nitrophenol produced, and one lipase activity unit was defined as the amount of enzyme needed to generate 1 μmol of p-nitrophenol. The invention is used for direct screening of lipase-producing bacteria with high ester synthesis activity, and will obtain lipase-producing bacteria with stronger purpose and more suitable for non-aqueous phase catalysis. Compared with the existing international methods for measuring and screening lipase synthetase activity, the method of the present invention does not need gas chromatography, liquid chromatography, expensive fluorescent substrates and fluorescent spectrophotometers.
Description
Technical field
The present invention relates to the rapid assay methods of lipase synthetase vigor in a kind of nonaqueous phase, can be used on the screening and the enzyme rapid determination alive of the fatty enzyme-producing bacteria that has high ester composite reactive in the nonaqueous phase, belong to the lipase-catalyzed technical field of nonaqueous phase.
Background technology
Lipase is as a kind of triglyceride hydrolysis enzyme, deepening continuously and develop along with the nonaqueous phase zymetology, become the effective catalyst that is used for organic synthesis in the nonaqueous phase biocatalysis process, effectively catalytic esterification and transesterification, successfully be used for many important compound productions, as aromatic ester, mono-glycerides, chipal compounds and biofuel or the like.The lipase kind is many, and catalysis characteristics varies, so the needed lipase of autotelic selection is the key of catalytic process.
The enzyme activity determination method is depended in the selection of enzyme, therefore formed the different lipase enzyme activity determination method of many kinds at different purposes, most lipase enzyme activity determination methods all are based on its hydrolysis properties, and what promptly measure is the hydrolysis vigor of lipase.Yet problem is the lytic enzyme of synthetase activity in the organic phase and the aqueous phase not corresponding (Wu that lives, Jaaskelainen et al., Enzyme and MicrobialTechnology, 1996,226), work is screened the lipase that obtains and is not suitable for esterification, transesterification and carries out catalysis in nonaqueous phase according to lytic enzyme.External many investigators all find this problem in research process, and have taked some means to solve this contradiction.Thereby some investigators have developed the measuring method that lipase synthetase is lived based on esterification and transesterification, the amount that these methods adopt GC, HPLC even titration measuring acid or ester is mostly lived to measure enzyme, and the shortcoming of these methods be when sample size greatly or will need a large amount of experiments and time loss when being used for screening operation.At above shortcoming, people such as Konarzycka-Bessler (Konarzycka-Bessler and Bornscheuer, AngewandteChemie-International Edition, 2003,1418) developed a kind of method that is used for lytic enzyme synthetase activity high flux screening, with people (Han such as the similar Min Su of its principle Han, Jung et al., Journal ofOrganic Chemistry, 2004,2583) also invented a kind of method that is used for measuring organic phase proteolytic enzyme transesterification vigor, both all adopt the method for fluorescence developing.Yet not all laboratory all possesses spectrophotofluorometer, and fluorogenic substrate costs an arm and a leg usually, and reaction solvent is required high.
Summary of the invention
(1) problem that will solve
The objective of the invention is to set up the rapid assay methods of lipase synthetase vigor in a kind of nonaqueous phase, this method is used for the screening of fatty enzyme-producing bacteria.Used substrate is selected p-nitrophenyl phenolic ester and ethanol, and it is cheap, and obtains easily, adopts common spectrophotometry to measure, and measuring method is simple, and most Laboratory Instruments can meet the demands.
(2) technical scheme
The present invention utilizes lipase transesterification between catalysis p-nitrophenyl phenolic ester and the alcohol in non-aqueous system, react after 5-30 minute, the reaction solution of drawing 50 μ L extracts in cuvette or porous microwell plate with the NaOH solution of 1ml 0.1M, measure absorbancy in the 410nm place to determine the growing amount of p-NP with colorimetry, a lipase enzyme is lived unit definition for generating the required enzyme amount of p-NP of 1 μ mol.Temperature of reaction is different for different lipase with the reaction times.The present invention can also be according to the different substrate of lipase and specificity changes the carbon chain lengths of p-nitrophenyl phenolic ester, is suitable for ester chain length C
4-C
16The synthetic enzyme vigor of the used lipase of transesterification of p-nitrophenyl phenolic ester measure.Can be used for investigating the substrate specificity of known lipase in nonaqueous phase, can be used as the specific lipase of screening specific substrates.
Reaction principle
Used substrate p-nitrophenyl phenolic ester is: the p-NP propionic ester, and the p-NP capronate, p-NP laurate or p-NP cetylate, used substrate alcohol is ethanol.
The rapid assay methods of lipase synthetase vigor in the nonaqueous phase of the present invention, the transesterification condition is: the p-NP ester concentration is 10mM, determining alcohol is 1M, temperature of reaction 30-60 ℃, reaction times 5-30 minute, 200 rev/mins of shaking speed, enzyme concn were 0.01-0.3U.
The rapid assay methods of lipase synthetase vigor in this nonaqueous phase, be used for having the screening of the fatty enzyme-producing bacteria of composite reactive at nonaqueous phase, fatty enzyme-producing bacteria cultivated be prepared into extraction in cuvette or porous microwell plate behind cell lyophilized powder or the fermented liquid lyophilized powder behind the certain hour, measure enzyme and live, filter out the purpose bacterial strain according to the enzyme height of living.Use 96 hole microwell plates to extract, measure enzyme and live, to improve the efficient of measuring and/or screening.
Below adopt p-NP cetylate (pNPP) and ethanol as substrate, commercialization lipase L-PS-C that derives from Brukholderia sp. and the full cellular fat enzyme (prepared in laboratory) that derives from Rhizopus chinenisis CCTCCM201021 are carried out more detailed research.
The feasibility of measuring method
Reagent and lipase
Used 5 kinds of commercialization lipase altogether and a kind ofly provided full cellular fat enzyme for oneself, be respectively:
L-G (Amano G, Penicillium sp.lipase) Amano Pharmaceutical company;
L-PS-C (Amano PS-C, Brukholderia sp.lipase) Amano Pharmaceutical company;
L-AY (Amano AY, Candida rugosa lipase) Amano Pharmaceutical company;
L-CR (lipase from Candida rugosa) Sigma company;
L-PP (lipase from Porcine Pancreas) Sigma company,
L-Rh (lipase from Rhizopus chinenisis, full cell lyophilized powder) prepared in laboratory, the preparation method referring to (Xu, Wang et al., Journal of Molecular Catalysis B-Enzymatic, 2002 (18), 29-37).
PNPP (p-NP cetylate) purchases the company in Sigma, and other reagent are analytical pure or chromatographically pure.
The canonical process of colorimetric method for determining synthetase activity: accurately take by weighing 189mg pNPP and be dissolved in 50mL in the dewatered normal heptane of 4 molecular sieves, be mixed with the solution of 10mM.Each lipase 2.5-10mg joins in the tool plug plastics tubing of 2mL.Add the above-mentioned pNPP n-heptane solution of 0.5ml, add 30 μ L then through the dewatered dehydrated alcohols of 4 molecular sieves (concentration is about 1M).200rmp, reaction is 5-30 minute under the 30-60 ℃ of condition, gets 50 μ L reaction solutions with 1mL 0.1M NaOH solution extraction, measures absorbancy in 410nm place, and is higher as if light absorption value, measures after then reaction solution being diluted finite concentration.Instrument is You Nike UNICOUV-2102 PC ultraviolet-visible pectrophotometer, 1mL glass or a plastics cuvette.The typical curve that the calculating that enzyme is lived is drawn according to pNP (p-NP) normal concentration solution calculates, and an enzyme is lived unit definition for generating the required enzyme amount of 1 μ mol pNP.
Be growing amount with the lipase synthetase vigour-testing method of present method contrast: get above-mentioned reaction solution 150 μ L with the gas chromatographic detection ethyl palmitate, with 3mL 0.1M NaOH solution extraction, centrifugal behind the mixing, get the interior mark of the 2-hexanol back vapor detection that supernatant liquid adds a certain amount of concentration.Chromatographic condition is 150 ℃ of initial temperatures, and 10 ℃/min rises to 220 ℃, keeps 10min, 250 ℃ of injector temperatures, 250 ℃ of detector temperatures.Gas-chromatography is Agilent 6820, and chromatographic column is AC20 (PEG20000), and detector is FID.
The lipase synthetase vigour-testing method that contrasts with present method is the synthetase activity mensuration of lipase-catalyzed synthetic ethyl octylate: sad and dehydrated alcohol is dissolved in normal heptane respectively, and concentration is respectively 0.6M and 0.72M.Get the 0.5mL reaction substrate respectively in tool plug plastics tubing, add about 20mg lipase, 40 ℃, oscillatory reaction 30min under the 150rmp.Centrifugal, get the 0.4mL supernatant liquor, add mark 2-hexanol in the 0.1mL, mixing, while blank, gas chromatography determination.The gas Chromatographic Determination condition is: 90 ℃ of initial temperatures, keep 1min, and 10 ℃/min is warming up to 200 ℃, keeps 5min, 250 ℃ of detector temperatures, 250 ℃ of sampler temperature.Gas-chromatography is Agilent 6820, and chromatographic column is AC20 (PEG20000), and detector is FID.Under condition determination, the enzyme amount that per minute transforms 1 μ mol ethyl octylate is defined as 1 lipase synthetase unit alive.
The optimum determining scope and the condition determination of measuring method of the present invention
Temperature of reaction: different lipase are different to the stability of temperature, will be much better than the water catalyzed reaction for the temperature stability of nonaqueous phase catalyzed reaction enzyme.This measuring method all is effective for most of lipase between 30 ℃-60 ℃ of temperature.
Rotating speed: because nonaqueous phase catalysis mostly is the solid-liquid two-phase system greatly, and for the solid-liquid biphasic system, the resistance to mass transfer of substrate certainly exists, particularly for immobilized enzyme.The first layer resistance to mass transfer derives from the solvent layer that solid catalyst surface is wrapped up, and also is referred to as the external diffusion resistance.When the speed of response of enzyme during much smaller than the substrate velocity of diffusion, the external diffusion resistance does not become limiting factor, and when the speed of response of enzyme was fast, the external diffusion resistance then was a restrictive factor.With lipase L-PS-C is example, and the speed of L-PS-C catalyzed reaction is very fast, is that the external diffusion resistance is controlled along with the fast reaction speed of rotating speed significantly improves this reaction of explanation, and when rotating speed reaches 200rmp when above, diffusional resistance disappears substantially.Rotating speed for this measuring method 200rmp can meet the demands.
Concentration of substrate: the required concentration of substrate of enzyme activity determination is widely different for different enzymes, be example with lipase L-PS-C and the full cellular fat enzyme of L-Rh respectively, canonical process by above-mentioned colorimetric method for determining synthetase activity is measured the speed of reaction of pNPP concentration in the 2.5-70mM scope, adopts the Lineweaver-Burk method to try to achieve the Km value.Take all factors into consideration for making this method have better generality to most lipase, the p-NP ester concentration that adopts 10mM is as reaction density, and alcohol concn is 1M.
Enzyme concn: enzyme concn also is vital for measuring method, and enzyme concn is crossed and low can be made the enzyme biopsy not detect, and enzyme concn is too high then to cause the institute's enzyme of surveying work inaccurate.Still be example with enzyme high lipase L-PS-C alive and the enzyme relatively low full cellular fat enzyme of L-Rh alive.By the canonical process of above-mentioned colorimetric method for determining synthetase activity, timing sampling is measured the conversion curve under the different enzyme concn conditions, and the enzyme that takes by weighing different mass joins in the reaction system, controls enzyme concn in the reaction system with this.With enzyme concn and speed of reaction curve plotting, investigate the suitableeest scope that adds enzyme concn.The result shows that can measure enzyme more exactly when enzyme concn between 0.01-0.3U lives.1U is the amount that generates the required lipase of 1 μ mol pNP.
In sum, it is as follows to draw the optimum determining condition (being the transesterification condition) of this measuring method: concentration of substrate p-nitrophenyl phenolic ester 10mM, ethanol 1M; 30 ℃-60 ℃ of temperature of reaction; Reaction times 5-30 minute, shaking speed 200rmp; Enzyme concn 0.01-0.3U.
(3) beneficial effect
The present invention has set up a kind of be applicable to lipase synthetase vigor rapid determination and screening method in the nonaqueous phase, can accurately measure its synthetase activity under the less situation of enzyme amount.Used substrate is easy to get, and measuring method is simple, can live by the rapid determination lipase synthetase.Adopt different synthetase activity measuring methods to detect to 6 kinds of lipase choosing arbitrarily, compare, proved the feasibility of this measuring method with this measuring method.Adopt this measuring method to study in great detail to a kind of commercialization enzyme L-PS-C and the full cellular fat enzyme of L-Rh, verified also that when determining optimum determining condition present method can effectively measure the synthetase activity in the lipase non-aqueous system.
The present invention compares with traditional lipase enzyme activity determination method, does not have this phenomenon of positive connection according to the composite reactive in lytic enzyme work and the nonaqueous phase, has given prominence to the catalysis characteristics of lipase in the nonaqueous phase.The present invention is used for the direct screening of high synthetase activity fat enzyme-producing bacteria, and it is stronger to obtain purpose, is more suitable in the catalytic fatty enzyme-producing bacteria of nonaqueous phase.The inventive method is compared with screening method with existing relevant fatty enzymic synthesis enzyme activity determination in the world, need not gas-chromatography, liquid chromatography, need not expensive fluorogenic substrate and spectrophotofluorometer.
Description of drawings
The degree of correlation of Fig. 1 the inventive method (method I) and method III
Method I: colorimetric method for determining synthetase activity; Method III: the synthetase activity of the synthetic ethyl octylate of lipase enzyme catalysis is measured.
Embodiment
Embodiment 1 usefulness the inventive method is verified 6 kinds of lipase
Choose and state 6 kinds of lipase, verify, and provide strong proof, the results are shown in Table 1 for this measuring method is used for the feasibility that lipase nonaqueous phase synthetic enzyme vigor measures with measuring method of the present invention.Because above reaction is not the result under best enzyme activity determination condition, so all represent the size of this enzyme catalysis synthesis of dynamic with transformation efficiency.Result (the method I in the table 1) degree of correlation of result of the growing amount of gas chromatographic detection ethyl palmitate (the method II in the table 1) and employing colorimetric method for determining is 90%, illustrate that measuring method of the present invention is feasible, and the detection method that the present invention adopted can effectively be measured lipase-catalyzed p-NP cetylate (pNPP) and alcoholic acid transesterification.The result (the method I in the table 1) of colorimetric method for determining and the synthetase activity measurement result (the method III in the table 1) of the synthetic ethyl octylate of lipase enzyme catalysis are compared, six kinds of lipase is measured the synthetase activity degree of correlation that obtains more than 80% with these two kinds of methods, and the reason that causes this result is the lipase substrate specificity difference of different sources, thereby caused measurement result difference to some extent, but in general, measuring method of the present invention can effectively be measured the synthetase activity in the lipase nonaqueous phase.
Table 1
| Lipase | Method I (transformation efficiency/%) | Method II (transformation efficiency/%) | Method III (transformation efficiency/%) |
| L-G L-PS-C L-CR L-PP L-Rh L-AY | 0 74.5 2.4 0.8 18.1 0 | 0 74.3 2.1 0.76 16.4 0 | 0.41 61.6 1.3 1.95 8.4 0.18 |
Method I: the transformation efficiency in the colorimetric method for determining 1 hour
Method II: the transformation efficiency of gas chromatographic detection ethyl palmitate
Method III: the transformation efficiency of the synthetic ethyl octylate of lipase enzyme catalysis
Embodiment 2 usefulness the inventive method are carried out L-PS-C lipase enzyme activity determination
Take by weighing 2.5mg L-PS-C lipase and place the tool plug plastics tubing of 2mL, add the pNPP n-heptane solution of 0.5mL 10mM and through the dewatered 30 μ L dehydrated alcohols of 4 molecular sieves, 200rmp, react 5min under 50 ℃ of conditions, get 50 μ L reaction solutions 1mL 0.1M NaOH solution extraction, measure absorbancy in the 410nm place, record the enzyme 140.1U/g of being alive.
Embodiment 3 usefulness the inventive method are carried out the full cellular fat enzyme of L-Rh enzyme activity determination
Take by weighing the full cellular fat enzyme of 10mg L-Rh and place the tool plug plastics tubing of 2mL, add the pNPP n-heptane solution of 0.5mL 10mM and through the dewatered 30 μ L dehydrated alcohols of 4 molecular sieves, 200rmp, react 30min under 40 ℃ of conditions, get 50 μ L reaction solutions 1mL 0.1M NaOH solution extraction, measure absorbancy in the 410nm place, record the enzyme 5.6U/g of being alive.
Embodiment 4 fatty enzyme-producing bacteria screenings
For fatty enzyme-producing bacteria, need screening to obtain a strain can have high composite reactive in nonaqueous phase bacterium, what employing gas phase or Liquid Detection demand side were right will be huge workload.From our strain improvement process, pick out 30 strain bacterium, be prepared into lyophilized powder, the preparation method referring to (Xu, Wang et al., Journal of MolecularCatalysis B-Enzymatic, 2002 (18), 29-37).The synthetase activity measuring method that adopts above-mentioned lipase enzyme catalysis to synthesize ethyl octylate is measured the synthetase activity of each bacterium, adopts the present invention's method to measure synthetase activity simultaneously, the results are shown in accompanying drawing 1.The result shows that two kinds of measuring methods have higher dependency, the degree of correlation is near 83%, show that spectrophotometry can be used for the mensuration of full cellular fat enzyme nonaqueous phase synthetase activity, and the primary dcreening operation that this method is used for high composite reactive lipase screening, with compare as the primary dcreening operation means with the work of lipase hydrolysis enzyme, can improve screening efficiency and accuracy greatly.
Embodiment 5
96 orifice plates are used for the mensuration of this measuring method, improve determination efficiency.8 * 12 types of employing, 96 microwell plates take by weighing a certain amount of enzyme powder in An-Fn (n=1,3,5,7,9,11) in the hole, perhaps the enzyme powder with solubility is dissolved in buffering, adds enzyme liquid by the enzyme amount that should add, standby after the freeze-drying, Am-Fm (m=2,4,6,8,10,12) add 250 μ L0.1M NaOH solution in the hole.An-Fn adds the pNPP n-heptane solution in the canonical process of the above-mentioned colorimetric method for determining synthetase activity of 200 μ L during reaction, add 12 μ L dehydrated alcohols, 8 passage pipettors are drawn 20 μ L reaction solutions and are joined in the adjacent hole that is added with 0.1M NaOH solution 410nm place mensuration absorbancy behind the reaction certain hour.
Claims (7)
1, the rapid assay methods of lipase synthetase vigor in a kind of nonaqueous phase, it is characterized in that utilizing lipase transesterification between catalysis p-nitrophenyl phenolic ester and the alcohol in non-aqueous system, react after 5-30 minute, the reaction solution of drawing 50 μ l extracts in cuvette or porous microwell plate with the NaOH solution of 1ml 0.1M, measure absorbancy in the 410nm place to determine the growing amount of p-NP with colorimetry, a lipase enzyme is lived unit definition for generating the required enzyme amount of p-NP of 1 μ mol.
2, the rapid assay methods of lipase synthetase vigor in the nonaqueous phase according to claim 1 is characterized in that used substrate p-nitrophenyl phenolic ester, is suitable for ester chain length C
4-C
16The synthetic enzyme vigor of the used lipase of transesterification of p-nitrophenyl phenolic ester measure.
3, the rapid assay methods of lipase synthetase vigor in the nonaqueous phase according to claim 1 and 2, it is characterized in that used substrate p-nitrophenyl phenolic ester is: the p-NP propionic ester, p-NP capronate, p-NP laurate or p-NP cetylate.
4, the rapid assay methods of lipase synthetase vigor in the nonaqueous phase according to claim 1 is characterized in that used substrate alcohol is ethanol.
5, the rapid assay methods of lipase synthetase vigor in the nonaqueous phase according to claim 1, it is characterized in that the transesterification condition is: the p-NP ester concentration is 10mM, determining alcohol is 1M, temperature of reaction 30-60 ℃, reaction times 5-30 minute, 200 rev/mins of shaking speed, enzyme concn are 0.01-0.3U.
6, according to the rapid assay methods of lipase synthetase vigor in claim 1,2,3, the 4 or 5 described nonaqueous phases, it is characterized in that being used for having the screening of the fatty enzyme-producing bacteria of composite reactive at nonaqueous phase, fatty enzyme-producing bacteria cultivated be prepared into extraction in cuvette or porous microwell plate behind cell lyophilized powder or the fermented liquid lyophilized powder behind the certain hour, measure enzyme and live, filter out the purpose bacterial strain according to the enzyme height of living.
7,, it is characterized in that used porous microwell plate is that 96 hole microwell plates extract, measure enzyme work, to improve the efficient of measuring and/or screening according to the rapid assay methods of lipase synthetase vigor in claim 1 or the 6 described nonaqueous phases.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101363043B (en) * | 2007-08-07 | 2012-05-23 | 北京北大维信生物科技有限公司 | Method for detecting lipase activity inhibition rate |
| CN103308510A (en) * | 2013-05-10 | 2013-09-18 | 浙江工业大学 | Detection method of transesterification activity of non-aqueous phase lipase |
| CN107727628A (en) * | 2017-11-22 | 2018-02-23 | 浙江工业大学 | A kind of fluorescence detection method of lipase alcoholysis vigor |
| CN108195782A (en) * | 2018-01-25 | 2018-06-22 | 江南大学 | It is a kind of to be simple and efficient the method for measuring beta amylase vigor |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1181187C (en) * | 2003-04-22 | 2004-12-22 | 江南大学 | A kind of lipase producing bacteria and its screening method and industrial application |
| CN1238469C (en) * | 2004-01-16 | 2006-01-25 | 清华大学 | Novel process for preparing biological diesel oil from grease catalyzed by lipase in the reaction system with organic substrate as medium |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363043B (en) * | 2007-08-07 | 2012-05-23 | 北京北大维信生物科技有限公司 | Method for detecting lipase activity inhibition rate |
| CN103308510A (en) * | 2013-05-10 | 2013-09-18 | 浙江工业大学 | Detection method of transesterification activity of non-aqueous phase lipase |
| CN107727628A (en) * | 2017-11-22 | 2018-02-23 | 浙江工业大学 | A kind of fluorescence detection method of lipase alcoholysis vigor |
| CN107727628B (en) * | 2017-11-22 | 2020-01-14 | 浙江工业大学 | Fluorescence detection method for alcoholysis activity of lipase |
| CN108195782A (en) * | 2018-01-25 | 2018-06-22 | 江南大学 | It is a kind of to be simple and efficient the method for measuring beta amylase vigor |
| CN108195782B (en) * | 2018-01-25 | 2020-01-21 | 江南大学 | Method for measuring activity of beta-amylase |
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