CN103308510A - Detection method of transesterification activity of non-aqueous phase lipase - Google Patents
Detection method of transesterification activity of non-aqueous phase lipase Download PDFInfo
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- CN103308510A CN103308510A CN2013101742451A CN201310174245A CN103308510A CN 103308510 A CN103308510 A CN 103308510A CN 2013101742451 A CN2013101742451 A CN 2013101742451A CN 201310174245 A CN201310174245 A CN 201310174245A CN 103308510 A CN103308510 A CN 103308510A
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Abstract
The invention provides a detection method of transesterification activity of non-aqueous phase lipase. The method comprises the following steps of: taking p-nitrophenol ester as shown in formula (I) and alkyl alcohol as shown in formula (II) as substrates, taking the lipase as a catalyst for performing transesterification in an organic solvent to generate p-nitrophenol and alkyl ester as shown in formula (III), performing organic dissolution and dilution, and then detecting the variation of an ultraviolet absorption value of a reaction solution at 310nm to determine the transesterification activity of the lipase. The beneficial effects of the detection method of the transesterification activity of the non-aqueous phase lipase, disclosed by the invention, are mainly represented as follows: the detection method is simple and feasible, the cost of raw materials is low, the time consumption in detection is low, the requirements for instruments are not high, and the detection method can be used for fast determination of esterification activity of the lipase, and is suitable for being applied to conventional biochemical laboratories.
Description
(1) technical field
The present invention relates to a kind of detection method of nonaqueous phase lipase transesterification vigor.
(2) background technology
Lipase (Lipase EC3.1.1.3) is the triacylglycerol Acyl-hydrolase, and its catalysis natural substrate grease hydrolysis generates fatty acid, glycerine and monoglyceride or diester, also can catalytic esterification, and transesterificationization, reactions such as transesterification and aminolysis.The triplet that most lipase actives center is made up of serine, aspartic acid, histidine.The lipase molecule is made up of hydrophilic segment and hydrophobic part, and the activated centre is near the molecule hydrophobic side.Lipase has special cap structure, the character of interface activation.Along with the develop rapidly of nearly more than 20 years nonaqueous phase living things catalysis, the basic theory of lipase and applied research are more and more.Lipase has been widely used in fields such as food, medicine, chemical materials and the energy.
Lipase need be measured its catalytic activity, i.e. the detection of enzyme activity as a kind of biocatalyst.The enzyme activity determination method of present most lipase all is the characteristic according to its hydrolysing activity, such as common olive oil emulsification titrimetry, is substrate spectrophotometer method etc. with the p-nitrophenyl phenolic ester.But the esterification activity of lipase is not fully can be corresponding with its hydrolysing activity.Along with the increase that the nonaqueous phase esterification of lipase is used, the assay method of setting up the lipase esterification activity has very important use value and significance.The assay method of the lipase esterification activity of now having reported, generally by GC, HPLC, large-scale instruments such as fluorescence detector, it is long to exist detection time, and instrument requires high, problems such as sample costliness
Teng Yun, Xu Yan etc. (1, Yun Teng, Yan Xu.A modified para-nitrophenyl palmitate assay for lipase synthetic activity determination in organic solvent[J] .Analytical Biochemistry, 2007,363:297-299; 2, Xu Yan; Teng Yun.The rapid assay methods of lipase synthetase vigor in a kind of nonaqueous phase. application (patent) number: CN200610086243.7) set up with p-nitrophenol palmitate and ethanol transesterification, detect the content of p-nitrophenol by 410nm behind the alkali lye extractive reaction liquid, thereby detect the esterification activity of lipase.Relate to the alkali lye extraction step in the testing process, may cause the spontaneous hydrolysis of p-nitrophenol palmitate and extract problems such as incomplete, will influence the accuracy of detection.
(L.Goujard such as Goujard, P.Villeneuve, B.Barea.A spectrophotometric transesterification-based assay for lipases in organic solvent[J] .Analytical biochemistry, 385 (1): 161-167) utilize the transesterification of lipase-catalyzed vinyl acetate and alkylol, detect the content of vinyl acetate by the 200nm wavelength to measure the enzyme activity of lipase.Reaction dissolvent, the acetaldehyde that substrate alkylol and the isomerization of product vinyl alcohol obtain all absorbs at the 200nm place, therefore all can have interference effect to testing result, and wherein acetaldehyde volatilizees easily, and the residual content in its reactant liquor is difficult to calculate.
(3) summary of the invention
The objective of the invention is in order to solve the deficiency of said method, set up a kind of simple to operately, instrument is less demanding, the detection method of the nonaqueous phase lipase transesterification vigor of good reproducibility.
The technical solution used in the present invention is:
A kind of detection method of nonaqueous phase lipase transesterification vigor, described method is to be substrate with alkylol shown in p-nitrophenyl phenolic ester shown in the formula (I) and the formula (II), be that catalyzer carries out Arrcostab shown in transesterification generation p-nitrophenol and the formula (III) in organic solvent with lipase, change to determine the transesterification vigor of lipase by the ultraviolet absorption value at detection reaction liquid 310nm place; The p-nitrophenyl phenolic ester that 1 enzyme activity unit is defined as every lmin catalysis 1 μ mol is converted into the enzyme amount of p-nitrophenol; Described organic solvent is one of following: normal hexane, cyclohexane, normal heptane;
Among formula (I), (II), (III):
R
1Alkyl for C2～C11;
R
2Alkyl for C1～C6.
Preferably, described p-nitrophenyl phenolic ester is the p-nitrophenol palmitate, and described alkylol is isopropyl alcohol, and described organic solvent is normal hexane.
Concrete, described method is as follows:
(1) typical curve is drawn: be solvent with ethanol, the p-nitrophenyl phenol solution of preparation gradient concentration detects solution at the ultraviolet absorption value at 310nm place, is that horizontal ordinate, ultraviolet absorption value are ordinate drawing standard curve with concentration;
In (2) 0.1～10mL organic solvents, add p-nitrophenyl phenolic ester, alkylol and lipase to be measured, p-nitrophenyl phenolic ester addition is 0.01mM～10mM, alkylol and p-nitrophenyl phenolic ester mol ratio are 5～200:1, lipase addition to be measured is 0.1～10mg, 50～200r/min, 10～60 ℃ of reaction 5～60min; React and get the 0.03ml supernatant after finishing with the ultraviolet absorption values at isopropyl alcohol or 100 times of detections of ethanol dilution 310nm places, the conversion ratio of reference standard curve calculation reaction, the enzyme live data of acquisition lipase.
Preferably, be reflected at described in the step (2) under the 200r/min shaking speed, 30 ℃ and carry out, the reaction time is 10min.
P-nitrophenyl phenolic ester addition is preferably 10mM, and the alkylol addition is preferably 1M, and lipase sample addition to be measured is preferably 1mg, and the organic solvent volume is preferably 2mL.
The detection method of nonaqueous phase lipase transesterification vigor of the present invention, reactant liquor can carry out the detection of ultraviolet absorption value by quartz colorimetric utensil or quartzy 96 orifice plates, detection volume was 2ml when wherein quartz colorimetric utensil detected, and detection volume was 0.2ml when quartzy 96 orifice plates detected.
Because the enzymatic transesterification need be got rid of the interference of moisture, used reagent and solvent utilize the 4A molecular sieve dehydration.
The typical reaction of nonaqueous phase lipase transesterification vigor detection method: contain 10mM nitrophenol acetic acid esters and 1M isopropyl alcohol in the 2mL normal hexane system, reaction 10min, shaking speed 200r/min, enzyme addition are 1mg, 30 ℃ of temperature of reaction.Get 100 times of 0.03ml supernatant dilutions after reaction finishes and detect 310nm place ultraviolet absorption value, the conversion ratio of reference standard curve calculation reaction.Instrument is multi-functional microplate reader or general ultraviolet spectrophotometer.
The beneficial effect of the detection method of nonaqueous phase lipase transesterification vigor of the present invention is mainly reflected in: the detection method simple possible, raw materials cost is low, detects consuming time fewly, and instrument is less demanding, can be used for the rapid screening of lipase esterification activity, be fit to the routine biochemistry laboratory applications.
(4) description of drawings
Fig. 1 is the full wavelength scanner figure of solvent with ethanol for the nitrophenol palmitate;
Fig. 2 is the full wavelength scanner figure of solvent with ethanol for p-nitrophenol;
Fig. 3 is the typical curve of solvent wavelength 310nm with ethanol for p-nitrophenol; Horizontal ordinate is concentration (Concentration); Ordinate is the absorbance (Absorbance at310nm) at 310nm place;
Fig. 4 is the typical curve of solvent wavelength 310nm with ethanol for the p-nitrophenol palmitate; Horizontal ordinate is concentration (Concentration); Ordinate is the absorbance (Absorbance at310nm) at 310nm place.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the full wavelength scanner figure that transesterification determination of activity and gas chromatography detection validation nitrophenol palmitate (pNPP), the p-nitrophenol (pNP) of 7 kinds of commercialization lipase with ethanol is solvent and the typical curve of wavelength 310nm are referring to Fig. 1～4.
2ml normal hexane reaction system, pNPP concentration 10mM, isopropyl alcohol concentration 1M, 0.01g lipase, 30 ℃, 200r/min shaking speed reaction 10min, 310nm surveyed the OD value after the 30 μ l that take a sample reaction supernatant diluted 100 times with ethanol.The typical curve of contrast p-nitrophenol calculates the conversion ratio of reaction, obtains the enzyme live data of lipase.
The conversion ratio computing formula:
A wherein
0, A
1Be the OD of blank and sample detection
310nm, ε
1, ε
2It is the molar extinction coefficient at pNPP and pNP310nm place.
Sampling simultaneously utilizes ethyl acetate to dilute laggard promoting the circulation of qi analysis of hplc.High performance liquid chromatograph method condition: Agilent6890, chromatographic column: the HP-5 post, the column temperature temperature: 150 ℃, flow rate of carrier gas=1ml/min, split ratio: 20:1, detecting device: fid detector.Get 100 times in 0.1ml dilution ethyl acetate, the peak area of detection reaction product p-nitrophenol.Draw the content of reaction product p-nitrophenol by the typical curve of gas chromatographic analysis p-nitrophenol, calculate the conversion ratio of reaction then.
The result is as shown in table 1.
Table 1
By table 1 as seen, commercialization lipase is utilized the ratio ester work of this ultraviolet spectro-photometric analysis method and the basically identical as a result that gas chromatography obtains, therefore detection method of the present invention is feasible.
Claims (4)
1. the detection method of a nonaqueous phase lipase transesterification vigor, described method is to be substrate with alkylol shown in p-nitrophenyl phenolic ester shown in the formula (I) and the formula (II), be that catalyzer carries out Arrcostab shown in transesterification generation p-nitrophenol and the formula (III) in organic solvent with lipase, change to determine the transesterification vigor of lipase by the ultraviolet absorption value at detection reaction liquid 310nm place; The p-nitrophenyl phenolic ester that 1 enzyme activity unit is defined as every lmin catalysis 1 μ mol is converted into the enzyme amount of p-nitrophenol; Described organic solvent is one of following: normal hexane, cyclohexane, normal heptane;
Among formula (I), (II), (III):
R
1Alkyl for C2～C11;
R
2Alkyl for C1～C6.
2. the method for claim 1 is characterized in that described p-nitrophenyl phenolic ester is the p-nitrophenol palmitate, and described alkylol is isopropyl alcohol, and described organic solvent is normal hexane.
3. method as claimed in claim 1 or 2 is characterized in that described method is as follows:
(1) typical curve is drawn: be solvent with ethanol, the p-nitrophenyl phenol solution of preparation gradient concentration detects solution at the ultraviolet absorption value at 310nm place, is that horizontal ordinate, ultraviolet absorption value are ordinate drawing standard curve with concentration;
In (2) 0.1～10mL organic solvents, add p-nitrophenyl phenolic ester, alkylol and lipase to be measured, p-nitrophenyl phenolic ester addition is 0.01mM～10mM, alkylol and p-nitrophenyl phenolic ester mol ratio are 5～200:1, lipase addition to be measured is 0.1～10mg, 50～200r/min, 10～60 ℃ of reaction 5～60min; React and get the 0.03ml supernatant after finishing with the ultraviolet absorption values at isopropyl alcohol or 100 times of detections of ethanol dilution 310nm places, the conversion ratio of reference standard curve calculation reaction, the enzyme live data of acquisition lipase.
4. method as claimed in claim 3 is characterized in that: be reflected at described in the step (2) under the 200r/min shaking speed, 30 ℃ and carry out, the reaction time is 10min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106940326A (en) * | 2017-05-11 | 2017-07-11 | 广州南侨食品有限公司 | The method for quick of ester exchange reaction degree |
| CN107727628A (en) * | 2017-11-22 | 2018-02-23 | 浙江工业大学 | A kind of fluorescence detection method of lipase alcoholysis vigor |
| CN108299163A (en) * | 2018-03-09 | 2018-07-20 | 兰州大学 | A kind of method that aromatic ester hydrolyzes in a mild condition |
| CN114813603A (en) * | 2022-03-09 | 2022-07-29 | 南京林业大学 | Method for rapidly representing halogenation conversion rate of phenolic hydroxyl compounds without standard substance |
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| EP1328795B1 (en) * | 2000-10-26 | 2009-09-09 | Imerys Minerals Limited | Processing of inorganic particulate materials |
| US20080286990A1 (en) * | 2003-12-02 | 2008-11-20 | Super Talent Electronics, Inc. | Direct Package Mold Process For Single Chip SD Flash Cards |
| CN101001959A (en) * | 2004-08-11 | 2007-07-18 | 西巴特殊化学品控股有限公司 | Enzyme-based time temperature indicator |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106940326A (en) * | 2017-05-11 | 2017-07-11 | 广州南侨食品有限公司 | The method for quick of ester exchange reaction degree |
| CN106940326B (en) * | 2017-05-11 | 2019-02-15 | 广州南侨食品有限公司 | The rapid detection method of ester exchange reaction degree |
| 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 |
| CN108299163A (en) * | 2018-03-09 | 2018-07-20 | 兰州大学 | A kind of method that aromatic ester hydrolyzes in a mild condition |
| CN108299163B (en) * | 2018-03-09 | 2021-10-12 | 兰州大学 | Method for hydrolyzing aromatic ester under mild condition |
| CN114813603A (en) * | 2022-03-09 | 2022-07-29 | 南京林业大学 | Method for rapidly representing halogenation conversion rate of phenolic hydroxyl compounds without standard substance |
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