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
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- lipase
- nitrophenol
- activity
- transesterification
- formula
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000004882 Lipase Human genes 0.000 title claims abstract description 48
- 108090001060 Lipase Proteins 0.000 title claims abstract description 48
- 239000004367 Lipase Substances 0.000 title claims abstract description 46
- 235000019421 lipase Nutrition 0.000 title claims abstract description 46
- 230000000694 effects Effects 0.000 title claims abstract description 32
- 238000001514 detection method Methods 0.000 title claims abstract description 22
- 238000005809 transesterification reaction Methods 0.000 title claims abstract description 21
- 239000008346 aqueous phase Substances 0.000 title claims description 8
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 20
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims abstract description 16
- -1 p-nitrophenol ester Chemical class 0.000 claims abstract description 15
- 125000005233 alkylalcohol group Chemical group 0.000 claims abstract description 12
- 239000003960 organic solvent Substances 0.000 claims abstract description 11
- 238000010521 absorption reaction Methods 0.000 claims abstract description 10
- 239000000758 substrate Substances 0.000 claims abstract description 5
- 125000005907 alkyl ester group Chemical group 0.000 claims abstract description 3
- 239000003054 catalyst Substances 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 31
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 11
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 8
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 6
- LVZSQWIWCANHPF-UHFFFAOYSA-N p-nitrophenyl palmitate Chemical group CCCCCCCCCCCCCCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 LVZSQWIWCANHPF-UHFFFAOYSA-N 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 4
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 230000032050 esterification Effects 0.000 abstract description 9
- 238000005886 esterification reaction Methods 0.000 abstract description 9
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 238000010790 dilution Methods 0.000 abstract 1
- 239000012895 dilution Substances 0.000 abstract 1
- 238000004090 dissolution Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 4
- 238000004817 gas chromatography Methods 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 239000010453 quartz Substances 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- GOOWMQLOYNJICV-UHFFFAOYSA-N (2-nitrophenyl) hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OC1=CC=CC=C1[N+]([O-])=O GOOWMQLOYNJICV-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 229920001567 vinyl ester resin Polymers 0.000 description 2
- MRCKRGSNLOHYRA-UHFFFAOYSA-N (2-nitrophenyl) acetate Chemical compound CC(=O)OC1=CC=CC=C1[N+]([O-])=O MRCKRGSNLOHYRA-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- IMROMDMJAWUWLK-UHFFFAOYSA-N Ethenol Chemical compound OC=C IMROMDMJAWUWLK-UHFFFAOYSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 235000002725 Olea europaea Nutrition 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000007098 aminolysis reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000005690 diesters Chemical class 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了一种非水相脂肪酶转酯化活力的检测方法，所述方法是以式（I）所示对硝基苯酚酯和式（II）所示烷基醇为底物，以脂肪酶为催化剂在有机溶剂中进行转酯化生成对硝基苯酚和式（III）所示烷基酯，通过有机溶解稀释后检测反应液310nm处的紫外吸收值变化来确定脂肪酶的转酯化活力；本发明所述非水相脂肪酶转酯化活力的检测方法的有益效果主要体现在：检测方法简单可行，原料成本低，检测耗时少，仪器要求不高，可以用于脂肪酶酯化活性的快速测定，适合常规生化实验室应用。
Description
（一）技术领域(1) Technical field
本发明涉及一种非水相脂肪酶转酯化活力的检测方法。The invention relates to a method for detecting transesterification activity of non-aqueous lipase.
（二）背景技术(2) Background technology
脂肪酶（Lipase，EC3.1.1.3）即三酰基甘油酰基水解酶，它催化天然底物油脂水解，生成脂肪酸、甘油和甘油单酯或二酯，也能催化酯化，转酯化，酯交换和胺解等反应。多数脂肪酶活性中心是由丝氨酸、天冬氨酸、组氨酸组成的三联体。脂肪酶分子由亲水部分和疏水部分组成，活性中心靠近分子疏水端。脂肪酶具有特殊的盖子结构，界面活化的性质。随着近20多年的非水相生物催化的飞速发展，脂肪酶的基础理论和应用研究越来越多。脂肪酶已经广泛应用于食品，医药，化工材料和能源等领域。Lipase (Lipase, EC3.1.1.3) is triacylglycerol acyl hydrolase, which catalyzes the hydrolysis of natural substrate oil to generate fatty acid, glycerol and monoglyceride or diester, and can also catalyze esterification, transesterification, esterification Exchange and aminolysis reactions. The active center of most lipases is a triplet composed of serine, aspartic acid and histidine. The lipase molecule consists of a hydrophilic part and a hydrophobic part, and the active center is close to the hydrophobic end of the molecule. Lipase has a special cap structure and the property of interfacial activation. With the rapid development of non-aqueous biocatalysis in the past 20 years, there are more and more basic theoretical and applied researches on lipase. Lipase has been widely used in the fields of food, medicine, chemical materials and energy.
脂肪酶作为一种生物催化剂，需要对其催化活性进行测定，即酶活力的检测。目前绝大部分脂肪酶的酶活测定方法都是根据其水解活性的特性，比如常见的橄榄油乳化滴定法，以对硝基苯酚酯为底物分光光度计法等。但是脂肪酶的酯化活性与其水解活性并非完全可以对应。随着脂肪酶的非水相酯化反应应用的增加，建立脂肪酶酯化活性的测定方法具有很重要的应用价值和意义。现已报道的脂肪酶酯化活性的测定方法，一般通过GC，HPLC，荧光检测器等大型仪器，存在着检测时间长，仪器要求高，样品昂贵等问题As a biocatalyst, lipase needs to be tested for its catalytic activity, that is, the detection of enzyme activity. At present, most lipase enzyme activity determination methods are based on the characteristics of its hydrolysis activity, such as common olive oil emulsification titration method, p-nitrophenol ester as substrate spectrophotometer method, etc. However, the esterification activity and hydrolysis activity of lipase are not completely corresponding. With the increasing application of non-aqueous phase esterification of lipase, it is of great application value and significance to establish a method for the determination of lipase esterification activity. The methods for measuring the esterification activity of lipases that have been reported are generally carried out by large-scale instruments such as GC, HPLC, and fluorescence detectors, and there are problems such as long detection time, high requirements for instruments, and expensive samples.
滕云，徐岩等（1、Yun Teng,Yan Xu.A modified para-nitrophenylpalmitate assay for lipase synthetic activity determination in organicsolvent[J].Analytical Biochemistry,2007,363:297-299；2、徐岩；滕云。一种非水相中脂肪酶合成酶活力的快速测定方法.申请（专利）号:CN200610086243.7）建立了以对硝基苯酚棕榈酸酯和乙醇转酯化反应，通过碱液萃取反应液后410nm检测对硝基苯酚的含量，从而检测脂肪酶的酯化活性。检测过程中涉及到碱液萃取步骤，可能导致对硝基苯酚棕榈酸酯的自发水解和萃取不完全等问题，将会影响检测的准确性。Teng Yun, Xu Yan et al. A rapid assay method for lipase synthetase activity in non-aqueous phase. Application (patent) number: CN200610086243.7) Established the transesterification reaction of p-nitrophenol palmitate and ethanol, and extracted the reaction solution with lye After that, the content of p-nitrophenol was detected at 410 nm, so as to detect the esterification activity of lipase. The detection process involves the lye extraction step, which may lead to problems such as spontaneous hydrolysis and incomplete extraction of p-nitrophenol palmitate, which will affect the accuracy of the detection.
Goujard等（L.Goujard,P.Villeneuve,B.Barea.A spectrophotometrictransesterification-based assay for lipases in organic solvent[J].Analyticalbiochemistry,385(1):161-167）利用脂肪酶催化乙烯酯和烷基醇的转酯化反应，通过200nm波长检测乙烯酯的含量以测定脂肪酶的酶活力。反应溶剂，底物烷基醇和产物乙烯醇异构化得到的乙醛在200nm处都吸收，因此都会对检测结果存在干扰作用，其中乙醛容易挥发，其反应液中的残留含量难以计算。Goujard et al. (L.Goujard, P.Villeneuve, B.Barea.A spectrophotometrictransesterification-based assay for lipases in organic solvent[J].Analyticalbiochemistry,385(1):161-167) using lipase to catalyze vinyl esters and alkyl alcohols In the transesterification reaction, the content of vinyl ester was detected by 200nm wavelength to determine the enzyme activity of lipase. The reaction solvent, the acetaldehyde obtained by the isomerization of the substrate alkyl alcohol and the product vinyl alcohol all absorb at 200nm, so they all interfere with the detection results. Among them, acetaldehyde is easy to volatilize, and its residual content in the reaction solution is difficult to calculate.
（三）发明内容(3) Contents of the invention
本发明的目的是为了解决上述方法的不足，建立一种操作简单，仪器要求不高，重复性好的非水相脂肪酶转酯化活力的检测方法。The purpose of the present invention is to solve the deficiency of the above-mentioned method, establish a kind of detection method of non-aqueous phase lipase transesterification activity with simple operation, low instrument requirement, good repeatability.
本发明采用的技术方案是：The technical scheme adopted in the present invention is:
一种非水相脂肪酶转酯化活力的检测方法，所述方法是以式（I）所示对硝基苯酚酯和式（II）所示烷基醇为底物，以脂肪酶为催化剂在有机溶剂中进行转酯化生成对硝基苯酚和式（III）所示烷基酯，通过检测反应液310nm处的紫外吸收值变化来确定脂肪酶的转酯化活力；1个酶活力单位定义为每lmin催化1μmol的对硝基苯酚酯转化为对硝基苯酚的酶量；所述有机溶剂为下列之一：正己烷，环己烷，正庚烷；A method for detecting the transesterification activity of non-aqueous lipase, the method uses p-nitrophenol ester represented by formula (I) and alkyl alcohol represented by formula (II) as substrates, and lipase as catalyst Perform transesterification in an organic solvent to generate p-nitrophenol and alkyl esters represented by formula (III), and determine the transesterification activity of lipase by detecting the change in the ultraviolet absorption value at 310nm of the reaction solution; 1 enzyme activity unit Defined as the enzyme amount that catalyzes 1 μmol of p-nitrophenol ester into p-nitrophenol per 1 min; the organic solvent is one of the following: n-hexane, cyclohexane, n-heptane;
式（I）、（II）、（III）中：In formula (I), (II), (III):
R1为C2～C11的烷基；R 1 is an alkyl group of C2-C11;
R2为C1～C6的烷基。R 2 is a C1-C6 alkyl group.
优选的，所述对硝基苯酚酯为对硝基苯酚棕榈酸酯，所述烷基醇为异丙醇，所述有机溶剂为正己烷。Preferably, the p-nitrophenol ester is p-nitrophenol palmitate, the alkyl alcohol is isopropanol, and the organic solvent is n-hexane.
具体的，所述方法如下：Specifically, the method is as follows:
（1）标准曲线绘制：以乙醇为溶剂，配制梯度浓度的对硝基苯酚溶液，检测溶液在310nm处的紫外吸收值，以浓度为横坐标、紫外吸收值为纵坐标绘制标准曲线；(1) Standard curve drawing: use ethanol as solvent, prepare p-nitrophenol solution with gradient concentration, detect the ultraviolet absorption value of the solution at 310nm, and draw the standard curve with the concentration as the abscissa and the ultraviolet absorption value as the ordinate;
（2）0.1～10mL有机溶剂中，添加对硝基苯酚酯、烷基醇和待测脂肪酶，对硝基苯酚酯添加量为0.01mM～10mM，烷基醇与对硝基苯酚酯摩尔比为5～200：1，待测脂肪酶添加量为0.1～10mg，50～200r/min、10～60℃反应5～60min；反应结束后取0.03ml上清液以异丙醇或乙醇稀释100倍检测310nm处的紫外吸收值，对照标准曲线计算反应的转化率，获得脂肪酶的酶活数据。(2) In 0.1-10mL organic solvent, add p-nitrophenol ester, alkyl alcohol and lipase to be tested, the amount of p-nitrophenol ester added is 0.01mM-10mM, and the molar ratio of alkyl alcohol to p-nitrophenol ester is 5~200:1, the amount of lipase to be tested is 0.1~10mg, 50~200r/min, 10~60℃ for 5~60min; after the reaction, take 0.03ml supernatant and dilute 100 times with isopropanol or ethanol The ultraviolet absorption value at 310nm was detected, and the conversion rate of the reaction was calculated according to the standard curve to obtain the enzyme activity data of the lipase.
优选的，步骤（2）中所述反应在200r/min摇床转速、30℃下进行，反应时间为10min。Preferably, the reaction in step (2) is carried out at 200 r/min shaker speed, 30° C., and the reaction time is 10 min.
对硝基苯酚酯添加量优选为10mM，烷基醇添加量优选为1M，待测脂肪酶样品添加量优选为1mg，有机溶剂体积优选为2mL。The added amount of p-nitrophenol ester is preferably 10 mM, the added amount of alkyl alcohol is preferably 1 M, the added amount of the lipase sample to be tested is preferably 1 mg, and the volume of the organic solvent is preferably 2 mL.
本发明所述的非水相脂肪酶转酯化活力的检测方法，反应液可以通过石英比色皿或者石英96孔板进行紫外吸收值的检测，其中石英比色皿检测时检测体积为2ml，石英96孔板检测时检测体积为0.2ml。In the detection method of the non-aqueous phase lipase transesterification activity of the present invention, the reaction solution can carry out the detection of the ultraviolet absorption value through a quartz cuvette or a quartz 96-orifice plate, wherein the detection volume of the quartz cuvette is 2ml, The detection volume of the quartz 96-well plate is 0.2ml.
由于酶催化转酯化反应需要排除水分的干扰，所用的试剂和溶剂利用4A分子筛脱水。Since the enzyme-catalyzed transesterification reaction needs to eliminate the interference of water, the reagents and solvents used are dehydrated by 4A molecular sieves.
非水相脂肪酶转酯化活力检测方法的典型反应：2mL正己烷体系中含有10mM硝基苯酚乙酸酯和1M异丙醇，反应10min，摇床转速200r/min，酶添加量为1mg，反应温度30℃。反应结束后取0.03ml上清液稀释100倍检测310nm处紫外吸收值，对照标准曲线计算反应的转化率。仪器为多功能酶标仪或普通紫外分光光度仪。Typical reaction of non-aqueous phase lipase transesterification activity detection method: 2mL n-hexane system contains 10mM nitrophenol acetate and 1M isopropanol, react for 10min, shaker speed is 200r/min, enzyme addition is 1mg, The reaction temperature is 30°C. After the reaction, 0.03ml of the supernatant was diluted 100 times to detect the UV absorbance at 310nm, and the conversion rate of the reaction was calculated against the standard curve. The instrument is a multifunctional microplate reader or an ordinary ultraviolet spectrophotometer.
本发明所述非水相脂肪酶转酯化活力的检测方法的有益效果主要体现在：检测方法简单可行，原料成本低，检测耗时少，仪器要求不高，可以用于脂肪酶酯化活性的快速筛选，适合常规生化实验室应用。The beneficial effects of the detection method of the non-aqueous phase lipase transesterification activity of the present invention are mainly reflected in: the detection method is simple and feasible, the raw material cost is low, the detection time is less, the instrument requirement is not high, and it can be used for the lipase esterification activity Rapid screening, suitable for routine biochemical laboratory applications.
（四）附图说明(4) Description of drawings
图1为硝基苯酚棕榈酸酯以乙醇为溶剂的全波长扫描图；Fig. 1 is that nitrophenol palmitate takes ethanol as the full-wavelength scanning figure of solvent;
图2为对硝基苯酚以乙醇为溶剂的全波长扫描图；Fig. 2 is the full-wavelength scanning figure that p-nitrophenol takes ethanol as solvent;
图3为对硝基苯酚以乙醇为溶剂波长310nm的标准曲线；横坐标为浓度（Concentration）；纵坐标为310nm处的吸光度值（Absorbance at310nm）；Figure 3 is the standard curve of p-nitrophenol with ethanol as the solvent wavelength 310nm; the abscissa is the concentration (Concentration); the ordinate is the absorbance value at 310nm (Absorbance at310nm);
图4为对硝基苯酚棕榈酸酯以乙醇为溶剂波长310nm的标准曲线；横坐标为浓度（Concentration）；纵坐标为310nm处的吸光度值（Absorbance at310nm）。Figure 4 is the standard curve of p-nitrophenol palmitate with ethanol as the solvent at a wavelength of 310nm; the abscissa is the concentration (Concentration); the ordinate is the absorbance value at 310nm (Absorbance at310nm).
（五）具体实施方式(5) Specific implementation methods
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:
实施例1：对7种商品化脂肪酶的转酯化活性测定和气相色谱检测验证硝基苯酚棕榈酸酯（pNPP）、对硝基苯酚（pNP）以乙醇为溶剂的全波长扫描图和波长310nm的标准曲线参见图1～4。Example 1: Determination of transesterification activity of 7 commercial lipases and gas chromatography detection and verification of full-wavelength scanning diagrams and wavelengths of nitrophenol palmitate (pNPP) and p-nitrophenol (pNP) using ethanol as a solvent Refer to Figures 1-4 for the standard curve of 310nm.
2ml正己烷反应体系，pNPP浓度10mM，异丙醇浓度1M，0.01g脂肪酶，30℃、200r/min摇床转速反应10min，取样30μl反应上清液用乙醇稀释100倍后310nm测OD值。对照对硝基苯酚的标准曲线计算反应的转化率，获得脂肪酶的酶活数据。2ml n-hexane reaction system, pNPP concentration 10mM, isopropanol concentration 1M, 0.01g lipase, 30°C, 200r/min shaker speed for 10min, sample 30μl reaction supernatant diluted 100 times with ethanol and measure OD value at 310nm. The conversion rate of the reaction was calculated according to the standard curve of p-nitrophenol, and the enzyme activity data of lipase was obtained.
转化率计算公式：其中A0、A1是空白对照和样品检测的OD310nm，ε1、ε2是pNPP和pNP310nm处的摩尔消光系数。Conversion rate calculation formula: Wherein A 0 , A 1 are the OD 310nm of the blank control and sample detection, ε 1 , ε 2 are the molar extinction coefficients of pNPP and pNP310nm.
同时取样利用乙酸乙酯稀释后进行气相色谱分析。高效液相色谱仪法条件：Agilent6890，色谱柱：HP-5柱，柱温温度：150℃，载气流速=1ml/min，分流比：20:1，检测器：FID检测器。取0.1ml稀释乙酸乙酯100倍，检测反应产物对硝基苯酚的峰面积。通过气相色谱分析对硝基苯酚的标准曲线得出反应产物对硝基苯酚的含量，继而计算反应的转化率。At the same time, samples were diluted with ethyl acetate and analyzed by gas chromatography. High performance liquid chromatography method conditions: Agilent6890, chromatographic column: HP-5 column, column temperature: 150°C, carrier gas flow rate = 1ml/min, split ratio: 20:1, detector: FID detector. Take 0.1ml of ethyl acetate diluted 100 times, and detect the peak area of the reaction product p-nitrophenol. The standard curve of p-nitrophenol was analyzed by gas chromatography to obtain the content of the reaction product p-nitrophenol, and then the conversion rate of the reaction was calculated.
结果如表1所示。The results are shown in Table 1.
表1Table 1
由表1可见，对商品化脂肪酶利用本紫外分光光度分析方法的比酯活和气相色谱分析法得到的结果基本一致，因此本发明所述检测方法可行。As can be seen from Table 1, the results obtained by utilizing the specific ester activity of the ultraviolet spectrophotometric analysis method and the gas chromatography analysis method for commercialized lipase are basically consistent, so the detection method of the present invention is feasible.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101742451A CN103308510A (en) | 2013-05-10 | 2013-05-10 | Detection method of transesterification activity of non-aqueous phase lipase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101742451A CN103308510A (en) | 2013-05-10 | 2013-05-10 | Detection method of transesterification activity of non-aqueous phase lipase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103308510A true CN103308510A (en) | 2013-09-18 |
Family
ID=49133967
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013101742451A Pending CN103308510A (en) | 2013-05-10 | 2013-05-10 | Detection method of transesterification activity of non-aqueous phase lipase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103308510A (en) |
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 | A method for quickly characterizing the conversion rate of halogenation reaction of phenolic hydroxyl compounds without standard |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1928116A (en) * | 2006-08-25 | 2007-03-14 | 江南大学 | Rapid determination method of lipase synthetase vitality in non-aqueous phase |
| 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 |
| US20080286990A1 (en) * | 2003-12-02 | 2008-11-20 | Super Talent Electronics, Inc. | Direct Package Mold Process For Single Chip SD Flash Cards |
| EP1328795B1 (en) * | 2000-10-26 | 2009-09-09 | Imerys Minerals Limited | Processing of inorganic particulate materials |
-
2013
- 2013-05-10 CN CN2013101742451A patent/CN103308510A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN1928116A (en) * | 2006-08-25 | 2007-03-14 | 江南大学 | Rapid determination method of lipase synthetase vitality in non-aqueous phase |
Non-Patent Citations (4)
| Title |
|---|
| L.GOUJARD ET AL: "A spectrophotometric transesterification-based assay for lipases in organic solvent", 《ANALYTICAL BIOCHEMISTRY》 * |
| 江慧芳等: "三种脂肪酶活力测定方法的比较及改进", 《化学与生物工程》 * |
| 纪建业: "脂肪酶活力测定方法的改进", 《通化师范学院学报》 * |
| 郑毅等: "脂肪酶活力测定研究进展", 《工业微生物》 * |
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 | A method for quickly characterizing the conversion rate of halogenation reaction of phenolic hydroxyl compounds without standard |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Biodiesel synthesis and conformation of lipase from Burkholderia cepacia in room temperature ionic liquids and organic solvents | |
| Zhang et al. | Simultaneous determination of ethyl carbamate and urea in alcoholic beverages by high-performance liquid chromatography coupled with fluorescence detection | |
| Cheng et al. | Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study | |
| Xu et al. | Experimental and theoretical evidence of enhanced catalytic performance of lipase B from Candida antarctica acquired by the chemical modification with amino acid ionic liquids | |
| CN107034205B (en) | A kind of method that utilizes surfactant to prepare high esterification active lipase | |
| CN103308510A (en) | Detection method of transesterification activity of non-aqueous phase lipase | |
| Paroul et al. | Solvent-free geranyl oleate production by enzymatic esterification | |
| Li et al. | Conformation studies on Burkholderia cenocepacia lipase via resolution of racemic 1-phenylethanol in non-aqueous medium and its process optimization | |
| Zheng et al. | A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay | |
| Zhang et al. | Rapid determination of histamine level in seafood using read-out strips based on high-performance thin layer chromatography modified with self-visualization nanomaterials | |
| Sun et al. | A chiral ligand exchange CE system for monitoring inhibitory effect of kojic acid on tyrosinase | |
| CN101430279A (en) | Method for screening catalysis non-aqueous phase system transesterification enzyme by fluorospectrophotometry | |
| Alimagham et al. | Real-time bioprocess monitoring using a mid-infrared fibre-optic sensor | |
| Zhang et al. | Enzyme powered self-assembly of hydrogel biosensor for colorimetric detection of metabolites | |
| CN106814153A (en) | The liquid chromatography mass screening technique of the double target spot anticoagulating active materials based on fibrin ferment and clotting factor | |
| Uchiyama et al. | Simultaneous Determination of C1− C4 Carboxylic Acids and Aldehydes Using 2, 4-Dinitrophenylhydrazine-Impregnated Silica Gel and High-Performance Liquid Chromatography | |
| Liu et al. | Improved performance of Yarrowia lipolytica lipase-catalyzed kinetic resolution of (R, S)-2-octanol by an integrated strategy of interfacial activation, bioimprinting and immobilization | |
| Li et al. | Preparation and properties of rhizopus oryzae lipase immobilized using an adsorption-crosslinking method | |
| Nioi et al. | The centrifugal partition reactor, a novel intensified continuous reactor for liquid–liquid enzymatic reactions | |
| Paroul et al. | Production of geranyl propionate by enzymatic esterification of geraniol and propionic acid in solvent‐free system | |
| Cornelio et al. | Cathepsin D immobilized capillary reactors for on-flow screening assays | |
| Daneshjoo et al. | Imidazolium chloride‐based ionic liquid‐assisted improvement of lipase activity in organic solvents | |
| Liu et al. | High throughput enzyme inhibitor screening by functionalized magnetic carbonaceous microspheres and graphene oxide-based MALDI-TOF-MS | |
| Pais et al. | Sequential injection system for the enzymatic determination of ethanol in alcoholic beverages with in-line dilution | |
| Tang et al. | Rapid on‐line system for preliminary screening of lipase inhibitors from natural products by integrating capillary electrophoresis with immobilized enzyme microreactor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130918 |