CN1928116B - A rapid assay method for lipase synthase activity in non-aqueous phase - Google Patents

Abstract

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 uses lipase to catalyze the transesterification reaction between p-nitrophenol ester and alcohol in a non-aqueous phase system. After a certain period of reaction, a certain volume of reaction liquid is drawn and extracted 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 lipase synthetase activity determination and screening methods, the method of the present invention does not need gas chromatography, liquid chromatography, expensive fluorescent substrates and fluorescent spectrophotometers.

Description

A rapid assay method for lipase synthase activity in non-aqueous phase

technical field

The invention relates to a rapid assay method for lipase synthetase activity in a non-aqueous phase, which can be used in the screening of lipase-producing bacteria with high ester synthesis activity in the non-aqueous phase and rapid assay of the enzyme activity, belonging to the non-aqueous phase lipase catalysis technology field.

Background technique

As a triglyceride hydrolase, lipase has become an efficient catalyst for organic synthesis in the process of non-aqueous biocatalysis with the continuous deepening and development of non-aqueous phase enzymology. It can effectively catalyze esterification and transesterification The reaction has been successfully used in the production of many important compounds, such as aromatic esters, monoglycerides, chiral compounds, and biodiesel. There are many kinds of lipases with different catalytic properties, so the key to the catalytic process is to select the desired lipases purposefully.

The choice of enzyme depends on the enzyme activity assay method, so many different lipase enzyme activity assay methods have been formed for different purposes. Most of the lipase enzyme activity assay methods are based on their hydrolysis characteristics, that is, Hydrolytic activity of lipase. However, the problem is that the synthetic enzyme activity in the organic phase does not correspond to the hydrolytic enzyme activity in the aqueous phase (Wu, Jaaskelainen et al., Enzyme and Microbial Technology, 1996, 226), and the lipase obtained by screening according to the hydrolytic enzyme activity is not suitable for Esterification, transesterification and catalysis in non-aqueous phases. Many foreign researchers have found this problem in the process of research, and have taken some measures to solve this contradiction. Therefore, some researchers have developed assay methods for lipase synthase activity based on esterification and transesterification reactions. Most of these methods use GC, HPLC or even titration to measure the amount of acid or ester to determine enzyme activity. The disadvantages of these methods are: The problem is that when the sample size is large or used for screening work, it will require a lot of experimentation and time consumption. In response to the above shortcomings, Konarzycka-Bessler et al. (Konarzycka-Bessler and Bornscheuer, Angewandte Chemie-International Edition, 2003, 1418) developed a method for high-throughput screening of hydrolase synthase activity, which is similar to its principle Min Su Han et al. (Han, Jung et al., Journal of Organic Chemistry, 2004, 2583) also invented a method for measuring the transesterification activity of protease in the organic phase, both of which adopt the method of fluorescent color development. However, not all laboratories have fluorescence spectrophotometers, and fluorescent substrates are usually expensive and require high reaction solvents.

Contents of the invention

(1) The problem to be solved

The purpose of the present invention is to establish a rapid assay method for lipase synthetase activity in the non-aqueous phase, and the method is used for the screening of lipase-producing bacteria. The substrates used are p-nitrophenol ester and ethanol, which are cheap and easy to obtain. The ordinary spectrophotometric method is used for determination. The determination method is simple and easy, and most laboratory instruments can meet the requirements.

(2) Technical solution

In the present invention, lipase is used to catalyze the transesterification reaction between p-nitrophenol ester and alcohol in a non-aqueous phase system. After reacting for 5-30 minutes, absorb 50 μL of reaction solution and use 1ml of 0.1M NaOH solution in a cuvette Or extract from a multi-well microplate, measure the absorbance at 410nm with a colorimetric method to determine the amount of p-nitrophenol produced, and one lipase activity unit is defined as the amount of enzyme required to generate 1 μmol of p-nitrophenol. Reaction temperature and reaction time are different for different lipases. The present invention can also specifically change the carbon chain length of p-nitrophenol ester according to different substrates of lipase, and is suitable for the lipase used in the transesterification reaction of p-nitrophenol ester with ester chain length C ₄ -C ₁₆ Synthetic enzyme activity assay. It can be used to investigate the substrate specificity of known lipases in non-aqueous phase, and can be used to screen lipases with specific substrate specificity.

Reaction principle

The substrate p-nitrophenol ester used is: p-nitrophenol propionate, p-nitrophenol hexanoate, p-nitrophenol laurate or p-nitrophenol palmitate, and the substrate alcohol used is ethanol.

In the rapid assay method of lipase synthase activity in the non-aqueous phase of the present invention, the transesterification reaction conditions are: p-nitrophenol ester concentration is 10mM, alcohol concentration is 1M, reaction temperature is 30-60°C, reaction time is 5 -30 minutes, the shaker speed is 200 rpm, and the enzyme concentration is 0.01-0.3U.

The rapid determination method of lipase synthetase activity in the non-aqueous phase is used for the screening of lipase-producing bacteria with synthetic activity in the non-aqueous phase, and the lipase-producing bacteria are cultured for a certain period of time to prepare cell freeze-dried powder or fermented After the liquid freeze-dried powder is extracted in a cuvette or porous microplate, the enzyme activity is determined, and the target strain is screened out according to the enzyme activity. Use a 96-well microplate for extraction and determination of enzyme activity to improve the efficiency of determination and/or screening.

Using p-nitrophenol palmitate (pNPP) and ethanol as substrates, the commercial lipase L-PS-C from Brukholderia sp. and the whole cell lipase from Rhizopus chinenisis CCTCCM201021 (prepared in the laboratory) Do more detailed research.

Feasibility of the assay method

Reagents and lipase

A total of 5 commercial lipases and one self-prepared whole-cell lipase were used, which are:

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;

L-PP (lipase from Porcine Pancreas) Sigma,

L-Rh (lipase from Rhizopus chinenisis, whole cell freeze-dried powder) was prepared in the laboratory, and the preparation method was referred to (Xu, Wang et al., Journal of Molecular Catalysis B-Enzymatic, 2002(18), 29-37).

pNPP (p-nitrophenol palmitate) was purchased from Sigma Company, and other reagents were analytical or chromatographically pure.

The typical process of colorimetric determination of synthetic enzyme activity: Accurately weigh 189mg pNPP and dissolve it in 50mL after 4 Prepare a 10 mM solution in n-heptane from which moisture has been removed by molecular sieves. Add 2.5-10mg of each lipase into a 2mL stoppered plastic tube. Add 0.5ml of the above pNPP n-heptane solution, then add 30μL of 4 Molecular sieve to remove water from absolute ethanol (concentration about 1M). 200rmp, 30-60℃, react for 5-30 minutes, take 50μL of the reaction solution and extract it with 1mL 0.1M NaOH solution, measure the absorbance at 410nm, if the absorbance value is too high, dilute the reaction solution to a certain concentration before measuring. The instrument is Unico UNICOUV-2102 PC UV-Vis spectrophotometer, 1mL glass or plastic cuvette. The calculation of enzyme activity is calculated according to the standard curve drawn from the pNP (p-nitrophenol) standard concentration solution, and one enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol of pNP.

The method for measuring the activity of lipase synthase compared with this method is to detect the amount of ethyl palmitate by gas chromatography: take 150 μL of the above reaction solution, extract with 3mL 0.1M NaOH solution, mix well and then centrifuge, take the supernatant and add a certain amount of The concentration of 2-hexanol internal standard was detected by gas phase. The chromatographic conditions are initial temperature 150°C, rising to 220°C at 10°C/min, holding for 10min, inlet temperature 250°C, detector temperature 250°C. The gas chromatograph is Agilent 6820, the chromatographic column is AC20 (PEG20000), and the detector is FID.

The lipase synthase activity assay method compared with this method is the assay of lipase catalyzed synthesis of octanoic acid ethyl ester: octanoic acid and absolute ethanol are dissolved in n-heptane respectively, and the concentrations are 0.6M and 0.72M respectively. Take 0.5 mL of the reaction substrate respectively into plastic tubes with stoppers, add about 20 mg of lipase, shake and react at 40°C and 150 rpm for 30 min. Centrifuge, take 0.4mL supernatant, add 0.1mL internal standard 2-hexanol, mix well, and at the same time blank control, gas chromatography determination. The gas chromatography measurement conditions are: initial temperature 90°C, keep for 1min, raise temperature to 200°C at 10°C/min, keep for 5min, detector temperature 250°C, injector temperature 250°C. The gas chromatograph is Agilent 6820, the chromatographic column is AC20 (PEG20000), and the detector is FID. Under the assay conditions, the amount of enzyme that converts 1 μmol ethyl octanoate per minute is defined as 1 lipase synthase activity unit.

Optimum measuring range and measuring condition of measuring method of the present invention

Reaction temperature: Different lipases have different stability to temperature, and the temperature stability of enzymes for non-aqueous catalyzed reactions is much better than that for water-phase catalyzed reactions. This assay method is effective for most lipases at temperatures between 30°C and 60°C.

Speed: Since non-aqueous catalysis is mostly a solid-liquid two-phase system, and for a solid-liquid two-phase system, the mass transfer resistance of the substrate must exist, especially for immobilized enzymes. The first layer of mass transfer resistance comes from the solvent layer wrapped on the surface of the solid catalyst, also known as the external diffusion resistance. When the reaction speed of the enzyme is much smaller than the diffusion speed of the substrate, the out-diffusion resistance does not become a limiting factor, but when the reaction speed of the enzyme is fast, the out-diffusion resistance becomes a limiting factor. Taking lipase L-PS-C as an example, L-PS-C catalyzes the reaction faster. As the speed increases, the reaction speed increases significantly, indicating that the reaction is controlled by external diffusion resistance. When the speed reaches 200rmp or more, the diffusion resistance basically disappeared. The rotational speed of 200rmp can meet the requirement for this determination method.

Substrate concentration: The substrate concentration required for the determination of enzyme activity is very different for different enzymes. Taking lipase L-PS-C and L-Rh whole-cell lipase as examples, the synthesis is determined according to the above colorimetric method Typical process of enzyme activity Measure the reaction rate of the pNPP concentration in the range of 2.5-70mM, and use the Lineweaver-Burk method to obtain the Km value. Comprehensive consideration In order to make this method have better versatility for most lipases, the concentration of p-nitrophenol ester is 10mM as the reaction concentration, and the concentration of ethanol is 1M.

Enzyme concentration: Enzyme concentration is also crucial to the determination method. If the enzyme concentration is too low, the enzyme activity will not be detected, while if the enzyme concentration is too high, the measured enzyme activity will be inaccurate. Still take lipase L-PS-C with high enzyme activity and L-Rh whole cell lipase with relatively low enzyme activity as examples. According to the typical process of measuring synthetic enzyme activity by the above-mentioned colorimetric method, samples are taken regularly to determine the conversion curve under different enzyme concentration conditions, and enzymes of different masses are weighed and added to the reaction system to control the enzyme concentration in the reaction system. Draw a curve with enzyme concentration and reaction rate to investigate the optimum range of enzyme concentration. The results showed that the enzyme activity could be measured more accurately when the enzyme concentration was between 0.01-0.3U. 1U is the amount of lipase required to generate 1 μmol pNP.

In summary, the optimal assay conditions (i.e. transesterification conditions) of this assay method are as follows: substrate concentration p-nitrophenol ester 10mM, ethanol 1M; reaction temperature 30°C-60°C; reaction time 5-30 Minutes, shaker speed 200rmp; enzyme concentration 0.01-0.3U.

(3) Beneficial effect

The invention establishes a method suitable for rapid determination and screening of lipase synthetase activity in the non-aqueous phase, which can accurately measure the synthetase activity under the condition that the enzyme amount is small. The substrate used is easy to obtain, the determination method is simple, and the lipase synthetase activity can be rapidly determined. Six kinds of lipases randomly selected were detected by different synthetic enzyme activity assay methods, and compared with this assay method, the feasibility of this assay method was proved. A commercial enzyme L-PS-C and L-Rh whole-cell lipase was studied in detail using this assay method. While determining the optimal assay conditions, it also verified that this assay method can effectively assay lipase non-aqueous phase systems synthetase activity in .

Compared with the traditional method for measuring lipase activity, the invention highlights the catalytic properties of the lipase in the non-aqueous phase according to the phenomenon that the activity of the hydrolytic enzyme has no necessary connection with the synthesis activity in the non-aqueous phase. By using the present invention for direct screening of lipase-producing bacteria with high synthetase activity, lipase-producing bacteria with stronger purpose and more suitable for non-aqueous phase catalysis will be obtained. Compared with the existing methods for measuring and screening lipase synthetase activity in the world, the method of the present invention does not need gas chromatography, liquid chromatography, expensive fluorescent substrates and fluorescent spectrophotometers.

Description of drawings

The degree of correlation between Fig. 1 method of the present invention (method I) and method III

Method I: colorimetric assay of synthetase activity; Method III: assay of synthetase activity of lipase-catalyzed synthesis of ethyl octanoate.

Detailed ways

Embodiment 1 Validate 6 kinds of lipases with the method of the present invention

Select above-mentioned 6 kinds of lipases, verify with assay method described in the present invention, and provide convincing proof for the feasibility that this assay method is used for lipase non-aqueous phase synthetase activity assay, the results are shown in Table 1. Since the above reactions are not the results under the optimal enzyme activity assay conditions, the conversion rate is used to represent the catalytic synthesis activity of the enzyme. The result (method II in table 1) of gas chromatography detection ethyl palmitate and the result (method I in table 1) that adopt colorimetry to measure are 90% correlation degree, illustrate that assay method of the present invention is feasible and the detection method adopted in the present invention can effectively measure the lipase-catalyzed transesterification reaction between p-nitrophenol palmitate (pNPP) and ethanol. The result of colorimetric assay (method I in table 1) is compared with the synthetase activity assay result (method III in table 1) of lipase enzymatically synthesizing octanoic acid ethyl ester, six kinds of lipases use these two kinds of methods The synthetase activity correlation that measures obtains is more than 80%, and the reason that causes this result is that the lipase substrate specificity of different sources is different, thereby has caused measurement result difference to some extent, but generally speaking, the determination of the present invention The method can effectively measure the synthetase activity in the lipase non-aqueous phase.

Table 1

Lipase Method I (conversion rate/%) Method II (conversion rate/%) Method III (conversion rate/%)   L-GL-PS-CL-CRL-PPL-RhL-AY 074.52.40.818.10 074.32.10.7616.40 0.4161.61.31.958.40.18

Method I: Colorimetric Determination of Conversion Rate in 1 Hour

Method II: Detection of conversion rate of ethyl palmitate by gas chromatography

Method III: Conversion of lipase-catalyzed synthesis of ethyl octanoate

Embodiment 2 carries out L-PS-C lipase enzyme activity assay with the inventive method

分子筛除去水分的30μL无水乙醇，200rmp，50℃条件下反应5min，取50μL反应液用1mL 0.1M NaOH溶液萃取，于410nm处测定吸光度，测得酶活为140.1U/g。Weigh 2.5mg of L-PS-C lipase and place it in a 2mL plastic tube with stopper, add 0.5mL of 10mM pNPP n-heptane solution and pass through 4

Molecular sieve removed 30 μL of absolute ethanol, 200 rpm, 50 °C for 5 minutes, 50 μL of the reaction solution was extracted with 1 mL of 0.1M NaOH solution, and the absorbance was measured at 410 nm. The enzyme activity was 140.1 U/g.

Embodiment 3 Carry out L-Rh whole cell lipase enzyme activity assay with the method of the present invention

分子筛除去水分的30μL无水乙醇，200rmp，40℃条件下反应30min，取50μL反应液用1mL 0.1M NaOH溶液萃取，于410nm处测定吸光度，测得酶活为5.6U/g。Weigh 10 mg of L-Rh whole cell lipase and place it in a 2 mL stoppered plastic tube, add 0.5 mL of 10 mM pNPP n-heptane solution and pass through 4

Molecular sieve removed 30 μL of absolute ethanol, 200 rpm, 40 °C for 30 minutes, 50 μL of the reaction solution was extracted with 1 mL of 0.1M NaOH solution, and the absorbance was measured at 410 nm. The enzyme activity was 5.6 U/g.

Example 4 Screening of lipase-producing bacteria

For lipase-producing bacteria, it is necessary to screen out a strain with high synthetic activity in the non-aqueous phase, and it will be a huge workload to use gas-phase or liquid-phase detection. Select 30 strains from our strain selection process and prepare them into freeze-dried powder. The preparation method is referred to (Xu, Wang et al., Journal of Molecular Catalysis B-Enzymatic, 2002 (18), 29-37). Adopt above-mentioned lipase enzyme to catalyze the synthetase activity assay method of synthesizing ethyl octanoate to measure the synthetase activity of each bacterium, adopt the method of the present invention to measure synthetase activity simultaneously, the results are shown in accompanying drawing 1. The results show that the two assay methods have a high correlation, and the correlation is close to 83%, indicating that the spectrophotometric method can be used for the determination of the synthetase activity in the non-aqueous phase of the whole cell lipase, and the method can be used for the synthesis of highly active fat The primary screening of enzyme screening can greatly improve the screening efficiency and accuracy compared with lipase hydrolysis enzyme activity as primary screening means.

Example 5

A 96-well plate is used for the determination of the assay method to improve the assay efficiency. Use 8×12 type 96 microwell plates, weigh a certain amount of enzyme powder into the wells of An-Fn (n=1, 3, 5, 7, 9, 11), or dissolve soluble enzyme powder in buffer, press The amount of enzyme to be added was added to the enzyme solution, freeze-dried for later use, and 250 μL of 0.1M NaOH solution was added to the wells of Am-Fm (m=2, 4, 6, 8, 10, 12). Add 200 μL of pNPP n-heptane solution during the reaction to An-Fn in the typical process of the above-mentioned colorimetric method for the determination of synthetic enzyme activity, add 12 μL of absolute ethanol, and after a certain period of reaction, 8-channel pipette absorbs 20 μL of the reaction solution and adds it to the adjacent The absorbance was measured at 410nm in the wells with 0.1M NaOH solution.

Claims (3)

1. a kind of synthetase active assay method of lipase in nonaqueous phase, it is characterized in that utilize lipase to catalyze the transesterification reaction between p-nitrophenol palmitate and ethanol in nonaqueous phase system, transesterification The chemical reaction conditions are: the concentration of p-nitrophenol palmitate is 10mM, the concentration of ethanol is 1M, the reaction temperature is 30-60°C, the reaction time is 5-30 minutes, the shaking table speed is 200 rpm, and the enzyme concentration is 0.01-0.3U After the reaction, draw 50 μl of the reaction solution and extract it in a cuvette or porous microplate with 1ml of 0.1M NaOH solution, and measure the absorbance at 410nm to determine the generation of p-nitrophenol by colorimetry. The enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol of p-nitrophenol. 2. the synthetase activity rapid assay method of lipase in the non-aqueous phase according to claim 1 is characterized in that being used for the screening of the lipase producing bacterium with synthetic activity in the non-aqueous phase, the lipase producing bacterium is cultivated After a certain period of time, prepare the freeze-dried powder of cells or the freeze-dried powder of fermentation broth, extract and measure the enzyme activity in a cuvette or a porous microporous plate, and screen out the target strain according to the enzyme activity. 3. the synthetase active assay method of lipase in non-aqueous phase according to claim 2 is characterized in that used porous microplate is 96 well microplates and extracts, measures enzymatic activity, to improve mensuration and/or screening efficiency.

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