CN109971735A - A kind of preparation method of cholesterol esterase - Google Patents

Abstract

The invention discloses a preparation method of cholesterol esterase, which belongs to the technical field of enzyme engineering. The cholesterol esterase in the present invention is prepared by using Burkholderia cepacia ZWS15 with deposit number CCTCC NO: M2017661. The enzyme has good acid-base stability and temperature stability, and the residual enzyme activity remains at 70% for 2 hours at 70 °C, and can withstand 5% high concentration of surfactants and 50% high concentration of organic solvents . The cholesterol esterase activity of cholesterol esterase prepared by the invention to cholesterol benzoate, cholesterol oleate, cholesterol linoleate and cholesterol octanoate is as high as 415U/L, 1038U/L, 996U/L and 294U respectively. /L. And it can degrade triacylglyceride and p-nitrophenyl ester substrates.

Description

A kind of preparation method of cholesterol esterase

technical field

The invention relates to a preparation method of cholesterol esterase, belonging to the technical field of enzyme engineering.

Background technique

Cholesterol esterase (EC 3.1.1.13), mainly acts on cholesterol esters in water-soluble media to hydrolyze them to cholesterol and fatty acids. It widely exists in mammalian tissues and microorganisms. Compared with the defects of low yield, complicated purification and unsatisfactory properties of mammalian-derived enzymes, the production cost of microbial-derived cholesterol esterases is low, and it has more advantages than animal-derived cholesterol esterases. A wide range of pH and temperature shows higher stability and substrate specificity, so it has gradually become a research hotspot.

Cholesterol esterase is usually used as a detection enzyme to detect the total cholesterol content in blood because it is related to lipid metabolism and cholesterol absorption in the human body; in addition, cholesterol esterase is also used in food, sewage treatment, pulp and paper industry, leather fabric degreasing and other fields. Broad application prospects. The enzymes used in the latter occasions require broad substrate specificity, high temperature tolerance and organic solvent tolerance in most cases. For example, the processing temperature in the pulp and paper industry is generally 60 ℃ ~ 80 ℃ °C.

Cholesterol esterase is a type of esterase, which belongs to the ester hydrolase family of α/β hydrolase (EC 3.1.1.X), which also includes lipase (EC 3.1.1.3). The biggest difference between cholesterol esterase and lipase lies in the different types of hydrolysis substrates. Lipase mainly acts on ester bonds formed by long-chain fatty acids, cholesterol esterase mainly acts on ester bonds formed by short-chain fatty acids, and a small number of cholesterol esterases act on ester bonds formed by short-chain fatty acids. It is also possible to hydrolyze lipase substrates.

In 1980, Smirnov et al. proposed that cholesterol esterases can be divided into two categories. One type specifically hydrolyzes cholesterol esters. For example, cholesterol esterase derived from Pseudomonas fluorescens has a significant preference for long-chain cholesterol esters and is derived from onion. Cholesterol esterase of Burkholderia ST-200 is highly specific for aromatic lipid substrates; another type has lipolytic ability (that is, can hydrolyze lipase substrates) while degrading cholesterol ester substrates For example, cholesterol esterase derived from Streptomyces X9 can also degrade triolein, and Ophiostoma piceae can also effectively degrade tributyrin.

In addition to a wide variety of hydrolysis substrates, studies have shown that some cholesterol esterases are also significantly tolerant to organic solvents and surfactants. Takeda et al. obtained cholesterol esterase from Burkholderia cepacia ST-200, which has good temperature tolerance (4℃～65℃), and the enzyme activity can be increased to 7.2 times in the presence of 20% methanol ; Sintawee et al. found that cholesterol esterase from Pseudomonas aeruginosa could still retain 70% of the enzyme activity after being placed at 37 ° C for 28 days; Hanna purified a cholesterol esterase from Melanocarpus albomyces, which was added to 5% Triton X -100 in the presence of. However, most of the current research only focuses on the tolerance of cholesterol esterase to hydrophilic organic solvents, while the tolerance of cholesterol esterase to hydrophobic organic solvents has been reported, and the tolerance of organic solvent concentrations is low , the temperature tolerance is still not enough to meet the needs of the industry.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides a preparation method of cholesterol esterase resistant to high temperature and organic solvent. The cholesterol esterase prepared by the method of the present invention has many kinds of hydrolysis substrates, and is resistant to high temperature, hydrophilic It has good resistance to organic and hydrophobic organic solvents and surfactants, and is suitable for food, sewage treatment, pulp and paper industry, fabric degreasing and other fields, providing reference and reference for its further industrial application.

The first object of the present invention is to provide a method for preparing a high temperature-resistant, organic solvent-resistant cholesterol esterase, which is prepared by using Burkholderia cepacia ZWS15 with a deposit number of CCTCC NO: M2017661 .

In one embodiment of the present invention, the preparation is to induce Burkholderia cepacia ZWS15 for fermentative production in a fermentation medium containing Triton X-100 using cholesterol esters as inducers.

In one embodiment of the present invention, the cholesterol esters are: cholesterol benzoate, cholesterol oleate, cholesterol linoleate or cholesterol octanoate.

In one embodiment of the present invention, the concentration of Triton X-100 is 0.34% (v/v).

In an embodiment of the present invention, the fermentation temperature is 25-30°C.

In one embodiment of the present invention, the formula of the fermentation medium is: per 1 L of distilled water, add peptone 10 g, yeast powder 5 g, K ₂ HPO ₄ 2 g, KCl 0.5 g, NaNO ₃ 2 g, TritonX-100 3.4 mL , MgSO4 100mM, glucose 0.1g, cholesterol oleate 2g, pH 7.0.

In one embodiment of the present invention, the method further comprises: precipitating the fermentation broth obtained by using Burkholderia cepacia ZWS15 for fermentative production, using ammonium sulfate to precipitate, dissolving the precipitate and purifying it with a DEAE ion exchange column , to obtain cholesterol esterase.

The second object of the present invention is to provide a kit or a sewage treatment agent for detecting serum total cholesterol, which contains the cholesterol esterase prepared in claim 1.

The third object of the present invention is to provide a method for preparing a kit for detecting serum total cholesterol, preparing a sewage treatment agent, making pulp and paper or degreasing leather fabrics, using the method to prepare cholesterol esterase, and then using the obtained cholesterol ester Enzyme preparation kits or sewage treatment agents for the detection of serum total cholesterol.

The fourth object of the present invention is to provide a method for degrading triacylglycerides and p-nitrophenyl esters, using the method to prepare cholesterol esterase and degrading the obtained cholesterol esterase.

In one embodiment of the present invention, the triacylglycerides are olive oil, triolein or tributyrin; the p-nitrophenyl esters are p-nitrophenyl palmitate or p-nitrophenyl acetate.

Beneficial effects of the present invention:

(1) The relative enzyme activity of the obtained cholesterol esterase of the present invention can reach more than 85% at pH 5.5-9.0; the residual enzyme activity remains 70% in 2 hours at 70°C, and has good acid-base stability and temperature stability.

(2) The cholesterol esterase obtained in the present invention can withstand 5% high concentration of surfactant, and the relative enzyme activity reaches 1.2 times in 5% Triton X-100; it has strong resistance to organic solvents , in the environment of 50% high-concentration organic solvent, the enzymatic hydrolysis ability of the enzyme is still not significantly weakened. In the presence of 50% high-concentration ethanol and cyclohexane, the relative enzyme activity can reach 1.25 and 1.21 times.

(3) The cholesterol esterase activity of cholesterol esterase of the present invention to different cholesterol esters: cholesterol benzoate, cholesterol oleate, cholesterol linoleate and cholesterol octanoate are as high as 415U/L and 1038U, respectively. /L, 996U/L, 294U/L.

(4) The obtained cholesterol esterase of the present invention has strong lipolytic ability and can degrade triacylglycerides (olive oil, triolein, tributyrin) and p-nitrophenyl esters (p-nitrophenyl esters). Nitrophenyl palmitate, p-nitrophenyl acetate) substrates, the deesterification activities were as high as 933U/L, 467U/L, 267U/L, 203165U/L, and 160U/L, respectively.

biomaterials

Burkholderia cepacia ZWS15 provided by the present invention, taxonomically named Burkholderia cepacia ZWS15 Burkholderia cepacia ZWS15, has been deposited in the China Center for Type Culture Collection on November 6, 2017 , the deposit number is CCTCC NO: M2017661, and the deposit address is Wuhan University, Wuhan, China.

Description of drawings

Figure 1: Results of cholesterol esterase production by Burkholderia cepacia ZWS15 in a 7.5L tank;

Figure 2: SDS-PAGE chart of purified cholesterol esterase; M: marker; 1: fermentation crude enzyme liquid; 2: enzyme sample after 70% ammonium sulfate precipitation; 3: enzyme sample after dialysis for 24 hours; 4: DEAE Purified enzyme samples.

Figure 3: Time-of-flight mass spectrometry results of the target band in SDS-PAGE; abscissa: mass-to-charge ratio of ions (representing the molecular size of the cleaved peptide); ordinate: ion current intensity of mass spectrometry (representing the peptide in the total sample) content).

Figure 4: Cholesterol esterase pH optimum.

Figure 5: pH stability of cholesterol esterase.

Figure 6: Optimum temperature for cholesterol esterase.

Figure 7: Cholesterol esterase temperature stability.

Detailed ways

(1) Cholesterol esterase enzyme activity

Definition of cholesterol esterase enzyme activity: the amount of enzyme that hydrolyzes cholesterol esters to produce 1 μmol of free cholesterol in 1 min under certain temperature and pH conditions.

In the present invention, the substrate for measuring the enzymatic activity of cholesterol esterase is: cholesterol ester (including cholesterol benzoate, cholesterol oleate, cholesterol linoleate, cholesterol octanoate, etc.).

Reaction solution A: 0.2mol/L potassium phosphate buffer 1.5mL, 0.6mmol/L cholesterol ester (specifically cholesterol benzoate, cholesterol oleate, cholesterol linoleate, cholesterol octanoate, etc.) 1mL, 0.087 mol/L 4-aminoantipyrine 0.05mL, 0.637mol/L phenol 0.1mL, 150U/mL horseradish peroxidase 0.1mL.

Reaction solution B: 0.1 mL of 300 U/mL cholesterol oxidase.

Cholesterol esterase enzyme activity assay method: take 2.75 mL of reaction solution A and incubate at 37 °C for 5 minutes, add 0.1 mL of reaction solution B, continue to incubate at 37 °C for 2 minutes, add 0.1 mL of the enzyme solution to be tested and invert After mixing, measure the change value within 3-4 minutes at 500nm, draw a curve and calculate the linear part of the OD change value per minute. The enzyme activity calculation formula is:

Vt: the total volume of the reaction system, 2.95mL;

Vs: sample volume, 0.1mL;

df: dilution factor;

(2) Lipolytic activity (lipase activity)

Definition of lipolytic activity: 1 mL of liquid enzyme, under certain temperature and pH conditions, hydrolyzes the substrate to produce 1 μmol of titratable fatty acids in 1 min, which is one unit of enzyme activity.

The substrates used for measuring lipolytic activity in the present invention are: triacylglycerides (olive oil, triolein, tributyrin), p-nitrophenyl ester (p-nitrophenyl palmitate, p- nitroacetate).

Example 1: Enzyme production by fermentation of Burkholderia cepacia ZWS15

(1) Slant culture: Inoculate Burkholderia cepacia ZWS15 with deposit number CCTCC NO: M2017661 on LB solid medium, and culture at 37° C. for 20-28 hours statically.

LB solid medium: add 5 g of yeast powder, 10 g of peptone, 10 g of NaCl, and 20 g of agar powder to each 1 L of distilled water.

(2) Seed cultivation: pick a single colony of the strains cultivated in step (1) under aseptic conditions and inoculate it into 50 mL of LB liquid medium, and shake for 9-13 hours in a shaker at 37°C and a rotating speed of 200 rpm.

LB liquid medium: add 5g of yeast powder, 10g of peptone and 10g of NaCl per 1L of distilled water.

(3) Fermentation culture: take the activated seed liquid in step (2), inoculate it into the fermentation medium according to the inoculum amount of 3%-10%, add cholesterol oleate as an inducer, induce the production of cholesterol esterase, and at the same time Triton X-100 was added to promote extracellular secretion of this enzyme. Shake culture for 20-28h in a shaker at 28°C with a rotation speed of 200rpm.

Fermentation medium: per 1L of distilled water, add peptone 10g, yeast powder 5g, K ₂ HPO ₄ 2g, KCl 0.5g, NaNO ₃ 2g, TritonX-100 3.4mL, MgSO4 100mM, glucose 0.1g, cholesterol oleate 2g, pH 7.0.

(4) Fermentation tank culture: take the activated seed liquid in step (2), inoculate it into the fermentation medium according to the inoculum amount of 3%-10%, and cultivate on a 7.5L fermenter, and the fermentation period is 40-44h. The results of cholesterol esterase production by Burkholderia cepacia ZWS15 in the 7.5L tank are shown in Figure 1. The highest yield of cholesterol esterase produced in the 7.5L fermentor with the enzyme activity of cholesterol oleate substrate is 1009U/L.

The fermentation medium is the same as above, and the formula of the feed medium is: add 20 g of glucose per 1 L of distilled water.

(5) Collection of cholesterol esterase: the fermentation broth obtained in step (3) was centrifuged at 8000rpm for 5-10min at 4°C, and the supernatant was taken as crude enzyme solution.

Example 2: Purification and mass spectrometry identification of Burkholderia cepacia ZWS15 cholesterol esterase

(1) after the fermentation crude enzyme liquid obtained in Example 1 was precipitated with ammonium sulfate, at 4°C, centrifuged at 10000rpm for 25min, and the precipitate was dissolved using the Tris-HCl buffer of pH 7.5, 50mmol/L, and the same The solution was dialyzed.

(2) after equilibrating the DEAE ion exchange column with the Tris-HCl buffer of pH 7.5, 50mmol/L, inject the dialyzed sample, and rinse the chromatography column with the Tris-HCl buffer of pH 7.5, 50mmol/L, Remove the impurity proteins that are not attached to the column, and finally use the Tris-HCl buffer containing 1mol/L NaCl, pH 7.5, 50mmol/L at a flow rate of 1mL/min to linearly elute the target protein bound to the column from the layer. eluted from the column, and the peak detected by UV was collected.

(3) The collected eluate was detected by SDS polyacrylamide gel electrophoresis containing 5% separating gel and 12% stacking gel, and it was found that the samples eluted when the NaCl concentration was 1 mol/L, were eluted in SDS - PAGE showed a single band with a molecular weight of about 37kDa (see Figure 2 lane 4), the single band on the SDS-PAGE of lane 4 in Figure 2 was cut for time-of-flight mass spectrometry (MALDI-TOF) identification (see Figure 2) 3). The mass spectrum peaks are the results obtained by time-of-flight mass spectrometry of the peptides formed by random digestion of the target protein by trypsin. For matching, the enzyme with high matching degree indicates high homology with the target protein. From the analysis of the mass spectrometry peaks and the system comparison results, it can be seen that the four peptides with molecular masses of 1206.6208, 1424.7186, 1706.8469 and 2138.1007 obtained from the decomposition of the target protein are similar to those in the lipase derived from Pseudomonas KWI-56, 1259.6136, 1423.7113, 1705.8396, 2137.0934 have a high match, and also have a high match with 1259.6136, 1423.7113, 1705.8396, 2137.0934 in the cholesterol esterase derived from Burkholderia cepacia. This enzyme is presumed to be cholesterol esterase.

Therefore, the sample eluted with the Tris-HCl buffer containing 1 mol/L NaCl and 50 mmol/L at pH 7.5 through DEAE ion exchange is the pure enzyme solution of Burkholderia cepacia ZWS15 cholesterol esterase. The liquid can be used for subsequent experiments.

Example 3: Cholesterol Esterase pH Optimum and pH Stability

(1) Determination of optimum pH for cholesterol esterase

Take 1.5mL 0.2mol/L sodium acetate buffer (pH 3.0～5.5), 0.2mol/L potassium phosphate buffer (pH5.5～7.5), 0.2mol/L Tris-HCl buffer (pH 7.5～7.5) 9.0), 0.2mol/L Na ₂ CO ₃ -NaHCO ₃ buffer solution (pH9.0～11.0), 0.2mol/L NaOH-NaCl buffer solution (pH 11.0～12.0), add the obtained in Example 2 Pure enzyme solution 0.1mL. 1 mL of 0.6 mmol/L cholesterol oleate was added as the substrate, and the activity of cholesterol esterase to degrade the substrate was measured at 37°C, and the final result was expressed as the percentage of the measured value to the highest enzyme activity. The relative enzyme activity changes measured are shown in Figure 4. The optimum reaction pH of the enzyme is between pH 7.5 and 9.0.

(2) pH stability of cholesterol esterase

Take 1.5 mL of the buffer in step (1), add 0.1 mL of the pure enzyme solution obtained in Example 2, incubate at 25°C for 16 hours, add 1 mL of 0.6 mmol/L cholesterol oleate as a substrate, and measure cholesterol respectively The activity of the esterase to degrade the substrate, the highest cholesterol esterase activity measured was the control (100%), and the enzyme activities in different pH systems are shown in Figure 5 (Table 1). Experiments show that the relative enzyme activity of the cholesterol esterase can reach more than 85% at pH 5.5-9.0.

Table 1 pH stability of cholesterol esterase

Example 4: Cholesterol esterase optimum temperature and temperature stability

(1) The optimum temperature for cholesterol esterase action

Take 1.5mL of 0.2mol/L phosphate buffer (pH 7.0), add 0.1mL of the pure enzyme solution obtained in Example 2, add 1mL of 0.6mmol/L cholesterol oleate as a substrate, and set different temperatures to carry out the enzyme. The final result was expressed as the percentage of the measured value to the highest enzyme activity. The results showed that the optimal reaction temperature of the enzyme was 40°C under the condition of pH 7.0 (as shown in Figure 6).

(2) Temperature stability of cholesterol esterase

Take 1.5mL of 0.2mol/L phosphate buffer (pH 7.0), add 0.1mL of the pure enzyme solution obtained in Example 2, place in a water bath at different temperatures for 2 hours, add 1mL of 0.6mmol/L cholesterol oleate Measure the residual enzyme activity, as shown in Table 2, at 20°C-70°C, the relative enzyme activity can reach more than 70%.

Table 2 Temperature stability of cholesterol esterase

Example 5: Effect of Surfactant on Cholesterol Esterase Activity

Get the pure enzyme liquid 0.1mL obtained in embodiment 2, and different kinds of surfactants (Triton x-100, Tween 20, Tween 40, Tween 60 and Tween 80), respectively in different concentrations (0 , 0.05%, 0.1%, 0.5%, 1%, 5%) (v/v), mixed and placed at 37 °C for 1 hour, and 1 mL of 0.6 mmol/L cholesterol oleate was added as the substrate. The results are shown in Table 3 shown. This indicates that cholesterol esterase derived from Burkholderia cepacia ZWS15 is more resistant to different surfactants. In a high concentration environment of 5%, compared with the control group, triton X-100, Tween 60 and Tween 80 still promotes the enzymatic hydrolysis ability of cholesterol esterase.

Table 3 Effects of surfactants on cholesterol esterase activity

Example 6: Influence of hydrophilic organic solvent on cholesterol esterase enzyme activity

Take 0.1 mL of the pure enzyme solution obtained in Example 2, mix it with 30% (v/v) different kinds of hydrophilic organic solvents (dimethyl sulfoxide, methanol, ethanol, acetone, isopropanol), add The pure enzyme solution of 30% (v/v) water was used as a control, and placed in a shaker at 37°C with a rotating speed of 200rpm for 1 hour, 12 hours and 24 hours, respectively, and added 1mL of 0.6mmol/L cholesterol oleate. The residual enzyme activity after mixing was determined, and the results are shown in Table 4. It indicated that among the five hydrophilic organic solvents, cholesterol esterase still showed higher enzymatic hydrolysis ability than the control group after being mixed with DMSO, methanol and ethanol for 24 hours.

Table 4 Effects of hydrophilic organic solvents on the stability of cholesterol esterase

Example 7: The effect of hydrophobic organic solvent on the enzymatic activity of cholesterol esterase

Take 0.1 mL of the pure enzyme solution obtained in Example 2, and 30% (v/v) different kinds of hydrophobic organic agents (ethyl acetate, petroleum ether, butanol, chloroform, benzene, toluene, p-xylene, 1 , 3,5-trimethylbenzene, cyclohexane, n-hexane, n-octane) were mixed, and the pure enzyme solution added with 30% (v/v) water was used as a control, and placed in a shaker at 37 ° C and a rotating speed of 200 rpm. 1 hour, 12 hours and 24 hours of shaking culture respectively, adding 1 mL of 0.6 mmol/L cholesterol oleate to measure the residual enzyme activity after mixing, the results are shown in Table 5. It shows that among the 11 kinds of hydrophobic organic solvents, cholesterol esterase still has higher enzymatic hydrolysis ability than the control group after being mixed with petroleum ether, benzene, toluene, cyclohexane, n-hexane and n-octane for 24 hours.

Table 5 Effects of hydrophobic organic solvents on the stability of cholesterol esterase

Example 8: Effects of different concentrations of organic solvents on the enzymatic activity of cholesterol esterase

3 kinds of hydrophilic organic solvents (DMSO, methanol, ethanol) that are more resistant to cholesterol esterase are selected from Example 6, and 6 kinds of hydrophobic organic solvents (petroleum) that are more resistant to cholesterol esterase are selected from Example 7 ether, benzene, toluene, cyclohexane, n-hexane and n-octane), mix it with 0.1 mL of the cholesterol esterase pure enzyme solution obtained in Example 2 at different ratios (0, 15%, 30%, 50% , v/v), incubate for 1 hour at 37°C in a shaker with a rotating speed of 200 rpm, using cholesterol oleate as a substrate to add (0, 15%, 30%, 50%, v/v) water The pure enzyme solution was used as a control, and the residual enzyme activity after mixing was determined. The results are shown in Table 6. It shows that the cholesterol esterase derived from Burkholderia cepacia ZWS15 has strong tolerance to organic solvents, and the enzymatic hydrolysis ability of the enzyme is still not significantly weakened in the environment of 50% high-concentration organic solvent.

Table 6 Effects of different concentrations of organic solvents on cholesterol esterase activity

Example 9: Substrate specificity of cholesterol esterase activity

Take 1.5mL of 0.2mol/L potassium phosphate buffer, add 1mL of 0.6mmol/L different cholesterol esters (cholesteryl benzoate, cholesterol oleate, cholesterol linoleate, cholesterol octanoate, etc.) It was mixed with 0.1 mL of the pure cholesterol esterase solution obtained in Example 2, and the enzymatic activities of cholesterol esterase on different substrates were measured.

Table 7 Substrate specificity of cholesterol esterase activity

Example 10: Determination of lipolytic activity with triacylglycerides as substrates

(1) When olive oil or tributyrin is used as the substrate, the lipolytic activity is calculated according to the following formula:

V1: The volume of sodium hydroxide standard solution consumed when titrating the sample, the unit is milliliter (mL);

V2: the volume of sodium hydroxide standard solution consumed when titrating blank, the unit is milliliter (mL);

c: concentration of sodium hydroxide standard solution, the unit is mole per liter (mol/L);

50: 1.00mL of 0.05mol/L sodium hydroxide solution is equivalent to 50μmol of fatty acid;

n1: the dilution factor of the sample;

0.05: Conversion factor for the concentration of sodium hydroxide standard solution;

t: reaction time 15min

(2) Prepare polyvinyl alcohol (PVA) 40g/L, heat in a boiling water bath, stir until all dissolved, filter for later use. Measure 150 mL of the above filtrate, add 50 mL of triacylglycerides (olive oil, tributyrin or tributyrin), and treat with a high-speed homogenizer for 6 min to obtain a milky white PVA emulsion. The solution is used now. Phosphate buffer solution (pH=7.5): Weigh 1.96 g of potassium dihydrogen phosphate and 39.62 g of disodium hydrogen phosphate dodecahydrate respectively, dissolve in water and dilute to 500 mL, and adjust the pH to 7.5±0.05. Sodium hydroxide standard solution 0.05mol/L, when using, dilute accurately 10 times.

(3) The reaction system contains 4.00 mL of substrate solution and 5.00 mL of phosphate buffer. Add 15.00 mL of 95% ethanol in advance to the blank, preheat it in a 40°C water bath for 5 minutes, then add 1.00 mL of the enzyme solution to be tested, and mix the reaction immediately. After 15 minutes, add 15.0 mL of 95% ethanol to the sample solution immediately to terminate the reaction, add two drops of phenolphthalein indicator solution to each of the blank and sample solution, and titrate with sodium hydroxide standard solution until it is reddish and does not fade for 30s as the titration end point. , record the volume of sodium hydroxide standard solution consumed.

Example 11: Determination of lipolytic activity with p-nitrophenyl ester as substrate

(1) When using p-nitrophenyl palmitate as a substrate, the lipolytic activity is calculated according to the following formula:

ΔODtest, ΔODblank: absorbance values of sample and blank at 410 nm;

K: the slope of the standard curve of p-nitrophenol;

Co: the slope of the standard curve of p-nitrophenol;

N: dilution ratio of enzyme solution;

V1: the volume of the reaction solution, mL;

V2: volume of enzyme solution, mL;

t: reaction time, min.

(2) This method needs to draw the standard curve of p-nitrophenol in advance: Weigh 0.08346g of p-nitrophenol, dissolve it with a small amount of 95% ethanol, and then dilute to 100 mL with water, and the concentration is 6 mmol/L. Different amounts of p-nitrophenol solution and 50 mmol/L Tris-HCl (pH 7.0) buffer were added, and the total volume was 2 mL. Then add 0.5 mL of 10% trichloroacetic acid and 0.5 mL of ₁₀ % _Na2CO3 solution to each tube for a total volume of 3 mL. The light absorption value was measured at 410 nm, and according to the results, the absorption value was taken as the abscissa and the p-nitrophenol content as the ordinate to draw a standard curve.

(3) Weigh 90 mg of p-nitrophenyl ester (p-nitrophenyl palmitate or p-nitrophenyl acetate) and dissolve it in 30 mL of isopropanol, configure 50 mmol/L Tris-HCl (PH 7.0), and in the reaction system Add 0.1mL of substrate and 1.8mL of Tris-HCl, after 37 ℃ water bath for 5min, add 0.1mL of enzyme solution, add 0.5ml of 10% trichloroacetic acid solution for 15min to stop the reaction, then continue to react in water bath for 10min, and then add 0.5 ml 10% Na ₂ CO ₃ solution developed color, and the final reaction solution was measured at 410 nm for the amount of p-nitrophenol produced.

Table 8 Determination of lipolytic ability of cholesterol esterase

Example 12: Application of Burkholderia cepacia ZWS15 cholesterol esterase in the determination of total cholesterol content in blood

The cholesterol esterase pure enzyme solution obtained in Example 2 was combined with cholesterol oxidase to measure the total cholesterol content in blood. Cholesterol esters in serum can be hydrolyzed into free cholesterol and free fatty acids by cholesterol esterase, and cholesterol is oxidized by cholesterol oxidase to generate cholestatrione and hydrogen peroxide. The total cholesterol content in serum can be determined finally, and the detection principle is as follows:

Example 13: Application of Burkholderia cepacia ZWS15 cholesterol esterase in pulp and paper making

The crude cholesterol esterase solution obtained in Example 1 was mixed with the pulp solution at a concentration of 1%, reacted at 50-70° C., pH 7.0, and 200 rpm for 2 hours, and mixed with 0.1% Triton X-100 to promote Mixing of enzyme solution and pulp solution. After the reaction was complete, the suspension was rinsed with deionized water. The paper is homogenized before using the pulp to prepare the paper. The properties of the prepared paper can be verified by measuring its ISO brightness, light scattering coefficient, draw index, tear index and contact angle with water.

Example 14: Application of Burkholderia cepacia ZWS15 cholesterol esterase to fabric degreasing

The polyester fabric was washed at 37°C with detergent and extracted with dichloromethane to remove oligomers. The treated polyester fabric was treated with the crude cholesterol esterase enzyme solution obtained in Example 1 at 37° C., pH 7.0, and 200 rpm for 1 hour, 2 hours and 24 hours, and the ratio of fabric to enzyme solution was 1:20. After the reaction, the treated fabric was rinsed with deionized water, and the performance of the treated fabric was verified by measuring the contact angle and the penetration time of water.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (10)

1. a preparation method of the cholesterol esterase of high temperature resistance, organic solvent resistance, is characterized in that, utilizes the Burkholderia cepacia (Burkholderia cepacia) ZWS15 that deposit number is CCTCC NO:M2017661 to prepare and produce. 2. method according to claim 1, is characterized in that, described preparation is to use cholesterol esters as inducer, in the fermentation medium containing Triton X-100, induce Burkholderia cepacia ZWS15 to carry out Fermentation production. 3. The method according to claim 2, wherein the cholesterol esters are: cholesterol benzoate, cholesterol oleate, cholesterol linoleate or cholesterol octanoate. The method according to claim 2, wherein the concentration of the Triton X-100 is 0.30-0.40% (v/v). 5 . The method according to claim 2 , wherein the fermentation temperature is 25-30° C. 6 . 6. according to the arbitrary described method of claim 1-5, it is characterized in that, described method also comprises: will utilize Burkholderia cepacia ZWS15 to carry out the fermentation liquid that fermented production obtains, use ammonium sulfate precipitation, will precipitation After dissolving, it was purified by DEAE ion exchange column to obtain cholesterol esterase. 7 . A kit for detecting serum total cholesterol or a sewage treatment agent, characterized in that it contains the cholesterol esterase prepared in claim 1 . 8 . 8. a method for preparing a test kit for detecting serum total cholesterol, preparing a sewage treatment agent, pulping paper or leather fabric degreasing, characterized in that, utilizing the method described in claim 1 to prepare cholesterol esterase, then with the cholesterol ester obtained Enzyme preparation kits or sewage treatment agents for the detection of serum total cholesterol. 9 . A method for degrading triacylglycerides and p-nitrophenyl esters, characterized in that, the method according to claim 1 is used to prepare cholesterol esterase, and the obtained cholesterol esterase is used for degradation. 10. The method according to claim 9, wherein the triacylglycerides are olive oil, triolein or tributyrin; the p-nitrophenyl esters are p-nitro Phenyl palmitate or p-nitrophenyl acetate.