CN108018273B

CN108018273B - Long-chain unsaturated fatty acid specific lipase mutant and application thereof

Info

Publication number: CN108018273B
Application number: CN201810097101.3A
Authority: CN
Inventors: 喻晓蔚; 徐岩
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-01-31
Filing date: 2018-01-31
Publication date: 2020-05-08
Anticipated expiration: 2038-01-31
Also published as: CN108018273A

Abstract

The invention discloses a long-chain unsaturated fatty acid-specific lipase mutant and an application thereof, belonging to the technical field of genetic engineering. According to the structural analysis of Rhizopus huai lipase (RCL), the invention carries out site-directed mutation to obtain a mutant enzyme with specific fatty acid change. Comparing the ability of wild-type lipase and mutant enzyme to catalyze the hydrolysis of soybean oil, the mutant enzyme HQL whose hydrolysis rate is much higher than that of wild-type lipase is obtained, and the hydrolysis rate reaches about 98%.

Description

Long-chain unsaturated fatty acid specific lipase mutant and application thereof

Technical Field

The invention relates to a long-chain unsaturated fatty acid specific lipase mutant and application thereof, belonging to the technical field of genetic engineering.

Background

Fatty acid is the most basic in the oil chemical industry and is one of the most widely used raw materials. Hydrolysis of natural oils and fats is one of the important ways to obtain free fatty acids. The production technology of fatty acid mainly comprises saponification, hydrolysis, steam cracking and enzymatic hydrolysis, wherein the enzymatic hydrolysis has the advantages of mild reaction conditions, difficult oxidation of unsaturated fatty acid and few byproducts, and thus becomes a research hotspot of the fatty acid preparation method. Fatty acids of different types, different carbon chain lengths and different proportions have different functions. The short chain fatty acid can be used as feed additive for promoting animal growth. The medium-chain fatty acid is widely applied to the medical, health-care edible oil and breeding industries. Long-chain saturated fatty acids such as palmitic acid and stearic acid are mainly used for producing fatty acid salts as emulsifiers, and are widely contained in animal oils such as pig, cattle and sheep, and the sum of the mass fractions of various saturated fatty acids is usually over 60%. The long chain unsaturated fatty acid has wide physiological activity in metabolism, and can be used for health promotion. The content of unsaturated fatty acid in vegetable oil is high, such as 84.34%, 77.70% and 81.43% in soybean oil, rapeseed oil and camellia oil.

Lipase (EC 3.1.1.3) is a kind of triglyceride hydrolase, which catalyzes hydrolysis or synthesis of ester bond at oil-water interface, and is an important industrial enzyme for processing and manufacturing green oil. Vegetable oil is an important source for preparing long-chain fatty acid, but most lipases have low catalytic efficiency on unsaturated fatty acid or tend to hydrolyze medium-chain fatty acid, so that the raw material utilization rate is reduced when the lipase is applied to hydrolysis of vegetable oil with higher content of long-chain unsaturated fatty acid. For example, Alves et al have shown that the hydrolysis rate of soybean oil catalyzed by single commercial enzymes (Novozym435, Lipozyme TL-IM and Lipozyme RM-IM) is lower than 50%, and that the combination of lipases with different substrate specificities (Lipozyme RM-IM and Novozym435) increases the hydrolysis rate to more than 80% (RSCA advances,2014,4: 6863-. Although the catalytic efficiency of the complex enzyme is remarkably improved, the current research cannot essentially solve the problem of low vegetable oil hydrolysis rate, such as improving the substrate specificity of the enzyme to unsaturated fatty acid, improving the long-chain fatty acid specificity and the like, thereby further improving the vegetable oil hydrolysis rate.

Disclosure of Invention

In order to solve the problems, the invention obtains the mutant enzyme with improved long-chain unsaturated fatty acid specificity by rationally designing the Rhizopus Chinensis (RCL) mutant site, and improves the hydrolysis rate of the soybean oil.

The first object of the present invention is to provide a lipase mutant comprising a change of one, two or three amino acid residues of a substrate binding pocket of a lipase, relative to a lipase having an amino acid sequence shown in SEQ ID NO. 1; the change increases pocket hydrophilicity, which is correlated with lipase specificity for long chain unsaturated fatty acids.

In one embodiment of the present invention, the lipase mutant is a mutant in which glutamine is inserted between histidine 284 of the sequence whose amino acid sequence is shown in SEQ ID NO.1 and leucine 285 of the sequence.

In one embodiment of the invention, the amino acid sequence of the lipase mutant is shown as SEQ ID No. 2.

In one embodiment of the invention, the mutant HQL is obtained by inserting glutamine between histidine at position 284 and leucine at position 285.

The second purpose of the invention is to provide a gene sequence for encoding the lipase mutant.

It is a third object of the invention to provide a plasmid or cell carrying the gene sequence.

In one embodiment of the invention, the cell is a bacterium, a fungus or an archaea.

The fourth purpose of the invention is to provide the application of the lipase mutant in the fields of food, health products and medicines.

In one embodiment of the invention, the application is the hydrolysis of vegetable fat by using the lipase mutant.

In one embodiment of the invention, the application comprises hydrolyzed soybean oil, corn oil, peanut oil, rapeseed oil, olive oil, cottonseed oil, walnut oil.

The invention has the beneficial effects that:

according to the invention, the mutant enzyme with changed fatty acid specificity is obtained by carrying out site-directed mutagenesis on the Rhizopus Chinensis Lipase (RCL) according to the structural analysis of the RCL. Comparing the capability of catalyzing and hydrolyzing soybean oil by wild lipase and mutant enzyme, the mutant enzyme HQL with hydrolysis rate far higher than that of wild lipase is obtained, and the hydrolysis rate reaches about 98%.

Description of the drawings:

FIG. 1 shows the fatty acid chain length specificity of lipases (C2: pNPC2, C4: pNPC4, C5: pNPC5, C8: pNPC8, C12: pNPC12, C14: pNPC14, C16: pNPC 16);

FIG. 2 is a graph showing the hydrolysis characteristics of lipase on saturated and unsaturated fatty acids in soybean oil;

FIG. 3 is a graph of the effect of temperature on lipase activity and stability, where A is the effect of temperature on activity and B is the effect of temperature on stability;

FIG. 4 is a graph of the effect of pH on lipase activity and stability, ApH on activity, B pH on stability;

FIG. 5 shows the lipase-catalyzed soybean oil hydrolysis rate.

Detailed Description

The saponification value of the raw soybean oil is determined by GB/T5534-2008.

Hydrolysis rate (AV)₀-AV)/(SV-AV)×100％

In the formula: AV (Audio video)₀The acid value of the hydrolyzed sample, mgKOH/g; AV and SV are respectively the acid value and saponification value of the raw material soybean oil, mgKOH/g.

Test method for hydrolyzing soybean oil by lipase: weighing 5g of soybean oil in each sample, adding a certain amount of 50mM potassium phosphate buffer solution into a 50mL triangular flask, and performing ultrasonic treatment to fully emulsify a substrate; the reaction conditions for soybean oil hydrolysis were set to 24 hours, the water-oil mass ratio was 1:1, the enzyme amount was 500U/g (oil weight), pH8.0, temperature was 40 ℃, after the reaction 10mL of 95% ethanol was added to terminate the reaction, and the acid value of the hydrolysate was determined. The method for measuring the acid value is described in GB/T5009.37-2003.

Fatty acid composition analysis: extracting the reacted grease mixture by using n-hexane, separating by Thin Layer Chromatography (TLC) to obtain free fatty acid, putting the free fatty acid into a 10ml colorimetric tube with a plug, adding 1ml of 2% H2SO 4-methanol solution, carrying out water bath at 80 ℃ for 30min, taking out the colorimetric tube, cooling to room temperature, adding 2ml of n-hexane, fully and uniformly mixing, adding a saturated NaCl solution to a bottle mouth, violently shaking, standing for centrifugal layering, collecting an upper n-hexane phase, adding a proper amount of anhydrous sodium sulfate, and carrying out nitrogen-blowing concentration sample injection analysis.

A chromatographic column: DB-Wax (30 m.times.250. mu.m.times.0.25 μm); sample introduction amount: 1 mu L of the solution; the split ratio is as follows: 50: 1; sample inlet temperature: 225 ℃; carrier gas: nitrogen, 30mL min-1; hydrogen, 45mL min-1; air, 450mL min-1; temperature programming: maintaining at 180 deg.C for 1.5min, heating to 210 deg.C at 10 deg.C/min-1, maintaining for 2min, and then heating to 220 deg.C at 5 deg.C/min-1, maintaining for 5 min; a detector: flame Ionization Detector (FID) temperature of 250 ℃. Each sample was tested for 13.5 min.

Calculating the percentage content of the fatty acid component of the soybean oil by adopting an area normalization method on an Agilent GC 6890N gas chromatograph workstation.

The method for determining the hydrolysis activity of the lipase comprises the following steps: the lipase hydrolyzes p-nitrophenol ester to generate p-nitrophenol and fatty acid, the p-nitrophenol shows yellow in aqueous solution and has maximum light absorption at 410nm, and the activity of the lipase can be measured by measuring the light absorption of the p-nitrophenol at 410 nm. The definition of enzyme activity is: the enzyme amount of 1 mu mol p-nitrophenol generated per minute under a certain reaction condition is an international unit of lipase hydrolase activity.

Example 1: site-directed mutagenesis of Rhizopus chinensis lipase

According to the analysis of the crystal structure of the rhizopus chinensis lipase and the comparison of the crystal structures of lipases with similar structures, site-directed mutagenesis and combined mutagenesis are carried out by utilizing the full plasmid PCR technology. Site-directed mutagenesis is carried out by combining a rhizopus chinensis lipase substrate with a pocket at a fixed point.

TABLE 1 primers required for the mutation sites

Factors such as the hydrophilicity and hydrophobicity of amino acids in the binding pocket of the enzyme substrate and the size of the side chain group may affect the substrate specificity of the lipase. Therefore, three mutants were designed from the perspective of increasing the hydrophilicity of the pocket, including L285Q, T286Q and the insertion of a hydrophilic amino acid Q between the sequences H284 and L285, the mutant being designated HQL; three mutation sites were designed from the perspective of increasing steric hindrance, including A116W, I281F, A116W/I281F.

Example 2: fatty acid specificity study of mutant enzymes

The fatty acid chain length specificity of the mutant enzyme of example 1 was examined at pH8.0 and at a temperature of 40 ℃ using p-nitrophenol fatty acid esters of different alkyl carbon chain lengths as substrates (pNPC2, pNPC4, pNPC5, pNPC8, pNPC12, pNPC14, pNPC 16).

As can be seen from fig. 1, the mutant enzyme HQL has enhanced specificity for long-chain fatty acids compared to the wild-type lipase, wherein the specificity for pNPC16 is the highest, and the hydrolysis activity is increased to 2.72 times that of the wild-type lipase. Both mutant enzymes a116W and L285Q had the highest specificity for pNPC16, with 1.23-fold and 1.50-fold hydrolytic activity compared to wild type, respectively. The mutant enzyme I281F widens the range of the length of the hydrolyzed fatty acid chain, increases the hydrolytic activity to the pNPC2, has the highest specificity to the pNPC12, and has the hydrolytic activity 2 times of that of the wild type. In addition, the mutant enzymes L285Q, T286Q, HQL, a116W, a116W/I281F failed to hydrolyze pNPC4, and all of the mutant enzymes had reduced hydrolytic activity against pNPC 8. Through site-directed mutagenesis of a rhizopus chinensis lipase substrate binding pocket, a series of mutant enzymes with changed fatty acid specificity are obtained, and particularly, the mutant enzymes with enhanced specificity to long-chain fatty acids are obtained.

Example 3: research on hydrolysis characteristics of mutant enzyme on fatty acid in soybean oil

In order to examine the substrate specificity of lipase to saturated and unsaturated long-chain fatty acids, measurement was performed using soybean oil rich in such triglycerides as a substrate. The fatty acid composition of soybean oil was first determined to contain 13.77% saturated fatty acids (palmitic acid 11.19%, stearic acid 2.58%), and 86.23% unsaturated fatty acids (oleic acid 25.21%, linoleic acid 54.67%, linolenic acid 6.34%). As can be seen from table 2 and fig. 2, the mutant enzyme HQL enhanced the specificity for unsaturated fatty acids, hydrolyzing unsaturated fatty acids 1.45 times as much as saturated fatty acids, whereas the wild type hydrolyzed unsaturated fatty acids only 1.10 times as much as the saturated fatty acids. The mutant enzyme HQL is more specific to unsaturated fatty acids, and therefore, it is expected that the hydrolysis rate of HQL to soybean oil will be higher than that of the wild-type enzyme. In addition, the mutant enzymes T286Q, I281F and A116W/I281F enhance the specificity to saturated fatty acid, the activity of hydrolyzing the saturated fatty acid is respectively 1.20, 1.83 and 1.34 times of that of unsaturated fatty acid, and the lipase mutants are more suitable for animal fat with high content of saturated fatty acid.

TABLE 2 mutant enzymes catalyze hydrolysis of soybean oil

Example 4: thermostability Studies of mutant enzymes

The activity of the lipase is detected by taking p-nitrophenol palmitate as a substrate (pNPC 16). The lipase activity of the enzyme solution is measured by a standard method at different temperatures (20-60 ℃), the highest enzyme activity is taken as the relative enzyme activity of 100%, and the influence of the temperature on the mutant enzyme activity is researched. And (3) measuring the lipase activity of the enzyme solution after heat preservation is carried out for 1h at different temperatures (20-60 ℃), calculating the residual enzyme activity by taking the enzyme activity without heat preservation as a control, and inspecting the influence of the temperature on the stability of the mutant enzyme.

As can be seen from FIG. 3A, the optimum temperature for all mutant enzyme-catalyzed reactions was 40 ℃ and was consistent with that of the wild type. The activity of the mutant enzyme T286Q can be maintained above 84% at 20-40 deg.C, and other mutant enzymes can retain above 65% at 20 deg.C. However, when the temperature is higher than 40 ℃, the enzyme protein is gradually denatured, the activity of all mutant enzymes is obviously reduced, when the temperature is increased to 60 ℃, the activity of the mutant enzyme HQL is still kept about 20%, and the activity of other mutant enzymes is reduced to below 10%.

FIG. 3B shows the temperature stability of lipase, at 45 deg.C, the activity of HQL and I281F can be maintained at about 80%, only the activity of A116W is reduced to about 50%, and the activity of other mutant enzymes is maintained between 69% -75%. When the temperature exceeds 55 ℃, the activity of the mutant enzyme is rapidly reduced.

Example 5: study of pH stability of mutant enzymes

pH affects the conformation of the enzyme and also affects the dissociation status of the groups involved in catalysis and the dissociation state of the substrate molecules. The activity of the enzyme is affected by the pH of the environment, at a certain pH the enzyme exhibits maximal activity, above or below which the enzyme activity decreases. Respectively preparing 0.05mol/L phosphate buffer solution (pH6.5-pH8), 0.05mol/L Tris-HCl buffer solution (pH8-pH9) and 0.05mol/L carbonate buffer solution (pH9-pH10), respectively adding the enzyme solution into the buffer solutions with different pH values, measuring the lipase activity under the standard condition, taking the highest enzyme activity as the relative enzyme activity of 100%, and researching the influence of the pH value on the mutant enzyme activity. And (3) keeping the temperature of the enzyme solution in the buffer solutions with different pH values for 1h at 25 ℃, measuring the lipase activity of the enzyme solution, calculating the residual enzyme activity by taking the enzyme activity without heat preservation as a control, and inspecting the influence of the pH value on the stability of the mutant enzyme.

As can be seen from FIG. 4A, the optimum pH values of all the mutant enzyme-catalyzed reactions were 8.0, which is consistent with the optimum pH value of the wild type. The pH range is between pH 7.5-9.0, the activity of only mutant enzyme HQLT and I28F can be maintained at above 50%, and the activity of other mutant enzymes is lower than 50% at pH 9.0. Under the conditions that the pH is less than 7.5 and the pH is greater than 9, the enzyme activity is obviously reduced, and the reaction pH obviously influences the activity of the mutant enzyme.

As shown in FIG. 4B, all mutant enzymes were most stable at optimum pH8.0. The mutant enzyme T286Q has poor stability relative to wild type, the enzyme activity can be maintained above 60% at pH 7.5-8.5, and other mutant enzymes can also retain above 50% at pH7.0 and pH 9.0. At pH <7 and pH >9, the stability decreased very rapidly, and only the mutant enzyme I281F maintained 22% activity at pH 10.0.

Example 6: comparison of hydrolytic capacities of soybean oil catalyzed by different mutagens

The reaction conditions for soybean oil hydrolysis were set to 24 hours, the water-oil mass ratio was 1:1, the enzyme amount was 500U/g (oil weight), pH8.0, and the temperature was 40 ℃ to examine the ability of the mutant enzyme to catalyze the hydrolysis of soybean oil. As can be seen from FIG. 5, the mutant enzyme L285Q showed a hydrolysis rate close to that of wild-type catalyzed soybean oil, and the hydrolysis rate was 82%. The mutant enzymes A116W, I281F and A116W/I281F have weaker soybean oil hydrolysis catalyzing ability than wild type enzymes, and the hydrolysis rate is as low as about 70%. The hydrolysis capacity of the mutant enzyme L285Q is slightly higher than that of the wild type, and the hydrolysis rate reaches 88%. The mutant enzyme HQL has the highest capability of catalyzing the hydrolysis of the soybean oil, and reaches about 98 percent. Because the content of unsaturated fatty acid in soybean oil is high and accounts for more than 80%, and the mutant enzyme HQL has stronger specificity to long-chain unsaturated fatty acid than to long-chain saturated fatty acid, the capability of catalyzing and hydrolyzing soybean oil is obviously improved.

Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and it is intended that the scope of the invention be defined by the appended claims.

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Claims

1. A lipase mutant, characterized in that the lipase mutant is between the histidine at position 284 and the leucine at position 285 in the amino acid sequence of the sequence shown in SEQ ID NO.1. Glutamine is inserted between; the amino acid sequence of the lipase mutant is shown in SEQ ID NO.2.

2 . The lipase mutant according to claim 1 , wherein glutamine is inserted between histidine at position 284 and leucine at position 285 to obtain mutant HQL. 3 .

3. A gene sequence encoding the lipase mutant of any one of claims 1-2.

4. A plasmid or non-plant cell carrying the gene sequence of claim 3.

5. The cell of claim 4, wherein the non-plant cell is a bacterium, a fungus or an archaea.

6. The application of any one of the lipase mutants of claims 1 to 2 in food and health care products.

7 . The application according to claim 6 , wherein the application is to hydrolyze vegetable oils and fats by utilizing the lipase mutant of any one of claims 1 to 2 .

8. The application according to claim 7, wherein the application comprises hydrolyzed soybean oil, corn oil, peanut oil, rapeseed oil, olive oil, cottonseed oil, and walnut oil.