CN108034647B - Saturated fatty acid specific lipase mutant and application thereof - Google Patents
Saturated fatty acid specific lipase mutant and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
Abstract
The invention discloses a saturated fatty acid specific lipase mutant and application thereof, belonging to the technical field of genetic engineering.A mutant enzyme with changed fatty acid specificity is obtained by carrying out site-directed mutagenesis on the mutant enzyme according to the structural analysis of rhizopus chinensis lipase (RC L). The mutant enzyme with hydrolysis rate far higher than that of wild lipase is obtained by comparing the abilities of the wild lipase and the mutant enzyme for catalyzing and hydrolyzing animal oil.compared with the wild type RC L, the mutant enzymes T286Q, I281F and A116W/I281F respectively improve the hydrolysis rate of lard from 55% to 85%, 90% and 86% due to the enhancement of the specificity on saturated fatty acid.
Description
Technical Field
The invention relates to a saturated 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. 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. Long-chain saturated fatty acids such as palmitic acid and stearic acid are widely present in animal oils of pigs, cattle and sheep, and are mainly used for producing fatty acid salts as emulsifiers and as stabilizers for commonly used plastic products. The sum of the mass fractions of various saturated fatty acids in the animal oil is usually more than 60%, the fatty acid chain length is mostly sixteen carbon and eighteen carbon, the sum can reach more than 90%, methyl ester products obtained by methyl esterification of fatty acids containing sixteen and eighteen carbon atoms have similar carbon atoms with mineral diesel oil, and meanwhile, the methyl ester products have similarity on a plurality of physicochemical properties, so that the fatty acids prepared by hydrolysis of the animal oil can be used as synthetic raw materials of the biodiesel.
Animal oil is an important source for preparing long-chain saturated fatty acid, but the hydrolysis rate of the lipase is not high, only about 60% (feed industry journal 2008, stage 12, P8-11). the possible reason is that the substrate specificity of the lipase to the saturated fatty acid cannot meet the requirements of the grease processing industry, and if the specificity of the lipase to the saturated fatty acid can be further improved, the hydrolysis efficiency of the animal oil can be expected to be improved.
Disclosure of Invention
In order to solve the problems, the invention obtains the mutant enzyme with improved saturated fatty acid specificity by rationally designing the mutation site of Rhizopus chinensis lipase (RC L), and improves the hydrolysis rate of animal 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 steric hindrance; the changes are related to the specificity of the lipase for saturated fatty acids.
In one embodiment of the present invention, the lipase mutant is obtained by mutating threonine at position 286 to glutamine, or isoleucine at position 281 to phenylalanine, or alanine at position 116 to tryptophan and isoleucine at position 281 to phenylalanine on the basis of the sequence having an amino acid sequence shown in SEQ ID No. 1.
In one embodiment of the invention, the amino acid sequence of the lipase mutant is shown as SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4.
In one embodiment of the invention, the mutation of threonine at position 286 to glutamine gives rise to the mutant T286Q; the mutant obtained by mutating isoleucine at the 281 th position into phenylalanine is I281F; a mutant obtained by mutating alanine at position 116 to tryptophan and isoleucine at position 281 to phenylalanine is A116W/I281F.
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 animal fat by using the lipase mutant.
In one embodiment of the invention, the use comprises hydrolyzing lard, tallow, mutton tallow, chicken oil or duck oil.
The invention has the beneficial effects that:
compared with wild type RC L, the mutant enzymes T286Q, I281F and A116W/I281F have the hydrolysis rate of lard respectively improved from 55% to 85%, 90% and 86% due to the enhancement of the specificity to saturated fatty acid
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, where A is the effect of pH on activity and B is the effect of 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.
A test method for hydrolyzing soybean oil with lipase comprises the steps of weighing 5g of soybean oil for each sample, adding a certain amount of 50mM potassium phosphate buffer solution into a 50m L triangular flask, performing ultrasonic treatment to fully emulsify a substrate, setting the reaction condition of soybean oil hydrolysis to be 24h, setting the mass ratio of water to oil to be 1:1, adding 500U/g enzyme (oil weight), pH to be 8.0, reacting at 40 ℃, adding 10m L95% ethanol to terminate the reaction, and determining the acid value of a hydrolysate, wherein the acid value determination method refers to GB/T5009.37-2003.
Analyzing the components of fatty acid, namely extracting the reacted oil mixture by using normal hexane, separating by thin layer chromatography T L C to obtain free fatty acid, placing the free fatty acid into a 10ml colorimetric tube with a plug, and adding 1ml of 2% H2SO4And (3) carrying out water bath on a methanol solution at the temperature of 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, centrifuging and layering, collecting an upper n-hexane phase, adding a proper amount of anhydrous sodium sulfate, and carrying out nitrogen blowing concentration and sample injection analysis.
DB-Wax (30m × 250 μm × 0.25 μm), sample injection amount of 1 μ L, split ratio of 50:1, injection port temperature of 225 deg.C, carrier gas of nitrogen gas, 30m L min-145m L min for hydrogen-1450m L min for air-1(ii) a Temperature programming: maintaining at 180 deg.C for 1.5min, and maintaining at 10 deg.C/min-1Raising the temperature to 210 ℃, keeping the temperature for 2min, and then raising the temperature to 5 ℃ min-1Heating to 220 deg.C and 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 on the substrate binding pocket site of rhizopus chinensis lipase.
TABLE 1 primers required for the mutation sites
Therefore, three mutants are designed from the perspective of increasing the hydrophilicity of the pocket, including L285Q, T286Q, and a hydrophilic amino acid Q is inserted between the sequences H284 and L285, the mutant is named as HQ L, and three mutation sites are designed from the perspective of increasing the 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, compared to the wild-type lipase, the mutant enzyme HQ L has enhanced specificity for long-chain fatty acids, wherein the specificity for pNPC16 is highest and the hydrolytic activity is increased to 2.72 times that of the wild-type, both mutant enzymes a116W and L285Q have highest specificity for pNPC16 and the hydrolytic activity is 1.23 times and 1.50 times that of the wild-type, respectively, mutant enzyme I281F widens the range of the hydrolytic fatty acid chain length, increases the hydrolytic activity for pNPC2, has highest specificity for pNPC12 and the hydrolytic activity is 2 times that of the wild-type, further, mutant enzymes L285Q, T286Q, HQ L, a116W, a 116W/281I 281F cannot hydrolyze pNPC4, and the hydrolytic activity for pNPC8 is reduced.
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, soybean oil rich in such triglycerides was measured as a substrate, and the fatty acid composition of soybean oil was first measured, containing 13.77% of saturated fatty acids (palmitic acid 11.19%, stearic acid 2.58%), and 86.23% of unsaturated fatty acids (oleic acid 25.21%, linoleic acid 54.67%, linolenic acid 6.34%), from table 2 and fig. 2, it can be seen that the mutant enzyme HQ L enhances the specificity to unsaturated fatty acids, the activity of hydrolyzing unsaturated fatty acids is 1.45 times that of saturated fatty acids, while the wild type hydrolyzes unsaturated fatty acids only 1.10 times that of saturated fatty acids, the mutant enzyme HQ L is more specific to unsaturated fatty acids, and therefore, the hydrolysis rate of HQ L to soybean oil can be expected to be higher than that of wild type enzyme, and further, the mutant enzymes T39286, I281 56 and a116W/I281F enhance the specificity to saturated fatty acids, and the activity of hydrolyzing saturated fatty acids is 1.20 times that of unsaturated fatty acids, respectively, and the content of saturated fatty acids is more suitable for animals.
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.
From FIG. 3A, it can be seen that the optimum temperature of all mutant enzyme catalytic reactions is 40 ℃, which is consistent with the optimum temperature of the wild type, within the range of 20-40 ℃, the activity of the mutant enzyme T286Q can be maintained above 84%, and the activity of other mutant enzymes can be maintained above 65% at 20 ℃.
FIG. 3B shows the temperature stability of lipase, at 45 deg.C, the activities of mutant HQ L and I281F can be maintained at about 80%, only the activity of mutant A116W is reduced to about 50%, while the activities of other mutant enzymes are maintained between 69% and 75%.
Example 5: study of pH stability of mutant enzymes
The enzyme activity is influenced by the environmental pH, the enzyme shows the maximum activity under a certain pH value, the enzyme activity is higher or lower than the pH value, the enzyme activity is reduced, 0.05 mol/L of phosphate buffer solution (pH6.5-pH8), 0.05 mol/L of Tris-HCl buffer solution (pH8-pH9) and 0.05 mol/L of carbonate buffer solution (pH9-pH10) are respectively prepared, the enzyme solution is respectively added into the buffer solutions with different pH values, the lipase activity is measured under the standard condition, the highest enzyme activity is 100 percent of relative enzyme activity, the influence of the pH on the mutant enzyme activity is researched, the lipase activity of the enzyme solution is measured after the enzyme solution is kept for 1h at 25 ℃, the non-heat-preserved enzyme activity is used as a reference to calculate the residual enzyme activity, and the influence of the pH on the mutant enzyme stability is investigated.
From FIG. 4A, it can be seen that the optimum pH values of all mutant enzyme catalyzed reactions are 8.0, which is consistent with the optimum pH value of the wild type, the pH range is between pH 7.5 and 9.0, only the activities of the mutant enzymes HQ L and I281F can be maintained above 50%, the activities of other mutant enzymes are all lower than 50% at pH9.0, and the enzyme activity is obviously reduced under the conditions of pH <7.5 and pH >9, which indicates that the reaction pH significantly affects the activities of the mutant enzymes.
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 soybean oil hydrolysis reaction conditions are set to be reaction time 24h, the water-oil mass ratio is 1:1, the enzyme adding amount is 500U/g (oil weight), the pH value is 8.0, the temperature is 40 ℃, and the capability of catalyzing the soybean oil hydrolysis by the mutant enzyme is examined, as can be seen from figure 5, the hydrolysis rate of the mutant enzyme L Q is close to that of the wild type catalyzed soybean oil, the hydrolysis rate is 82%, the capability of catalyzing the soybean oil hydrolysis by the mutant enzyme A116W, I281F and A116W/I281F is weaker than that of the wild type, the hydrolysis rate is as low as about 70%, the hydrolysis capability of the mutant enzyme L285Q is slightly higher than that of the wild type, the hydrolysis rate reaches 88%, the capability of catalyzing the soybean oil hydrolysis by the mutant enzyme HQ L is highest and reaches about 98%, the content of unsaturated fatty acid in the soybean oil is higher than 80%, and the specificity of the mutant enzyme HQ L to the long-chain unsaturated fatty acid is stronger than that of the long-chain saturated fatty acid, so the.
Example 7: comparison of the ability of different mutants to catalyze hydrolysis of lard
The reaction conditions of lard hydrolysis are set as the reaction time of 24h, the water-oil mass ratio is 1:1, the enzyme adding amount is 500U/g (oil weight), the pH value is 8.0, the temperature is 40 ℃, and the capability of mutant enzymes for catalyzing lard hydrolysis is examined.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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<120> saturated fatty acid specific lipase mutant and application thereof
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Claims (13)
1. A lipase mutant is characterized in that on the basis of a sequence with an amino acid sequence shown as SEQ ID NO.1, threonine at the 286 th position is mutated into glutamine,
or isoleucine at position 281 is mutated to phenylalanine,
or alanine at position 116 to tryptophan and isoleucine at position 281 to phenylalanine.
2. The lipase mutant as claimed in claim 1, wherein the amino acid sequence of the lipase mutant is shown as SEQ ID No. 2.
3. The lipase mutant as claimed in claim 1, wherein the amino acid sequence of the lipase mutant is shown as SEQ ID No. 3.
4. The lipase mutant as claimed in claim 1, wherein the amino acid sequence of the lipase mutant is shown as SEQ ID No. 4.
5. The lipase mutant according to claim 1, wherein threonine at position 286 is mutated to glutamine to give mutant T286Q; the mutant obtained by mutating isoleucine at the 281 th position into phenylalanine is I281F; a mutant obtained by mutating alanine at position 116 to tryptophan and isoleucine at position 281 to phenylalanine is A116W/I281F.
6. A gene sequence encoding the lipase mutant according to any one of claims 1 to 5.
7. A plasmid carrying the gene sequence of claim 6.
8. A microbial cell carrying the gene sequence of claim 6.
9. The microbial cell of claim 8, wherein the microbial cell is a bacterium, a fungus, or an archaea.
10. The lipase mutant as claimed in any one of claims 1 to 5, which is used in the fields of food and health care products.
11. Use of the lipase mutant of any one of claims 1 to 5 in the medical field for preparing a medicament for catalyzing esterification, alcoholysis, ammonolysis, transesterification or reverse synthesis of lipids.
12. The use according to claim 10, wherein the lipase mutant according to any one of claims 1 to 4 is used for hydrolyzing animal fat.
13. Use according to claim 12, characterized in that it comprises hydrolysed lard, tallow, mutton tallow, chicken fat or duck fat.
Priority Applications (1)
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