CN108034647B - A saturated fatty acid specific lipase mutant and its application - Google Patents

Abstract

The invention discloses a saturated fatty acid specific lipase mutant and an application thereof, belonging to the technical field of genetic engineering. According to the structure analysis of Rhizopus sinensis lipase (RCL), the invention carries out site-directed mutation to obtain a mutant enzyme whose fatty acid specificity is changed. Comparing the ability of wild-type lipase and mutant enzyme to catalyze the hydrolysis of animal oil, the mutant enzyme whose hydrolysis rate is much higher than that of wild-type lipase was obtained. Compared with the wild-type RCL, the mutant enzymes T286Q, I281F and A116W/I281F enhanced their specificity for saturated fatty acids, and their hydrolysis rates of lard increased from 55% to 85%, 90% and 86%, respectively.

Description

A saturated fatty acid specific lipase mutant and its application

technical field

The invention relates to a saturated fatty acid specific lipase mutant and its application, and belongs to the technical field of genetic engineering.

Background technique

Fatty acid is one of the most basic and widely used raw materials in oleochemical industry. Hydrolysis of natural oils is one of the important ways to obtain free fatty acids. The production technologies of fatty acids mainly include saponification, hydrolysis, steam cracking and enzymatic hydrolysis. Among them, enzymatic hydrolysis has the advantages of mild reaction conditions, unsaturated fatty acids are not easily oxidized, and by-products are few, and it has become the research method of fatty acid preparation. hot spot. Different types, different carbon chain lengths and different proportions of fatty acids have different functions. Short-chain fatty acids can be used as feed additives to promote animal growth. Medium-chain fatty acids are widely used in medicine, health-care edible oil and aquaculture. Long-chain unsaturated fatty acids have a wide range of physiological activities in metabolism and are used in health care. The content of unsaturated fatty acids in vegetable oils and fats is relatively high, such as soybean oil, rapeseed oil and camellia oil with unsaturated fatty acid content of 84.34%, 77.70% and 81.43%, respectively. Long-chain saturated fatty acids such as palmitic acid and stearic acid are widely present in animal oils such as pigs, cattle and sheep, and are mainly used to produce 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 animal oil is usually more than 60%, and the fatty acid chain length is mostly 16 carbons and 18 carbons, which can reach more than 90% in total. The fatty acids containing 16 and 18 carbon atoms are The methyl ester product obtained after methyl esterification has a similar number of carbon atoms as mineral diesel, and at the same time has similarities in many physical and chemical properties. Therefore, fatty acids can be prepared by hydrolysis of animal oil as a synthetic raw material for biodiesel.

Lipase (Triacylglycerol hydrolase, Lipase, EC 3.1.1.3) is a class of triglyceride hydrolase, which catalyzes the hydrolysis or synthesis of ester bonds at the oil-water interface, and is an important industrial enzyme for the processing and manufacture of "green" oils. Animal oil is an important source for the preparation of long-chain saturated fatty acids, but the hydrolysis rate of lipase is not high, only about 60% (Journal of Feed Industry 2008, No. 12, P8-11). The possible reason is that the substrate specificity of lipase for saturated fatty acids cannot meet the needs of the oil processing industry. If the specificity of lipase for saturated fatty acids can be further improved, the hydrolysis efficiency of animal fats can be expected to be improved.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention obtains a mutant enzyme with improved specificity of saturated fatty acids by rationally designing the mutation site of Rhizopus chinensis lipase (RCL), and improves the hydrolysis rate of animal oil.

The first object of the present invention is to provide a lipase mutant, which, relative to the lipase having the amino acid sequence shown in SEQ ID NO: 1, comprises one or two lipase substrate binding pockets or three amino acid residue changes; the changes increase pocket steric hindrance; the changes correlate with the specificity of the lipase for saturated fatty acids.

In one embodiment of the present invention, the lipase mutant is based on the amino acid sequence shown in SEQ ID NO. 1, by mutating threonine at position 286 to glutamine, or by Isoleucine at position 281 was mutated to phenylalanine, or alanine at position 116 was mutated to tryptophan at the same time as isoleucine at position 281 was mutated to phenylalanine.

In one embodiment of the present invention, the amino acid sequence of the lipase mutant is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

In one embodiment of the present invention, the mutant T286Q is obtained by mutating threonine at position 286 to glutamine; the mutant obtained by mutating isoleucine at position 281 to phenylalanine is I281F; The mutant obtained by mutating alanine at position 116 to tryptophan and isoleucine at position 281 to phenylalanine is A116W/I281F.

The second object of the present invention is to provide a gene sequence encoding the lipase mutant.

The third object of the present invention is to provide plasmids or cells carrying the gene sequence.

In one embodiment of the invention, the cells are bacteria, fungi or archaea.

The fourth object of the present invention is to provide the application of the lipase mutant in the fields of food, health products and medicine.

In one embodiment of the present invention, the application is the hydrolysis of tallow using the lipase mutant.

In one embodiment of the invention, the application comprises hydrolyzing lard, tallow, suet, chicken or duck fat.

Beneficial effects of the present invention:

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. Compared with wild-type RCL, the mutant enzymes T286Q, I281F and A116W/I281F have enhanced specificity for saturated fatty acids, and their hydrolysis rates of lard increased from 55% to 85%, 90% and 86%, respectively

Description of drawings:

Figure 1 shows the fatty acid chain length specificity of lipases (C2:pNPC2, C4:pNPC4, C5:pNPC5, C8:pNPC8, C12:pNPC12, C14:pNPC14, C16:pNPC16);

Fig. 2 is the hydrolysis characteristic of lipase to saturated fatty acid and unsaturated fatty acid in soybean oil;

Figure 3 is the effect of temperature on the activity and stability of lipase, wherein A is the effect of temperature on activity, and B is the effect of temperature on stability;

Figure 4 is the effect of pH on lipase activity and stability, wherein A is the effect of pH on activity, and B is the effect of pH on stability;

Figure 5 shows the hydrolysis rate of soybean oil catalyzed by lipase.

Detailed ways

The determination of the saponification value of raw soybean oil refers to GB/T 5534-2008.

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

In the formula: AV ₀ is the acid value of the hydrolyzed sample, mgKOH/g; AV and SV are the acid value and saponification value of the raw soybean oil, mgKOH/g, respectively.

Lipase hydrolysis soybean oil test method: Weigh 5g soybean oil for each sample, add a certain amount of 50mM potassium phosphate buffer into a 50mL conical flask, and ultrasonically emulsify the substrate; the reaction conditions for soybean oil hydrolysis are set to the reaction time 24h, the mass ratio of water to oil is 1:1, the amount of enzyme added is 500U/g (oil weight), pH 8.0, temperature 40°C, after the reaction, 10 mL of 95% ethanol is added to terminate the reaction, and the acid value of the hydrolyzate is determined. The acid value determination method refers to GB/T 5009.37-2003.

Analysis of fatty _acid composition: Extract the reacted oil mixture with n _- hexane, and separate free fatty acids by thin layer chromatography TLC. 30min, take out the colorimetric tube, cool to room temperature, add 2ml of n-hexane, mix well, add saturated NaCl solution to the bottle mouth, shake vigorously, stand and centrifuge for stratification, collect the upper n-hexane phase, add an appropriate amount of anhydrous sulfuric acid Sodium, nitrogen blowing concentration injection analysis.

Chromatographic column: DB-Wax (30m×250μm×0.25μm); injection volume: 1μL; split ratio: 50:1; injection port temperature: 225°C; carrier gas: nitrogen, 30mL·min ^-1 ; hydrogen, 45mL ·min ^-1 ; Air, 450mL ·min ^-1 ; temperature program: 180°C for 1.5min, rise to 210°C at 10°C·min ^-1 , hold for 2min, then rise to 220°C at 5°C·min ^-1 5min; detector: flame ionization detector (FID), the temperature is 250 ℃. Each sample was tested for 13.5 minutes.

The percentages of fatty acid components in soybean oil were calculated by area normalization on an Agilent GC 6890N gas chromatograph workstation.

Determination method of lipase hydrolysis activity: lipase hydrolyzes p-nitrophenol ester to produce p-nitrophenol and fatty acid, p-nitrophenol is yellow in aqueous solution, and has the maximum light absorption at 410nm, by measuring p-nitrophenol at 410nm The light absorption of lipase can be measured. The definition of enzyme activity is: the amount of enzyme that produces 1 μmol of p-nitrophenol per minute under certain reaction conditions is one international unit of lipase hydrolase activity.

Example 1: Site-directed mutagenesis of Rhizopus sinensis lipase

According to the crystal structure analysis of Rhizopus huai lipase and the comparison of crystal structure of lipase with similar structure, site-directed mutagenesis and combinatorial mutagenesis were carried out by using whole plasmid PCR technology. By site-directed mutagenesis of the Rhizopus sinensis lipase substrate-binding pocket site.

Table 1 Primers required for mutation sites

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

Example 2: Fatty acid specificity studies of mutant enzymes

Using p-nitrophenol fatty acid esters with different alkyl carbon chain lengths as substrates (pNPC2, pNPC4, pNPC5, pNPC8, pNPC12, pNPC14, pNPC16), at pH 8.0 and temperature of 40 ℃, the mutant enzymes of Example 1 were detected. Fatty acid chain length specificity.

It can be seen from Figure 1 that compared with the wild-type lipase, the specificity of the mutant enzyme HQL for long-chain fatty acids is enhanced, among which the specificity of pNPC16 is the highest, and the hydrolysis activity is increased to 2.72 times that of the wild-type. Mutant enzymes A116W and L285Q both had the highest specificity for pNPC16, and their hydrolysis activities were 1.23 and 1.50 times higher than those of the wild type, respectively. The mutant enzyme I281F broadened the range of hydrolyzed fatty acid chain length, increased the hydrolytic activity of pNPC2, and had the highest specificity to pNPC12, and the hydrolytic activity was twice that of the wild type. In addition, the mutant enzymes L285Q, T286Q, HQL, A116W, A116W/I281F could not hydrolyze pNPC4, and all the mutant enzymes had reduced hydrolytic activity on pNPC8. We obtained a series of mutant enzymes with altered specificity of fatty acids by site-directed mutagenesis of the substrate-binding pocket of Rhizopus chinensis lipase, especially those with enhanced specificity for long-chain fatty acids.

Example 3: Hydrolysis properties of mutant enzymes on fatty acids in soybean oil

In order to investigate the substrate specificity of lipase for saturated and unsaturated long-chain fatty acids, soybean oil rich in these triglycerides was used as the substrate for the assay. First, the fatty acid composition of soybean oil was determined, which contained 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%) . It can be seen from Table 2 and Figure 2 that the mutant enzyme HQL has enhanced specificity for unsaturated fatty acids, and the activity of hydrolyzing unsaturated fatty acids is 1.45 times that of saturated fatty acids, while the wild-type hydrolysis of unsaturated fatty acids is only 1.10 times that of saturated fatty acids. The mutant enzyme HQL is more specific for unsaturated fatty acids, therefore, it can be expected that the hydrolysis rate of soybean oil by HQL will be higher than that of the wild-type enzyme. In addition, the mutant enzymes T286Q, I281F and A116W/I281F have enhanced specificity for saturated fatty acids, and the hydrolysis activities of saturated fatty acids are 1.20, 1.83 and 1.34 times that of unsaturated fatty acids, respectively. These lipase mutants will be more suitable for saturated fatty acids. Animal fats high in fatty acids.

Table 2 Mutated enzymes catalyze the hydrolysis of soybean oil

Example 4: Thermostability studies of mutant enzymes

The lipase activity was detected using p-nitrophenol palmitate as a substrate (pNPC16). The lipase activity of the enzyme solution was measured at different temperatures (20°C-60°C) by standard methods, and the highest enzyme activity was taken as the relative enzyme activity of 100% to study the effect of temperature on the mutant enzyme activity. After incubation at different temperatures (20℃-60℃) for 1 h, the lipase activity of the enzyme solution was determined. The enzyme activity without incubation was used as a control to calculate the residual enzyme activity, and the effect of temperature on the stability of the mutant enzyme was investigated.

It can be seen from Figure 3A that the optimum temperature for all mutant enzymes catalyzed reactions is 40°C, which is consistent with the optimum temperature of the wild type. In the range of 20-40℃, the activity of the mutant enzyme T286Q can keep more than 84%, and other mutant enzymes can keep more than 65% of the activity at 20℃. However, when the temperature was higher than 40°C, the enzyme protein gradually denatured, and the activities of all mutant enzymes decreased significantly. When the temperature increased to 60°C, the activities of the mutant enzyme HQL remained at about 20%, and the activities of other mutant enzymes decreased. to less than 10%.

Figure 3B shows the temperature stability of lipase. At 45 °C, the activities of mutant enzymes HQL and I281F can be maintained at about 80%, only the activity of mutant enzyme A116W decreased to about 50%, while the activities of other mutant enzymes remained Between 69%-75%. When the temperature exceeded 55℃, the activity of the mutant enzyme decreased rapidly.

Example 5: pH stability studies of mutant enzymes

pH affects the conformation of the enzyme, as well as the dissociation state of catalysis-related groups and the dissociation state of substrate molecules. The activity of the enzyme is affected by the pH of the environment. At a certain pH, the enzyme exhibits the maximum activity, and the activity of the enzyme decreases above or below this pH. Prepare 0.05mol/L phosphate buffer (pH6.5-pH8), 0.05mol/L Tris-HCl buffer (pH8-pH9), and 0.05mol/L carbonate buffer (pH9-pH10), respectively, The enzyme solution was added to the above buffers with different pH, and the lipase activity was determined under standard conditions. The highest enzyme activity was taken as the relative enzyme activity of 100% to study the effect of pH on the mutant enzyme activity. The lipase activity of the enzyme solution was measured after the enzyme solution was incubated at 25°C for 1 h in the above-mentioned buffers with different pH.

It can be seen from Fig. 4A that the optimum pH value of all mutant enzymes catalyzed reactions is 8.0, which is consistent with the optimum pH value of the wild type. The pH range is between pH 7.5-9.0. Only the mutant enzymes HQL and I281 F can maintain more than 50% activities, and the other mutant enzymes are all less than 50% active at pH 9.0. In the case of pH<7.5 and pH>9, the enzyme activity decreased significantly, indicating that the reaction pH significantly affected the activity of the mutant enzyme.

As shown in Figure 4B, all mutant enzymes were the most stable at the optimum pH 8.0. The mutant enzyme T286Q is less stable than the wild type, and the enzyme activity can keep more than 60% at pH 7.5-8.5, while other mutant enzymes can also retain more than 50% activity at pH 7.0 and pH 9.0. In the case of pH<7 and pH>9, the stability decreased rapidly, and only the mutant enzyme I281F could maintain 22% activity at pH10.0.

Example 6: Comparison of different mutant enzymes catalyzing the hydrolysis of soybean oil

The reaction conditions of soybean oil hydrolysis were set as reaction time 24h, water-oil mass ratio 1:1, enzyme amount 500U/g (oil weight), pH 8.0, temperature 40℃, and the ability of mutant enzyme to catalyze soybean oil hydrolysis was investigated. It can be seen from Figure 5 that the mutant enzyme L285Q catalyzes the hydrolysis rate of soybean oil close to that of the wild type, and the hydrolysis rate is 82%. The ability of mutant enzymes A116W, I281F and A116W/I281F to catalyze the hydrolysis of soybean oil is weaker than that of the wild type, and the hydrolysis rate is as low as about 70%. The hydrolysis capacity of the mutant enzyme L285Q was slightly higher than that of the wild type, and the hydrolysis rate reached 88%. The mutant enzyme HQL had the highest ability to catalyze the hydrolysis of soybean oil, reaching about 98%. Due to the high content of unsaturated fatty acids in soybean oil, accounting for more than 80%, and the mutant enzyme HQL is more specific for long-chain unsaturated fatty acids than long-chain saturated fatty acids, so the ability to catalyze the hydrolysis of soybean oil is significantly improved.

Example 7: Comparison of different mutant enzymes catalyzing the hydrolysis of lard

The reaction conditions of lard hydrolysis were set as reaction time 24h, water-oil mass ratio 1:1, enzyme amount 500U/g (oil weight), pH 8.0, temperature 40℃, to investigate the ability of mutant enzyme to catalyze lard hydrolysis. The results showed that compared with the wild-type RCL, the mutant enzymes T286Q, I281F and A116W/I281F enhanced their specificity for saturated fatty acids, and their lard hydrolysis rates increased from 55% to 85%, 90% and 86%, respectively.

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.

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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.

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