Phospholipase a1 mutant and use thereof

Abstract

The application discloses a phospholipase A1 mutant and application thereof. In the application, the PLA1 mutant is obtained by modifying the PLA1, wherein compared with the wild-type PLA1, the optimal temperature of the PLA1 mutant remains unchanged, and the PLA1 mutant has higher phospholipase A1 activity between 40-50 DEG C; the optimal pH of the PLA1 mutant is changed from 3 to 8; the PLA1 mutant has high phospholipase activity at pH 3-9, and the phospholipase activity is higher than that of the wild-type PLA1 under the optimal condition; and the pH adaptability range of the PLA1 is widened. Therefore, the PLA1 mutant has better application prospect, and meets the requirement of the enzyme performance in phospholipid processing, and can be applied to oil degumming, production of lysophosphatidylcholine, and application in the fields of biology, food, medicine, beauty, agriculture and industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of enzyme engineering, in particular to a phospholipase A1 mutant and application thereof. BACKGROUND

[0002] In the processing of animal and vegetable oils, the crude oil obtained by pressing, leaching and other processes is called crude oil. The main components of crude oil are glycerides and fatty acids. In addition, crude oil also contains phospholipids, proteins, mucilage and other impurities. Among these impurities, the highest content and the most difficult to remove is phospholipid, which is commonly known as "glue" or "glue". If not removed, not only will it reduce the quality of oil products, but also will greatly increase the cost of later oil decolorization, deodorization and other processes, and also cause problems such as filtration difficulty, equipment failure and shutdown. Therefore, oil degumming refers to the removal of gum from crude oil, which is an essential step in oil refining.

[0003] Traditional degumming technologies, such as hydration degumming and acid degumming, have limited degumming effect and cannot achieve ideal results, and still require other processing methods. Although membrane degumming has high degumming efficiency, it has defects such as high production cost and difficulty in replacement, which seriously limits the large-scale industrial application of this method. Therefore, efficient and green enzyme degumming is particularly important. Enzyme degumming is a new type of oil degumming technology that uses enzyme preparations with substrate specificity to convert non-hydrated phospholipids into hydrophilic lysophospholipids that are easy to remove, thereby achieving efficient degumming. Compared with other degumming methods, enzyme degumming can greatly save the consumption of chemicals and almost no waste water is produced, which has potential advantages in environmental protection and quality.

[0004] The main enzymes for enzyme degumming at present include phospholipase A1 (PLA1), phospholipase A2 and phospholipase C. Among them, PLA1 specifically hydrolyzes the fatty acid ester bond at sn-1 position of phospholipid molecules, and PLA2 accurately cuts off the fatty acid chain at sn-2 position, both of which generate a lysophospholipid and a free fatty acid. The highly hydrophilic lysophospholipid is easily dissolved in the aqueous phase, thereby rapidly separating from the oil, which can be easily removed by centrifugation. Phospholipase C cuts off the entire hydrophilic phosphate group (such as phosphocholine, phosphoethanolamine, etc.) from the phospholipid molecule to generate a diacylglycerol and a phosphate (such as phosphocholine), and the phosphate is a water-soluble substance that enters the aqueous phase and is removed. SUMMARY

[0005] The present application aims to overcome the shortcomings and deficiencies of the prior art, and provides a phospholipase A1 mutant.

[0006] Another object of the present application is to provide the application of the above-mentioned phospholipase A1 mutant.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] A phospholipase A1 mutant, the amino acid sequence of which is shown as SEQ ID NO. 3.

[0009] The coding gene of the phospholipase A1 mutant, the nucleotide sequence of which is obtained according to the codon coding rule.

[0010] The coding gene of the phospholipase A1 mutant is shown as SEQ ID NO. 4.

[0011] The phospholipase A1 mutant is obtained by knocking out the amino acids at positions 276 to 304 of phospholipase A1.

[0012] A recombinant expression vector comprising the coding gene of the phospholipase A1 mutant.

[0013] The vector skeleton of the recombinant expression vector is pPICZ alpha A.

[0014] An engineering bacterium comprising the recombinant expression vector.

[0015] The starting strain of the engineering bacterium is Pichia pastoris X-33.

[0016] The application of the phospholipase A1 mutant in oil degumming.

[0017] The present application has the following advantages and effects relative to the prior art:

[0018] In the present application, the PLA1 mutant is obtained by modifying PLA1, and the PLA1 mutant has the same optimum temperature as the wild-type PLA1, i.e. 40-50 DEG C, has higher phospholipase A1 activity, the optimum pH is changed from 3 to 8, the PLA1 mutant has high phospholipase activity at pH 3-9, and the phospholipase activity is higher than that of the wild-type PLA1 under the optimum conditions, thereby widening the pH adaptability range of PLA1. Therefore, the PLA1 mutant has better application prospect, and meets the requirements of enzyme performance in phospholipid processing, and can be used in oil degumming, production of lysophosphatidylcholine, and application in the fields of biology, food, medicine, beauty, agriculture, industry, etc. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the plasmid map of the vector pPICZ alpha A-PLA1 in Example 1 of the present application.

[0020] Figure 2 It is the electrophoresis detection diagram of the purified PLA1 mutant in Example 1 of the present application.

[0021] Figure 3Figure for the results of the determination of the enzyme activity of the purified PLA1 mutant in Example 3 of the present application.

[0022] Figure 4 Figure for the results of the influence of pH on the lipase activity of PLA1-D276-304 in Example 3 of the present application.

[0023] Figure 5 Figure for the results of the influence of temperature on the lipase activity of PLA1-D276-304 in Example 3 of the present application.

[0024] Figure 6 Figure for the results of the degumming effect of PLA1-D276-304 in Example 4 of the present application. DETAILED DESCRIPTION

[0025] The present application will be further described in conjunction with the examples and the accompanying drawings, but the embodiments of the present application are not limited thereto.

[0026] In the following examples, if the specific test conditions are not specified, the test conditions are generally in accordance with the conventional test conditions or in accordance with the test conditions suggested by the reagent companies. The materials, reagents, etc. used, if not specifically stated, are reagents and materials obtained from commercial channels.

[0027] Example 1 Construction of PLA1 mutant vector

[0028] 1.1 Design of mutant primers

[0029] In order to improve the activity of PLA1 enzyme, the terminal part of amino acids of PLA1 is removed by designing primers to obtain a truncated mutant of PLA1, or a point mutation mutant is obtained by designing a mutation primer. The specific mutants and primers are shown in Table 1.

[0030] Table 1 Primers used for constructing mutants

[0031]

[0032] Note: PLA1-D276-304, PLA1-D273-310, PLA1-D267-310 are mutants in which the amino acids in the corresponding range are knocked out, for example, PLA1-D276-304 is a mutant in which amino acids at positions 277-304 are knocked out; PLA1-N87Q / W88R, PLA1-S82G / S83G / S84G, PLA1-L145Y, PLA1-L145T are point mutation mutants, for example, PLA1-N87Q / W88R is a mutant in which N at position 87 is mutated to Q and W at position 88 is mutated to R.

[0033] 1.2 Construction of mutant vector

[0034] The vector carries the wild-type gene of phospholipase PLA1, the amino acid sequence of which is shown as SEQ ID NO. 1, and the nucleotide sequence of which is shown as SEQ ID NO. 2), and PCR amplification is performed using the primers shown in Table 1, and the PCR amplification reaction system is as follows: 1 μL of each forward primer F, 1 μL of each reverse primer R, 1 μL of plasmid pPICZαA-PLA1, 12.5 μL of 2x PrimeStar Max, 9.5 μL of H2O, and a total system of 25 μL. Figure 1

[0035] The PCR reaction program is as follows: 98 ℃ pre-denaturation for 3 min; 98 ℃ denaturation for 15 s, 55 ℃ annealing for 15 s, 72 ℃ extension for 3 min, 30 cycles; and 72 ℃ extension for 5 min.

[0036] The PCR product is detected by 1% agarose gel electrophoresis, and after the PCR product is confirmed, Dpn I is added to remove the original template chain with methylation by enzyme digestion. The enzyme digestion product is transformed into E. coli Top10, and after overnight culture at 37 ℃, a single colony is picked and cultured in LB liquid medium, and the plasmid is extracted for gene sequence determination to determine that the mutation site is correct, and a phospholipase PLA1 mutant vector is designed and successfully constructed.

[0037] Example 2 Preparation and purification of PLA1 mutant enzyme protein

[0038] 2.1 Transformation of mutant vector

[0039] The PLA1 mutant vector constructed in Example 1 is transformed into Pichia pastoris X-33 by using the electroporation method to obtain an engineering strain.

[0040] 2.2 Culture of engineering strain

[0041] The engineering strain is inoculated into YPG culture medium for expansion culture, and when the OD value reaches 1.6-2, a primary seed liquid is obtained, which is transferred to YPG culture medium and cultured for 12-16 hours to obtain a secondary seed liquid.

[0042] The secondary seed liquid is inoculated into a fermentation tank culture medium at a ratio of 1:10 for high-density fermentation, and when the wet weight of the bacterial cells reaches 150-180 g / L, induction expression is performed, the inducer is 1% methanol, and the bacteria are collected after induction for 72-108 hours. The bacterial liquid is centrifuged at 10,000 rpm for 20 min, and the supernatant is collected, which is the crude enzyme liquid.

[0043] 2.3 Purification of PLA1 mutant enzyme protein

[0044] ​The PLA1 mutant crude enzyme solution was concentrated and desalted with 20 mM pH 7.4 PBS buffer, and then loaded onto a Ni-NTA agarose gel column at a flow rate of 2 mL / min, and then eluted with 200 mM pH 7.4 PBS buffer containing 300 mM NaCl to obtain the purified PLA1 mutant enzyme protein.

[0045] 2.4 Electrophoretic identification of PLA1 mutant enzyme protein

[0046] To stabilize the protein for storage, the protein prepared in 2.3 was exchanged to 20 mM PBS buffer at pH 7.4 to obtain PLA1 mutant with a purity of more than 90%.

[0047] The prepared protein was detected by SDS-PAGE, and the results are shown in Figure 2 The lanes are Maker, PLA1-WT, PLA1-D276-304, PLA1-D273-310, PLA1-D267-310, Maker, PLA1-WT, PLA1-N87Q / W88R, PLA1-S82G / S83G / S84G, PLA1-L145Y, and PLA1-L145T, respectively. It can be seen that PLA1 and PLA1 mutants have good purification effect, and the molecular weight of the protein is about 33.77 kDa, 30.6 kDa, 29.70 kDa, 28.77 kDa, 33.77 kDa, 33.77 kDa, 33.77 kDa, 33.77 kDa, and 33.77 kDa, respectively.

[0048] 2.5 Determination of the concentration of the target protein

[0049] 20 μL of the protein solution to be tested was mixed with 200 μL of Bradford reagent, and the A595 was measured after reaction at room temperature for 5 min. The protein concentration of the PLA1 mutant was calculated to be 0.9 mg / mL, 0.28 mg / mL, 0.35 mg / mL, 0.58 mg / mL, 0.65 mg / mL, 0.78 mg / mL, 0.6 mg / mL, 0.81 mg / mL, and 0.92 mg / mL, respectively, according to the standard curve.

[0050] Example 3 Performance determination of PLA1 mutant lipase

[0051] 3.1 Definition of enzyme activity and calculation of enzyme activity

[0052] Definition of enzyme activity: Under certain reaction conditions, the amount of enzyme required to catalyze the hydrolysis of 1 μmol of fatty acid per minute is defined as one unit of enzyme activity, represented by U, i.e. 1 U.

[0053] Enzyme activity was calculated by the following formula:

[0054] X = [(V1-V0) x 50] / (t x c x v) t x c x v )

[0055] Wherein: X is specific enzyme activity, U / mg; V1: the volume of sodium hydroxide consumed by the experimental group, mL; V0: the volume of sodium hydroxide consumed by the control group, mL; t: reaction time, min; c: protein concentration of the reaction enzyme solution, mg / mL; v: the volume of enzyme solution added, mL.

[0056] 3.2 Enzyme activity determination method

[0057] Olive oil emulsion preparation: weigh polyvinyl alcohol (PVA) 40 g, add water 800 mL, heat in boiling water bath, stir until completely dissolved, cool to volume to 1000 mL. Filter with clean double gauze, take the filtrate for use. Take 150 mL of the above filtrate, add 50 mL of olive oil, process with a high-speed homogenizer for 6 minutes, and obtain a milky white olive oil emulsion. This solution is prepared fresh.

[0058] Soybean lecithin emulsion preparation: weigh soybean lecithin 4 g, Triton X-100 25 g, add water to dissolve and make up to 100 mL, process with a high-speed homogenizer for 6 minutes, and obtain a soybean lecithin emulsion. This solution is prepared fresh.

[0059] In a 50 mL stoppered flask, add 4 mL of soybean lecithin emulsion (substrate for phospholipase activity determination) or olive oil emulsion (substrate for lipase activity determination) and 5 mL of 20 mM phosphate buffer, preheat in a constant temperature water bath shaker for 5 min, add 1 mL of PLA1 wild type or PLA1 mutant pure enzyme solution to the experimental group, add 1 mL of corresponding 100°C inactivated enzyme solution to the control group, react at 200 rpm for 5 min, then add 15 mL of 95% ethanol to terminate the reaction.

[0060] After the reaction is completed, add 2 drops of 1% phenolphthalein solution, titrate with 0.05 mol / L NaOH standard solution, calculate the volume of NaOH consumed, repeat each experiment three times, and then calculate the phospholipase or lipase activity units.

[0061] 3.3 Determination of optimum pH

[0062] The emulsion of soybean lecithin was used as the substrate, and the phospholipase and lipase activities were determined in 20 mM reaction buffer with pH values of 3.0, 4.0, 5.0, 6.0 and 7.0 (pH 3.0-5.0: citric acid-sodium phosphate buffer, pH 6.0-7.0: sodium phosphate-sodium dihydrogen phosphate buffer) at 45°C, and each group of experiments was repeated three times. The pH value was used as the horizontal coordinate, and the specific enzyme activity was used as the vertical coordinate to draw a curve.

[0063] 3.4 Determination of optimal reaction temperature

[0064] The emulsion of soybean lecithin was used as the substrate, and the phospholipase and lipase activities were determined in 20 mM reaction buffer with pH values of 3.0, 4.0, 5.0, 6.0 and 7.0 (pH 3.0-5.0: citric acid-sodium phosphate buffer, pH 6.0-7.0: sodium phosphate-sodium dihydrogen phosphate buffer) at 45°C, and each group of experiments was repeated three times. The pH value was used as the horizontal coordinate, and the specific enzyme activity was used as the vertical coordinate to draw a curve.

[0065] 3.5 Determination results

[0066] The determination results are shown in Table 2 and FIG. 2. Figure 3 As shown in Table 2 and FIG. 2, compared with the wild-type PLA1, the optimal pH of the mutant PLA1-D276-304 changed the most, and the phospholipase activity remained high in the pH range of 3-9 and was higher than that of the wild type, while the lipase activity in the pH range of 3-9 was close to 0. In addition, the phospholipase activity of PLA1-D276-304 under the optimal conditions was 56.67 U / mg, which was 1.66 times that of the wild type, and was higher than that of other mutants. The phospholipase activity of other mutants did not increase and the pH change was not obvious.

[0067] In addition, the experimental results showed that only PLA1-D276-304 exhibited lipase activity loss, and other mutants still had certain lipase activity. In the oil degumming process, if the enzyme itself still retains certain lipase catalytic activity, it will degrade the oil components that need to be retained in the raw material, reduce the yield and easily produce impurities. Therefore, reducing the lipase activity is a key indicator for the enzyme process of oil degumming. Therefore, according to the above results, it can be seen that the mutant PLA1-D276-304 not only improves the pH tolerance, but also completely removes its lipase activity, and the total phospholipase activity is also improved, which is the most suitable mutant for the oil degumming process.

[0068] Table 2 Determination results of enzyme activities of wild-type PLA1 and mutants

[0069]

[0070] The optimal pH determination results are shown in Table 2 and FIG. 2. Figure 4As shown in the table, the optimal reaction pH of PLA1-D276-304 of the present application is 8.0, compared with the optimal pH of 3.0 of the wild type PLA1, which is more suitable for alkaline environment, and the phospholipase activity of the PLA1 mutant remains high at pH 3-9, indicating that the PLA1-D276-304 of the present application is suitable for a wide pH range and can be applied to various conditions.

[0071] The optimal reaction temperature determination results are as shown in the table Figure 5 As shown in the table, the optimal reaction temperature of PLA1-D276-304 of the present application is 50℃, which is not much different from the optimal temperature (45℃) of the wild type PLA1, indicating that the PLA1-D276-304 of the present application can achieve good catalytic effect at medium temperature.

[0072] Example 4: Detection of the effect of PLA1-D276-304 for oil degumming

[0073] 4.1 Experimental method

[0074] Take 300 g of soybean crude oil and walnut crude oil, heat to 70℃ in a water bath, then add 0.18 mL of 45wt% citric acid, and homogenize at 10000 rpm for 1 min. After stirring at 70℃ for 25 min, cool to 40℃. Add a certain amount of 4% NaOH solution to adjust the pH of the system to 4, add 3% water and 20000 U / kg enzyme solution, homogenize at 10000 rpm for 1 min, then react at 500 rpm for 5 h. After the reaction is completed, warm to 90℃, and inactivate the enzyme for 10 min. Centrifuge at 10000 rpm for 10 min, collect the upper oil phase, and measure the phosphorus content. The analysis method of phosphorus content in oil and fat refers to the molybdenum blue colorimetric method in GB5537-1985.

[0075] 4.2 Experimental results

[0076] The experimental results are as shown in the table Figure 6As shown, it can be seen that, compared with the wild type PLA1, the degumming of crude oil using the mutant PLA1-D276-304 can reduce the phosphorus content in soybean crude oil from 197.0 mg / kg to 13.8 mg / kg and in walnut crude oil from 138.0 mg / kg to 20.4 mg / kg under the same reaction time and reaction conditions, while the PLA1-D276-304 of the application can reduce the phosphorus content in soybean crude oil from 197.0 mg / kg to 3.5 mg / kg and in walnut crude oil from 138.0 mg / kg to 6.7 mg / kg, meeting the requirement of phospholipid content less than 10 mg / kg, and achieving good degumming effect of soybean crude oil and walnut crude oil, showing that it has application value and prospect, and can be applied to the degumming of soybean oil, walnut oil, rapeseed oil, corn oil and other oils.

[0077] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be included in the protection scope of the present application.

Claims

1. A phospholipase A1 mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. The phospholipase A1 mutant according to claim 1, characterized in that: The gene encoding the phospholipase A1 mutant is shown in SEQ ID NO.

4.

3. The phospholipase A1 mutant according to claim 1, characterized in that: The phospholipase A1 mutant is obtained by knocking out amino acids from position 276 to 304 of phospholipase A1.

4. A recombinant expression vector, characterized in that: Includes the encoding gene of the phospholipase A1 mutant as described in claim 2.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector has a vector backbone of pPICZαA.

6. An engineered bacterium, characterized in that: Includes the recombinant expression vector as described in claim 4 or 5.

7. The engineered bacteria according to claim 6, characterized in that: The starting strain of the engineered bacteria is Pichia pastoris X-33.

8. The application of the phospholipase A1 mutant according to any one of claims 1 to 3 in oil degumming. The oil is at least one of crude walnut oil and crude soybean oil.

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