Phospholipase D mutant and application thereof

Abstract

The invention belongs to the field of biological enzymes, and particularly relates to a phospholipase D mutant and application thereof, and the phospholipase D mutant is an amino acid sequence with at least one mutation of T346V, K653L, S697Y, T346H, S697P, T346L and K653A in a wild amino acid sequence as shown in SEQ ID NO.1. The phospholipase D mutant is a phospholipase D mutant with at least one mutation of T346V, K653L, S697Y, T346H, S697P, T346L and K653A. According to the mutant disclosed by the invention, the stability and catalytic activity of enzyme are remarkably improved, the reaction condition range is widened, and the process control difficulty is reduced. The affinity and catalytic efficiency to a substrate are remarkably improved, the production and application cost of the enzyme is reduced, and the method is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme proteins, and particularly relates to a field of phospholipase D mutants. Background Art

[0002] Phosphatidylserine (PS), as an important component of cell membranes, plays an important role in promoting brain function, promoting brain development, enhancing memory, treating depression, and sports health care, etc., and has a broad market prospect. At present, there are mainly the following two methods for obtaining phosphatidylserine: (1) Extraction method, which extracts PS from plant cells and lecithin of animals. Since the PS content in plants is relatively low, it is usually extracted from animal sources such as bovine brain and porcine brain. This method has low extraction efficiency because the PS concentration in natural raw materials is not high, and a large amount of raw materials need to be processed to obtain a certain amount of PS, resulting in high costs; moreover, due to problems such as mad cow disease, the safety of PS from animal sources is questioned; at the same time, this method requires the use of organic chemical reagents, resulting in a series of problems such as by-products and environmental unfriendliness; (2) Enzymatic catalytic synthesis, phospholipases are widely present in animal, plant and microbial cells and mainly act on phospholipid compounds. Among them, the action site of phospholipase D (PLD; EC 3.1.4.4) lies in the phosphodiester bond of phospholipid substrates, and it can catalyze two types of reactions: hydrolysis of phosphodiester bonds and transacylation reactions. The substrates that can be catalyzed include phosphatidylcholine, phosphatidylethanolamine, and other soluble phospholipids. Therefore, the biosynthesis of PS mediated by phospholipase D (PLD) has attracted much attention due to its advantages such as simple operation, mild reaction conditions, and environmental friendliness, and is more suitable for the large-scale production of PS. Therefore, phospholipase D has become an important raw material in the production process of PS and has become a research hotspot for major scientific research institutions and enterprises.

[0003] However, considering the existing methods for enzymatic catalytic synthesis of phosphatidylserine, the main problems are as follows:

[0004] (1) Existing enzyme sources generally have relatively low transphosphatidylation activity. Due to the cytotoxicity of recombinant phospholipase D to Escherichia coli, its overexpression in Escherichia coli will exacerbate the hydrolysis of the main components of the cell membrane, such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC), and thus lead to a significant increase in the content of phosphatidic acid (PA) in the cell, exacerbating the death of the host cell;

[0005] (2) The production and manufacturing cost of the enzyme during the reaction process is too high, which is not conducive to industrial large-scale production and large-scale application of products;

[0006] (3) The catalytic performance problems of the enzyme are mainly reflected in poor stability. The reaction process requires a relatively high temperature of 40 - 60 °C for the reaction, and the enzyme is easily inactivated, resulting in a low catalytic synthesis yield of PS and high costs, etc.

[0007] To improve the effect of PLD, there are also some mutant improvement schemes in the prior art. For example, a Chinese patent document with the publication number CN116855474A discloses an L346M mutant. A Chinese patent document with the publication number CN111004787A discloses Streptomyces phospholipase D mutants of Q357F and Y405A.

[0008] In summary, although there are some mutant means of PLD in the prior art, there are a wide variety of wild-type PLDs, and there are numerous sites that can be mutated in theory for wild-type PLDs. The functions and effects of different sequence mutations cannot be predicted. Therefore, developing mutants with high activity and high stability still belongs to an industry-wide problem. Summary of the Invention

[0009] Aiming at the deficiencies of the prior art, the first object of the present invention is to provide a novel phospholipase D mutant, aiming to provide a novel PLD mutant that takes into account excellent stability and phosphatidylation reaction activity.

[0010] The second object of the present invention is to provide a method for phosphatidylation reaction using the phospholipase D mutant, aiming to efficiently synthesize phosphatidylated products using the novel mutant.

[0011] There are a wide variety of wild-type PLDs, and the number of amino acids in the amino acid sequences of each wild-type PLD is numerous. It is extremely difficult to find a highly active mutant from this huge mutation method. In view of the current situation of PLD mutation, the present invention has conducted in-depth research and provides the following solutions:

[0012] A phospholipase D mutant having an amino acid sequence with at least one mutation of T346V, K653L, S697Y, T346H, S697P, T346L, K653A in the wild-type amino acid sequence shown in SEQ ID NO.1.

[0013] The research of the present invention shows that innovatively mutating the said sites of the wild-type amino acid sequence shown in SEQ ID NO.1 can unexpectedly improve the enzyme catalytic activity and stability. Using it in the enzymatic phosphatidylation reaction can effectively improve the reaction conversion rate.

[0014] In the present invention, T346V means that the T at position 346 of the wild-type amino acid sequence shown in SEQ ID NO.1 is mutated to V; K653L means that the K at position 653 of the wild-type amino acid sequence is mutated to L; S697Y means that the S at position 697 of the wild-type amino acid sequence is mutated to Y; T346H means that the T at position 346 of the wild-type amino acid sequence is mutated to H; S697P means that the S at position 697 of the wild-type amino acid sequence is mutated to P; T346L means that the T at position 346 of the wild-type amino acid sequence shown in SEQ ID NO.1 is mutated to L; K653A means that the K at position 653 of the wild-type amino acid sequence is mutated to A.

[0015] The phospholipase D mutant of the present invention is preferably an amino acid sequence having 1 to 3 mutations among T346V, K653L, S697Y, T346H, S697P, T346L, and K653A in the wild-type amino acid sequence shown in SEQ ID NO.1.

[0016] Research in the present invention shows that single-point mutation of K653L in the wild-type amino acid sequence shown in SEQ ID NO.1 is expected to further improve the performance of the phospholipase D mutant compared with other single-point mutations.

[0017] Preferably, the phospholipase D mutant is an amino acid sequence having a K653L mutation in the wild-type amino acid sequence shown in SEQ ID NO.1 and further comprising at least one mutation among T346H and S697P. Research in the present invention shows that on the basis of the K653L mutation, further combined double-site or three-site mutations of T346H and S697P contribute to the combined synergy based on the mutation position and mutation method, and contribute to further improving the enzyme activity and enzyme-catalyzed reaction effect of the mutant.

[0018] Preferably, the phospholipase D mutant has the amino acid sequences shown in SEQ ID NO.2 to 7; preferably has the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.7. Research shows that the preferred mutant amino acid sequence can obtain better mutant enzyme activity and stability.

[0019] The phospholipase D mutant of the present invention can be prepared based on known mutation means.

[0020] The present invention also provides an application of the phospholipase D mutant, using it as an enzyme catalyst for phosphatidylation reaction.

[0021] In the present invention, a phosphatidylation product is obtained by carrying out a phosphatidylation reaction on a phospholipid substrate and an alcohol substrate under the catalysis of an enzyme catalyst.

[0022] In the present invention, the excellent biocatalytic ability of the phospholipase D mutant can be utilized to catalyze the transesterification reaction between the phospholipid group in the phospholipid substrate and the alcohol substrate.

[0023] For example, as an alternative, the phosphatidylation product can specifically be obtained by subjecting the substrate of Formula 1 and the substrate of Formula 2 to a phosphatidylation reaction under the catalysis of an enzyme catalyst to obtain the phosphatidylation product of Formula 3;

[0024] Formula 1;

[0025] Formula 2;

[0026] Formula 3;

[0027] The R1 and R2 are each independently a saturated or partially unsaturated carbon chain having 2 to C 20 ; substituents are allowed on the carbon chain.

[0028] The R3 is an alkyl group having 1 to C 10 ; substituents are allowed on the alkyl group.

[0029] The R4 is a straight-chain alkyl group, a branched-chain alkyl group, a cycloalkyl group, or an alkyl group with substituents.

[0030] The substituents can be at least one of a hydroxyl group, an amino group, and a carboxyl group.

[0031] As an alternative, Formula 1 can be lecithin. The Formula 2 can be at least one of ethanolamine, serine, inositol, and glycerol.

[0032] The molar ratio of the phospholipid substrate to the alcohol substrate is 1:1 to 1:5.

[0033] In the present invention, the pH of the phosphatidylation reaction is 3.5 to 7; preferably 4 to 5. Research shows that with the innovative use of the mutant and further combined control of the pH of the enzymatic reaction, it is expected to further synergistically improve the enzymatic reaction activity of the present invention and improve the effect of the phosphatidylation reaction.

[0034] In the present invention, the temperature of the phosphatidylation reaction is 20 to 60 °C, preferably 35 to 50 °C; further preferably 35 to 45 °C. Research shows that with the innovative use of the mutant and further combined control of the temperature of the enzymatic reaction, it is expected to further synergistically improve the enzymatic reaction activity of the present invention and improve the effect of the phosphatidylation reaction.

[0035] In the present invention, the usage equivalent of the enzyme catalyst relative to the phospholipid substrate is 2 - 10 U / g. That is, 2 - 10 U of the enzyme catalyst is used per gram of the phospholipid substrate. Considering the cost, the usage equivalent of the enzyme catalyst relative to the phospholipid substrate can further be 4 - 6 U / g.

[0036] In the present invention, the time of the phosphatidylation reaction can be 2 - 8 h; considering the reaction efficiency, it can further be 2.5 - 5 h.

[0037] In the present invention, the catalytic reaction is carried out under stirring, and the stirring speed can be reasonably adjusted according to common knowledge. For example, it can be above 100 rpm, and further can be 140 - 250 rpm.

[0038] Using lecithin as the substrate, the phosphatidylation reaction is carried out under the action of the enzyme catalyst to obtain phosphatidylethanolamine (substrate lecithin + ethanolamine), phosphatidylserine (substrate lecithin + serine), phosphatidylinositol (substrate lecithin + inositol), phosphatidylglycerol (substrate lecithin + glycerol), etc.

[0039] Beneficial effects

[0040] The present invention provides a phospholipase D mutant with a completely new mutation site and mutation mode, which can unexpectedly exhibit better enzymatic activity compared with the wild type, can significantly reduce the manufacturing cost of enzyme production and application, and is suitable for industrial scale production and application.

[0041] For example, innovatively applying the mutant described in the present invention to the synthesis of PS can obtain excellent conversion rate and product purity. Description of the drawings

[0042] Figure 1 It is the HPLC peak map of the catalytic conversion synthesis of phosphatidylserine by phospholipase D in Example 3. Detailed implementation manners

[0043] Example 1: Construction of an Escherichia coli recombinant expression strain of phospholipase D (SrPLD) derived from Streptomyces racemochromogenes

[0044] Download the amino acid sequence of phospholipase D (SrPLD, SEQ ID NO.1) from Streptomyces racemochromogenes from the NCBI database (Sequence ID: BAJ15265.1). After optimizing the codons for E. coli expression, submit it to General Biology (Anhui) Co., Ltd. for synthesis. The gene has an NdeI restriction site at the 5' end and an XhoI restriction site at the 3' end, and the 3' end of the gene carries a nucleotide sequence with 6 His tags. Clone the synthesized gene sequence into the prokaryotic expression plasmid pET30a(+), and construct the recombinant expression plasmid pET30a(+)-SrPLD. After the recombinant plasmid is verified by sequencing, transfer it into the E. coli expression host E. coli BL21(DE3), and the E. coli recombinant strain containing the phospholipase D gene is obtained.

[0045] Carefully pick a single colony of the recombinant strain containing the SrPLD gene with a sterilized pipette tip on an LB solid medium plate (containing 50 μg / mL kanamycin), and inoculate it into a triangular flask containing 20 mL of LB liquid medium. Incubate overnight at 37 °C with shaking at 200 r / min. The next day, inoculate the shake flask culture into a triangular flask containing 100 mL of TB liquid medium at an inoculation amount of 1%. Incubate at 37 °C with shaking at 220 r / min, and measure the OD value of the culture medium every 1 h. When the OD value of the culture medium = 1.5, supplement lactose with a final concentration of 1% (m / v), and continue to incubate at 25 °C with shaking at 220 rpm for 4 h - 6 h. Stop the culture, collect the cells, and break them to obtain the crude enzyme solution of phospholipase D.

[0046] The hydrolysis activity of phospholipase D is measured under the following conditions:

[0047] Reaction system: 100 μL, containing 60 μL of substrate solution (substrate solution: 10 mg / mL lecithin, 0.1% (v / v) Triton X-100, 15 mM CaCl2, 40 mM Tris-HCl (pH 7.5)) and 40 μL of enzyme sample. The reaction temperature is 40 °C and the reaction time is 20 minutes. Terminate the reaction: Boil for 5 minutes with 50 mL of EDTA solution (50 mM) to terminate the reaction. After cooling, add 500 μL of colorimetric solution (40 mM Tris-HCl, 0.75 U choline oxidase, 0.5 U peroxidase), and heat at 37 °C for 30 min. Measure the absorbance of the reaction solution at 505 nm. Obtain the calibration curve by replacing the enzyme solution with a choline chloride standard solution. Each experiment is repeated three times.

[0048] The hydrolytic activity unit (U) of phospholipase D is defined as the amount of enzyme that produces 1 μmol of choline per minute under the experimental conditions.

[0049] Example 2: Construction and screening of an error-prone mutant library of phospholipase D (SrPLD) from Streptomyces racemochromogenes

[0050] Extract the plasmid pET30a(+)-SrPLD of phospholipase D. After being detected and analyzed by 0.8% agarose gel electrophoresis without error, using this plasmid as a template, perform error-prone PCR reaction. After the error-prone PCR reaction product is used for the construction of recombinant plasmid DNA, it is transformed into Escherichia coli cells to obtain an error-prone mutant library containing different phospholipase D mutants. High-throughput screening is carried out on this phospholipase D error-prone mutant library. Use a sterilized pipette tip to pick single colonies of the mutant library into a 96-well culture plate (the culture plate is added with LB liquid medium), place the 96-well cell culture plate in a constant temperature shaker at 37 °C and 700 rpm for 6 hours, then use an 8-channel pipette to take 50 μL and store it in a new 96-well plate as a seed solution. Then add lactose with a final concentration of 1% (m / v) to each well, and induce culture at 25 °C and 250 rpm for 8 hours. After the induction culture is completed, put the 96-well cell culture plate into an ultra-low temperature refrigerator at -86 °C for 2 hours, take it out and place it at room temperature for half an hour, centrifuge at 4000 r / min and 4 °C for 20 minutes, and take 50 μL of the supernatant from each well. Place the 96-well plate containing 50 μL of the supernatant in a constant temperature incubator at 50 °C for 60 min, and then add 100 μL of substrate solution (substrate solution: 10 mg / mL lecithin, 0.1% (v / v) Triton X-100, 15 mM CaCl2, 40 mM Tris-HCl (pH 7.5)) to each well, and incubate at 40 °C for 30 - 60 min. Use 50 mL of EDTA solution (50 mM) to boil for 5 minutes to terminate the reaction. After cooling, add 500 μL of colorimetric solution (40 mM Tris-HCl, 0.75 U choline oxidase, 0.5 U peroxidase), and heat at 37 °C for 30 min.

[0051] And use an enzyme-labeled instrument (detection wavelength 505 nm) for detection and analysis. Select the wells with high absorbance values for further analysis and verification, measure the activity, sequence and analyze the nucleotide and amino acid mutations and conduct comparative analysis. The results are shown in Table 1.

[0052]

[0053] By screening a library of more than 100,000 error-prone mutants of phospholipase D, three beneficial mutants shown in Table 1 were obtained, and their thermal stability and enzyme activity were significantly improved compared with the wild type. To further enhance their activity and stability, the strains of mutants SrPLD-T346V, SrPLD-K653L, and SrPLD-S697Y were selected as the starting strains, plasmids were extracted, and on this basis, the construction and screening of overlapping saturation mutation libraries at positions K653, T346, and S697 were carried out respectively. The design of saturation mutation primers is shown in Table 2:

[0054]

[0055] Based on the mutants SrPLD-T346V, SrPLD-K653L, and SrPLD-S697Y, high-throughput screening of the overlapping saturation mutation library was carried out, and the amino acid sequences and enzyme properties of the obtained beneficial mutants were analyzed. The results are shown in Table 3:

[0056]

[0057] It can be seen from Table 3 that after the overlapping mutation, the most obvious effect is the SrPLD-K653L-T346H mutant, whose activity can reach 1.82 U / mL, and the thermal stability at 50 °C has no obvious decrease.

[0058] Example 3: Verification of phosphatidylserine synthesis After the above mutants were fermented and cultured, the same amount of bacterial cells was taken, the phospholipase D protein of each mutant was isolated and purified, and the synthesis of phosphatidylserine was studied. The reaction system is as follows: Weigh 48 g of soybean phospholipids (PC content of 50%), add 4 g of anhydrous CaCl2, stir and dissolve in 160 mL of 0.2 M acetic acid-sodium acetate buffer solution with pH 5.0 to an emulsified state, then add 50 g of serine, 100 U of phospholipase D samples of different mutants, the reaction system is controlled to 500 mL, the reaction temperature is controlled to 40 °C, the reaction pH is 5.0, the stirring speed is set to 150 rpm, and the reaction time is 3 h. During the process, the yield of phosphatidylserine is monitored (the detection method of HPLC refers to the method described in Appendix A (Determination method of phosphatidylserine content) of the National Light Industry Standard QB / T5821-2023 Phosphatidylserine of the People's Republic of China). And other properties of the above mutant enzymes were measured, and the results are shown in Table 4:

[0059]

[0060] Note: T346L mentioned above means that the T at position 346 of the wild-type amino acid sequence shown in SEQ ID NO.1 is mutated to L; K653A means that the K at position 653 of the wild-type amino acid sequence shown in SEQ ID NO.1 is mutated to A.

[0061] As can be seen from Table 4, there are differences in the performance of different mutant enzymes. Among them, the specific activity of the mutant SrPLD-K653L has a significant increase, with a specific activity of 3.59 U / mL, and the Kcat / Km (mM -1 ·S -1 ) also has a significant increase. In terms of phosphatidylserine synthesis, the synthesis experimental data are shown in Table 5:

[0062]

[0063] As can be seen from the above experiments, for the mutants of phospholipase D, under the condition of the same enzyme dosage, there are also obvious differences in their catalytic synthesis of phosphatidylserine. Among them, the mutants SrPLD-K653L and SrPLD-K653L-T346H show better performance and are more suitable for industrial scale production.

[0064] Example 4: Optimization of phosphatidylserine synthesis conditions

[0065] Select the optimal mutant SrPLD-K653L-T346H in Example 3 as the research object to further optimize the reaction parameters. Adjust the reaction system. The adjusted reaction system is as follows: The total reaction system is set to 1 L. Weigh 96 g of soybean phospholipid (PC content of 50%), add 8 g of anhydrous CaCl2, dissolve it into an emulsified state in different pH buffer solutions, and then set different reaction temperatures, reaction speeds, pH and other parameters to optimize the reaction system. The results are shown in Table 6.

[0066]

[0067] As can be seen from Table 6, the synthesis method described in the present invention has excellent process stability. And compared with Example 3, even if the processing volume is increased, there will be no obvious deterioration effect of the amplification effect, and a comparable synthesis effect can still be obtained, indicating that the technical solution has good process stability.

[0068] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone 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

1. A phospholipase D mutant, characterized in that: The amino acid sequence has at least one mutation among T346V, K653L, S697Y, T346H, S697P, T346L, and K653A in the wild amino acid sequence shown in SEQ ID NO.

1.

2. The phospholipase D mutant according to claim 1, characterized in that: The amino acid sequence has a K653L mutation in the wild amino acid sequence shown in SEQ ID NO. 1, and further includes at least one of T346H and S697P mutations.

3. The phospholipase D mutant according to claim 1, characterized in that: It has the amino acid sequence shown in SEQ ID NO.2 ~ 7.

4. The phospholipase D mutant according to claim 1, characterized in that: It has the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.

7.

5. Use of the phospholipase D mutant according to any one of claims 1 to 4, characterized in that: It is used as an enzyme catalyst for phosphatidyl transfer reactions.

6. The use of the phospholipase D mutant according to claim 5, characterized in that: A phospholipid substrate and an alcohol substrate are subjected to a phosphatidyl reaction under the catalysis of an enzyme catalyst to obtain a phosphatidyl product.

7. The use of the phospholipase D mutant according to claim 6, characterized in that: The phospholipid substrate includes lecithin; The alcohol substrate includes at least one of ethanolamine, serine, inositol, and glycerol; The molar ratio of the phospholipid substrate to the alcohol substrate is 1:1 to 1:

5.

8. The use of the phospholipase D mutant according to claim 6, characterized in that: The pH of the phosphatidyl transfer reaction is 3.5 ~7.

9. The use of the phospholipase D mutant according to claim 6, characterized in that: 20 ~ 60℃ for phosphatidylcholine reaction.

10. The use of the phospholipase D mutant according to claim 6, characterized in that: The enzyme catalyst is used in an equivalent amount of 2 to 10 U / g relative to the phospholipid substrate.

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