Phospholipase D mutant and its application

Abstract

The present invention belongs to the field of biological enzymes and specifically relates to phospholipase D mutants and their applications. The phospholipase D mutants have an amino acid sequence containing at least one of the following mutations: T346V, K653L, S697Y, T346H, S697P, T346L, and K653A, in the wild-type amino acid sequence shown in SEQ ID NO. 1. The mutants described in the present invention significantly improve enzyme stability and catalytic activity, broaden the range of reaction conditions, and reduce the difficulty of process control. They also significantly enhance substrate affinity and catalytic efficiency, reduce enzyme production and application costs, and are suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme proteins, and in particular relates to the field of phospholipase D mutation. 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, and has a broad market prospect. Currently, phosphatidylserine is mainly obtained by the following two methods: (1) Extraction method, which is to extract PS from plant cells and animal lecithin. Since the content of PS in plants is relatively low, it is usually extracted from animal sources such as cow brain and pig brain. This method has low extraction efficiency because the concentration of PS in natural raw materials is not high. A large amount of raw materials needs to be processed to obtain a certain amount of PS, which is costly. Moreover, due to problems such as mad cow disease, the safety of PS from animal sources has been questioned. At the same time, this method requires the use of organic chemical reagents, which has a series of problems such as by-products and environmental unfriendliness. (2) Enzymatic synthesis. Phospholipases are widely present in animal, plant and microbial cells and mainly act on phospholipid compounds. Among them, phospholipase D (PLD; EC 3.1.4.4) acts on the phosphodiester bond of phospholipid substrates and can catalyze two types of reactions: phosphodiester bond hydrolysis and transacylation. The substrates that can be catalyzed include phosphatidylcholine, phosphatidylethanolamine and other soluble phospholipids. Therefore, the bioenzymatic synthesis 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. It is more suitable for large-scale production of PS. Therefore, phospholipase D has become an important raw material in the PS production process and has become a research hotspot for major scientific research institutions and enterprises.

[0003] However, the main problems of the existing enzymatic synthesis method of phosphatidylserine are:

[0004] (1) Existing enzyme sources generally have low transphosphatidyltransferase activity. Due to the cytotoxicity of recombinant phospholipase D to Escherichia coli, its overexpression in Escherichia coli will aggravate the hydrolysis of major components of the cell membrane, such as phospholipids acetylethanolamine (PE) and phosphatidylcholine (PC), leading to a significant increase in the content of intracellular phosphatidic acid (PA), exacerbating the death of host cells.

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

[0006] (3) The catalytic performance problem of the enzyme is mainly reflected in its poor stability. The reaction process requires a high temperature of 40~60℃, and the enzyme is easily inactivated, resulting in a low catalytic synthesis yield of PS and high cost.

[0007] To improve the effectiveness of PLD, several mutation-based improvements have been proposed. For example, Chinese patent publication CN116855474A discloses an L346M mutant. Chinese patent publication CN111004787A discloses a Streptomyces phospholipase D mutant with Q357F and Y405A.

[0008] In summary, although there are some methods for PLD mutation in the existing technology, there are many types of wild-type PLD, and there are many sites at which wild-type PLD can theoretically mutate. The functions and effects of mutations in different sequences are unpredictable. Therefore, the development of highly active and stable mutants remains an industry-wide challenge. Summary of the Invention

[0009] In view of the shortcomings of the existing technology, the first purpose of the present invention is to provide a new phospholipase D mutant, aiming to provide a new PLD mutant with excellent stability and phosphatidyl transferase activity.

[0010] The second purpose of the present invention is to provide a method for performing a phospholipases D mutant phospholipases D mutant reaction, aiming to utilize the novel mutant to efficiently synthesize phosphatidyl products.

[0011] There are many different wild-type PLDs, and the number of wild-type PLD amino acid sequences is enormous. Finding highly active mutants from this vast array of mutations is extremely challenging. In light of the current state of PLD mutations, the present invention, after in-depth research, provides the following solutions:

[0012] A phospholipase D mutant has an amino acid sequence having 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.

[0013] The present invention shows that the innovative special mutation of the site of the wild amino acid sequence shown in SEQ ID NO.1 can unexpectedly improve the catalytic activity and stability of the enzyme, and its use in enzymatic phosphatidyl transfer reactions can effectively improve the reaction conversion rate.

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

[0015] The phospholipase D mutant of the present invention preferably has an amino acid sequence in which one to three mutations among T346V, K653L, S697Y, T346H, S697P, T346L, and K653A exist in the wild amino acid sequence shown in SEQ ID NO.1.

[0016] The present invention shows that a single point mutation of K653L in the wild 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 comprises an amino acid sequence having the K653L mutation in the wild-type amino acid sequence of SEQ ID NO. 1, and further comprising at least one of the following mutations: T346H and S697P. Studies in the present invention have shown that, in addition to the K653L mutation, the combined use of two or three mutations of T346H and S697P promotes synergistic effects based on the position and pattern of the mutations, further improving the enzymatic activity and enzymatic reaction performance of the mutant.

[0018] Preferably, the phospholipase D mutant has the amino acid sequence shown in SEQ ID NOs. 2 to 7, preferably the amino acid sequence shown in SEQ ID NOs. 3, 5, and 7. Studies have shown that preferred mutant amino acid sequences can achieve better mutant enzyme activity and stability.

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

[0020] The present invention also provides an application of the phospholipase D mutant as an enzyme catalyst for phosphatidyl transfer reaction.

[0021] In the present invention, a phospholipid substrate and an alcohol substrate are subjected to a phosphatidyl acylation reaction under the catalysis of an enzyme catalyst to obtain a phosphatidyl acylation product.

[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 optional solution, the phospholipid acylation product can be obtained by subjecting the substrate of Formula 1 and the substrate of Formula 2 to a phospholipid acylation reaction under the catalysis of an enzyme catalyst to obtain the phospholipid acylation product of Formula 3;

[0024] Formula 1;

[0025] Formula 2;

[0026] Formula 3;

[0027] The R1 and R2 are independently C2~C 20 A saturated or partially unsaturated carbon chain; the carbon chain is allowed to have a substituent.

[0028] The R3 is C1~C 10 wherein the alkyl group is allowed to have a substituent.

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

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

[0031] As an optional solution, Formula 1 may be lecithin. Formula 2 may be at least one of ethanolamine, serine, inositol, and glycerol.

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

[0033] In the present invention, the pH of the phosphatidyl transfer reaction is 3.5 to 7, preferably 4 to 5. Studies have shown that the innovative use of the mutants, combined with the combined control of the pH of the enzymatic reaction, is expected to further synergistically improve the enzymatic reaction activity and the phosphatidyl transfer reaction effect.

[0034] In the present invention, the phosphatidyl transfer reaction temperature is 20-60°C, preferably 35-50°C, and further 35-45°C. Studies have shown that the innovative use of the mutants, combined with the combined control of the enzymatic reaction temperature, is expected to further synergistically improve the enzymatic reaction activity and the phosphatidyl transfer reaction effect.

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

[0036] In the present invention, the phospholipid acylation reaction time may be 2 to 8 hours; considering the reaction efficiency, it may be further 2.5 to 5 hours.

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

[0038] Using lecithin as a substrate, the phosphatidyl acylation reaction is carried out under the action of an 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 unexpectedly exhibits better enzymatic activity than the wild type, can significantly reduce the production and application costs of the enzyme, and is suitable for industrial-scale production and application.

[0041] For example, the mutants described in the present invention are innovatively used in the synthesis of PS to obtain excellent conversion rate and product purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the HPLC peak diagram of the synthesis of phosphatidylserine catalyzed by phospholipase D in Example 3. DETAILED DESCRIPTION

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

[0044] The amino acid sequence of phospholipase D (SrPLD) from Streptomyces racemochromogenes (SEQ ID NO. 1) was downloaded from the NCBI database (Sequence ID: BAJ15265.1). After codon optimization for E. coli expression, the amino acid sequence was submitted to General Biotechnology (Anhui) Co., Ltd. for synthesis. The gene sequence contained an NdeI restriction site at the 5' end, an XhoI restriction site at the 3' end, and a nucleotide sequence carrying six His tags at the 3' end. The synthesized gene sequence was cloned into the prokaryotic expression plasmid pET30a(+) to construct the recombinant expression plasmid pET30a(+)-SrPLD. The recombinant plasmid was verified by sequencing and transformed into the E. coli expression host strain BL21(DE3), resulting in a recombinant E. coli strain containing the phospholipase D gene.

[0045] Carefully pick a single colony of the recombinant strain containing the SrPLD gene from a LB solid medium plate (containing 50 µg / mL kanamycin) using a sterile pipette tip and inoculate it into a flask containing 20 mL of LB liquid medium. Incubate the culture at 37°C, 200 rpm, and shake overnight. The next day, inoculate 1% of the inoculum from the shake flask into a flask containing 100 mL of TB liquid medium. Incubate the culture at 37°C, 220 rpm, and measure the OD value of the culture every hour. When the OD value reaches 1.5, add lactose to a final concentration of 1% (m / v). Continue incubating at 25°C, 220 rpm, and continue incubation for 4–6 hours. Cultivation is then terminated, and the cells are harvested and crushed to obtain the crude phospholipase D enzyme solution.

[0046] The hydrolysis activity of phospholipase D was determined under the following conditions:

[0047] The reaction system consisted of a 100 μL volume containing 60 μL of substrate solution (10 mg / mL phosphatidylcholine, 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 was 40°C for 20 minutes. The reaction was terminated by boiling with 50 mL of 50 mM EDTA solution for 5 minutes. After cooling, 500 μL of colorimetric solution (40 mM Tris-HCl, 0.75 U choline oxidase, 0.5 U peroxidase) was added and heated at 37°C for 30 minutes. The absorbance of the reaction solution was measured at 505 nm. A calibration curve was generated using a choline chloride standard solution in place of the enzyme solution. Each experiment was repeated three times.

[0048] The hydrolytic activity unit (U) of phospholipase D was 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 a Library of Error-Prone Mutants of Phospholipase D (SrPLD) from Streptomyces racemochromogenes

[0050] The phospholipase D plasmid pET30a(+)-SrPLD was extracted and analyzed by 0.8% agarose gel electrophoresis. Error-prone PCR reactions were then performed using this plasmid as a template. Recombinant plasmid DNA was constructed from the error-prone PCR products and transformed into Escherichia coli cells to generate an error-prone mutant library containing various phospholipase D mutants. This library of phospholipase D error-prone mutants was screened using high-throughput screening. Single colonies from the mutant library were picked with a sterile pipette tip and transferred to a 96-well culture plate containing LB liquid medium. The 96-well cell culture plate was incubated in a thermostatic shaker at 37°C and 700 rpm for 6 hours. Then, 50 µL of the culture medium was transferred using an 8-channel pipette to a new 96-well plate as seed stock. Lactose was then added to each well to a final concentration of 1% (m / v) and induced for 8 hours at 25°C and 250 rpm. After induction, the 96-well cell culture plate was placed in a -86°C ultra-low temperature freezer for 2 hours, then removed and allowed to stand at room temperature for half an hour. The plates were then centrifuged at 4000 rpm for 20 minutes at 4°C, and 50 μL of supernatant was collected from each well. The 96-well plate containing 50 μL of supernatant was placed in a 50°C incubator for 60 minutes. Then, 100 μL of substrate solution (10 mg / mL phosphatidylcholine, 0.1% (v / v) Triton X-100, 15 mM CaCl2, 40 mM Tris-HCl (pH 7.5)) was added to each well and incubated at 40°C for 30–60 minutes. The reaction was terminated by boiling with 50 mL of 50 mM EDTA solution for 5 minutes. After cooling, 500 μL of colorimetric solution (40 mM Tris-HCl, 0.75 U choline oxidase, 0.5 U peroxidase) was added and heated at 37°C for 30 min.

[0051] The detection and analysis were performed using a microplate reader (detection wavelength 505 nm), and the wells with high absorbance values ​​were selected for further analysis and verification. The activity was measured, and the nucleotide and amino acid mutations were analyzed by sequencing and compared. The results are shown in Table 1.

[0052]

[0053] By screening a library of error-prone phospholipase D mutants from over 100,000 strains, we obtained the three favorable mutants shown in Table 1. Their thermostability and enzyme activity were significantly improved compared to the wild type. To further enhance their activity and stability, we selected the mutants SrPLD-T346V, SrPLD-K653L, and SrPLD-S697Y as starting strains. Plasmids were extracted, and saturation mutation libraries were constructed and screened at the K653, T346, and S697 sites, respectively. Primers for saturation mutations were designed as shown in Table 2.

[0054]

[0055] Based on the mutants SrPLD-T346V, SrPLD-K653L and SrPLD-S697Y, a high-throughput screening of the superimposed saturation mutation library was performed. The amino acid sequence and enzyme property analysis of the obtained favorable mutants were performed. The results are shown in Table 3:

[0056]

[0057] As can be seen from Table 3, after superimposed mutations, the most obvious effect is the SrPLD-K653L-T346H mutant, whose activity can reach 1.82 U / mL and its thermal stability at 50 ℃ does not decrease significantly.

[0058] Example 3: Verification of Phosphatidylserine Synthesis After the above mutants were fermented and cultured, the same amount of cells were taken, and the phospholipase D protein of each mutant was isolated and purified, and the synthesis of phosphatidylserine was studied. The reaction system was as follows: 48 g of soybean lecithin (PC 50% content) was weighed, 4 g of anhydrous CaCl2 was added, and stirred and dissolved in 160 mL of 0.2 M pH 5.0 acetic acid-sodium acetate buffer to an emulsified state, and then 50 g of serine and 100 U of different mutant phospholipase D samples were added. The reaction system was controlled to 500 mL, the reaction temperature was controlled to 40 ° C, the reaction pH was 5.0, the stirring speed was set to 150 rpm, the reaction time was 3 h, and the yield of phosphatidylserine was monitored during the process (the HPLC detection method refers to the "Light Industry Standard of the People's Republic of China QB / T5821-2023"). The method described in Appendix A (Determination of Phosphatidylserine Content) of "Phosphatidylserine" was used. Other enzyme performance tests were also performed on the mutant enzymes. The results are shown in Table 4:

[0059]

[0060] Note: T346L refers to the mutation of T at position 346 of the wild amino acid sequence shown in SEQ ID NO.1 to L; K653A refers to the mutation of K at position 653 of the wild amino acid sequence shown in SEQ ID NO.1 to A;

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

[0062]

[0063] From the above experiments, it can be seen that the mutants of phospholipase D have obvious differences in their catalytic synthesis of phosphatidylserine under the conditions of identical enzyme dosage. 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] The optimal mutant SrPLD-K653L-T346H from Example 3 was selected for further optimization of reaction parameters. The reaction system was adjusted to the following: 96 g of soybean lecithin (50% PC) was weighed into a 1 L reaction system, and 8 g of anhydrous CaCl₂ was added. The mixture was dissolved in buffer solutions of varying pH values ​​until emulsified. The reaction system was then optimized using various parameters, including reaction temperature, reaction speed, and pH. The results are shown in Table 6.

[0066]

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

[0068] Although the present invention has been disclosed above in terms of 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 scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A phospholipase D mutant, characterized in that: The amino acid sequence of the phospholipase D mutant is the amino acid sequence shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.

6.

2. A use of the phospholipase D mutant according to claim 1, characterized in that: It is used as an enzyme catalyst for phosphatidyl transfer reactions.

3. The use of the phospholipase D mutant according to claim 2, characterized in that: A phospholipid substrate and an alcohol substrate are subjected to a phosphatidyl acylation reaction under the catalysis of an enzyme catalyst to obtain a phosphatidyl acylated product.

4. The use of the phospholipase D mutant according to claim 3, wherein: 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.

5. The use of the phospholipase D mutant according to claim 3, characterized in that: The pH of the phosphatidylserine reaction is 3.5 ~ 7.

6. The use of the phospholipase D mutant according to claim 3, characterized in that: The temperature of the phosphatidyl transfer reaction is 20~60℃.

7. The use of the phospholipase D mutant according to claim 3, wherein: The enzyme catalyst is used in an equivalent amount of 2 to 10 U / g relative to the phospholipid substrate.

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