Phospholipase d mutant and use thereof

Abstract

The present application aims to solve the technical defects in the prior art that phospholipase D has low catalytic activity on phosphatidyl ethanolamine, especially low activity when phosphatidyl ethanolamine is converted into phosphatidyl serine. Meanwhile, in order to make full use of soybean processing by-product phosphatidyl ethanolamine, the present application provides a protein-engineered phospholipase D. The mutant phospholipase D is obtained by mutating amino acids at positions 59, 178, 231 and 409 based on the amino acid sequence of phospholipase D from Streptomyces halstedii. Compared with the original enzyme, the activity of the mutant in catalyzing PE to synthesize PS is significantly improved, with an increase of 206.5%. In the reaction of the mutant phospholipase D catalyzing PE to synthesize PS established in the present application, the product yield can be as high as 82.35%.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a highly efficient phospholipase D that catalyzes the production of phosphatidylserine from phosphatidylethanolamine and its applications. Background Technology

[0002] Phosphatidylserine (PS) is an important functional phospholipid with significant physiological activities such as improving memory, relieving stress, and repairing brain damage. It is widely used in health foods, pharmaceuticals, and cosmetics.

[0003] Currently, the main methods for preparing PS include solvent extraction, chemical synthesis, and enzyme catalysis. Among these, PS prepared by solvent extraction and chemical synthesis has been gradually phased out due to low purity and residual organic solvents. Enzyme catalysis, with its advantages of mild reaction conditions, good selectivity, and high conversion rate, is currently the primary method for PS preparation. However, industrial production of PS via enzyme catalysis still faces certain obstacles. First, commercially available phospholipase D (PLD) is expensive, while PLD produced through microbial fermentation suffers from long preparation cycles, low yields, and poor enzyme activity, all of which hinder the widespread adoption of industrial PS production. Second, due to PLD's unique catalytic mechanism—interfacial activation—a "cap" structure exists above the active site of PLD, which requires a hydrophobic interface to open, thereby activating PLD's transphosphatidyl activity (transesterification activity).

[0004] There are two pathways for phospholipase D to catalyze the synthesis of phosphatidylserine: one uses phosphatidylcholine as an acyl donor and L-serine as an acceptor; the other uses phosphatidylethanolamine as a substrate. However, the activity of existing phospholipase D in catalyzing the conversion of phosphatidylethanolamine to phosphatidylserine is far lower than its activity in catalyzing the conversion of phosphatidylcholine to phosphatidylserine. This severely restricts the development of processes for producing phosphatidylserine using phosphatidylethanolamine as a substrate. Furthermore, phosphatidylethanolamine, as one of the main byproducts of soybean oil processing, is abundant and inexpensive, but currently, a large amount of phosphatidylethanolamine is not utilized at a high value and is often treated as waste or low-value feed. Therefore, developing a highly efficient phospholipase D mutant enzyme for the preparation of phosphatidylserine from phosphatidylethanolamine would not only solve the problem of high substrate costs in phosphatidylserine synthesis but also achieve high-value conversion of soybean byproducts, resulting in significant economic and environmental benefits.

[0005] When phosphatidylethanolamine is used as a substrate, due to its poor dispersibility in the aqueous phase and the low recognition efficiency of phosphatidylethanolamine by phospholipase D, it is difficult to achieve efficient and green preparation of phosphatidylserine series products with existing technologies. Summary of the Invention

[0006] This invention aims to address the low catalytic activity of phospholipase D for phosphatidylethanolamine in existing technologies, particularly its extremely low activity during the conversion of phosphatidylethanolamine to phosphatidylserine. Furthermore, to fully utilize phosphatidylethanolamine, a byproduct of soybean processing, this invention provides a protein-engineered phospholipase D. The phospholipase D constructed in this invention not only exhibits significantly enhanced catalytic activity but also enables highly efficient catalysis of phosphatidylserine synthesis using phosphatidylethanolamine as an acyl donor. Based on these findings, this invention has been completed.

[0007] In a first aspect, the present invention provides a phospholipase D mutant that efficiently catalyzes the synthesis of phosphatidylserine from phosphatidylethanolamine. The phospholipase D mutant is obtained by mutating any one or more of the four sites, namely, position 59, position 178, position 231, or position 409, based on the amino acid sequence shown in SEQ ID NO.1.

[0008] Furthermore, the phospholipase D mutant is based on phospholipase D with an amino acid sequence as shown in SEQ ID NO.1, by mutating the amino acid at positions 59, 178, 231, or 409 to leucine, tyrosine, alanine, or asparagine.

[0009] Furthermore, the mutation is as follows: Replace the alanine at position 59 of phospholipase D, as shown in SEQ ID NO.1, with leucine; The leucine at position 178 of phospholipase D, as shown in SEQ ID NO.1, is replaced with tyrosine. Replace tryptophan at position 231 of phospholipase D, as shown in SEQ ID NO.1, with alanine; Replace the arginine at position 409 of phospholipase D, as shown in SEQ ID NO.1, with asparagine; Replace the alanine at position 59 of phospholipase D, as shown in SEQ ID NO.1, with leucine, the leucine at position 178 with tyrosine, the tryptophan at position 231 with alanine, or the arginine at position 409 with asparagine.

[0010] In one specific embodiment of the present invention, the phospholipase D mutant is formed by a combined mutation at positions 59, 178, 231, and 409 of phospholipase D, as shown in SEQ ID NO. 1. The mutation type is that alanine at position 59 is replaced with leucine, leucine at position 178 is replaced with tyrosine, tryptophan at position 231 is replaced with alanine, and arginine at position 409 is replaced with asparagine. The amino acid sequence of the phospholipase D mutant is shown in SEQ ID NO. 2.

[0011] In a second aspect, the present invention provides a plasmid loaded with the gene encoding the phospholipase D mutant as described in the first aspect.

[0012] Furthermore, the phospholipase D mutant is based on phospholipase D with an amino acid sequence as shown in SEQ ID NO.1, by mutating the amino acid at positions 59, 178, 231, or 409 to leucine, tyrosine, alanine, or asparagine.

[0013] Furthermore, the mutation is as follows: Replace the alanine at position 59 of phospholipase D, as shown in SEQ ID NO.1, with leucine; The leucine at position 178 of phospholipase D, as shown in SEQ ID NO.1, is replaced with tyrosine. Replace tryptophan at position 231 of phospholipase D, as shown in SEQ ID NO.1, with alanine; Replace the arginine at position 409 of phospholipase D, as shown in SEQ ID NO.1, with asparagine; Replace the alanine at position 59 of phospholipase D, as shown in SEQ ID NO.1, with leucine, the leucine at position 178 with tyrosine, the tryptophan at position 231 with alanine, or the arginine at position 409 with asparagine.

[0014] In one specific embodiment of the present invention, the phospholipase D mutant is formed by a combined mutation at positions 59, 178, 231, and 409 of the phospholipase D amino acid sequence as shown in SEQ ID NO. 1. The mutation type is that alanine at position 59 is replaced with leucine, leucine at position 178 is replaced with tyrosine, tryptophan at position 231 is replaced with alanine, and arginine at position 409 is replaced with asparagine. The amino acid sequence of the phospholipase D mutant is shown in SEQ ID NO. 2, and the gene encoding the phospholipase D mutant is shown in SEQ ID NO. 4.

[0015] Thirdly, the present invention provides an engineered bacterium expressing the phospholipase D mutant described in the first aspect, wherein the genome of the engineered bacterium contains the coding gene of the phospholipase D mutant, or the engineered bacterium contains a plasmid loaded with the coding gene of the phospholipase D mutant.

[0016] Furthermore, the chassis strain of the engineered bacteria is yeast or bacteria.

[0017] Furthermore, the chassis strain of the engineered bacteria is selected from one of the following: Saccharomyces cerevisiae, Pichia pastoris, Yersinia lipolytica, Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis.

[0018] Fourthly, the present invention provides the application of the phospholipase D mutant described in the first aspect in the biocatalytic synthesis of phosphatidylserine from phosphatidylethanolamine.

[0019] Furthermore, the biocatalysis includes enzyme catalysis and whole-cell catalysis.

[0020] Beneficial effects This invention provides a mutant phospholipase D, which is obtained by mutating amino acids 59, 178, 231, and 409 based on the amino acid sequence of phospholipase D derived from Streptomyces halstedii. Compared with the original enzyme, the mutant exhibits a significantly enhanced activity in catalyzing the synthesis of PS from PE, with an increase of up to 206.5%. In the reaction catalyzing the synthesis of PS from PE using the mutant phospholipase D established by this invention, the product yield can reach as high as 82.35%. Attached Figure Description

[0021] Figure 1 This is the initial HPLC chromatogram of PE, the substrate in this embodiment of the invention.

[0022] Figure 2 This is an HPLC chromatogram of the synthesis of PS from PE catalyzed by the enzyme solution of the recombinant Bacillus subtilis phospholipase D mutant in an embodiment of the present invention. Detailed Implementation

[0023] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments can be purchased through conventional commercial channels unless otherwise specified.

[0025] Example 1: Preparation of phospholipase D mutant Using the gene sequence of phospholipase D derived from Streptomyces halstedii as a template, a mutation site was introduced using site-directed mutagenesis. The amino acid sequence of phospholipase D derived from Streptomyces halstedii is shown in SEQ ID NO.1.

[0026] SEQ ID NO.1, the amino acid sequence of phospholipase D derived from Streptomyces halstedii (abbreviated as ShPLD): *; The nucleotide sequence encoding phospholipase D derived from Streptomyces halstedii is shown in SEQ ID NO.17.

[0027]

[0028] Based on phospholipase D derived from Streptomyces halstedii, site-directed mutations were performed at amino acid positions 59, 178, 231, and 409 to obtain the following phospholipase D mutants: PLD-1, with the amino acid sequence shown in SEQ ID NO.2, is obtained by replacing the alanine at position 59 of phospholipase D (as shown in SEQ ID NO.1) with leucine; it is abbreviated as A59L. *; PLD-2, with the amino acid sequence shown in SEQ ID NO.3, is a modified version of phospholipase D with the amino acid sequence shown in SEQ ID NO.1 having the leucine at position 178 replaced by a tyrosine, abbreviated as L178Y. *; PLD-3, with the amino acid sequence shown in SEQ ID NO.4: PLD-3 is formed by replacing tryptophan at position 231 of phospholipase D as shown in SEQ ID NO.1 with alanine, abbreviated as W231A. *; PLD-4, with the amino acid sequence shown in SEQ ID NO.5, is a modified version of phospholipase D with the amino acid sequence shown in SEQ ID NO.1 having arginine at position 409 replaced by asparagine, abbreviated as R409N. *; PLD-5, with the amino acid sequence shown in SEQ ID NO.6, is a modified version of phospholipase D with the amino acid sequence SEQ ID NO.1 having alanine at position 59 replaced by leucine and tyrosine at position 178 replaced by leucine. It is abbreviated as A59L / L178Y. *; PLD-6, with the amino acid sequence shown in SEQ ID NO.7, is a modified version of phospholipase D with the amino acid sequence SEQ ID NO.1, where alanine at position 59 is replaced with leucine and tryptophan at position 231 is replaced with alanine. It is abbreviated as A59L / W231A. *; (7) PLD-7, with the amino acid sequence shown in SEQ ID NO.8, wherein PLD-7 is obtained by replacing alanine at position 59 of phospholipase D (as shown in SEQ ID NO.1) with leucine and arginine at position 409 with asparagine, abbreviated as A59L / R409N. *; (8) PLD-8, with the amino acid sequence shown in SEQ ID NO.9, wherein PLD-8 is obtained by replacing leucine at position 178 of phospholipase D (as shown in SEQ ID NO.1) with tyrosine and tryptophan at position 231 with alanine, abbreviated as L178Y / W231A. *; (9) PLD-9, the amino acid sequence of which is shown in SEQ ID NO.10. PLD-9 is obtained by replacing leucine at position 178 of phospholipase D (as shown in SEQ ID NO.1) with tyrosine and arginine at position 409 with asparagine, abbreviated as L178Y / R409N. *; PLD-10, with the amino acid sequence shown in SEQ ID NO.11, is a modified version of phospholipase D with the amino acid sequence SEQ ID NO.1 showing the substitution of tryptophan at position 231 with alanine and arginine at position 409 with asparagine, abbreviated as W231A / R409N. *; PLD-11, with the amino acid sequence shown in SEQ ID NO.12, is a modified version of phospholipase D shown in SEQ ID NO.1, where position 59 (alanine) is replaced with leucine, position 178 (leucine) is replaced with tyrosine, and position 231 (tryptophan) is replaced with alanine. It is abbreviated as A59L / L178Y / W231A. *; PLD-12, with the amino acid sequence shown in SEQ ID NO.13, is a modified version of phospholipase D with the amino acid sequence SEQ ID NO.1 showing the substitution of alanine at position 59 with leucine, leucine at position 178 with tyrosine, and arginine at position 409 with asparagine. It is abbreviated as A59L / L178Y / R409N. *; PLD-13, with the amino acid sequence shown in SEQ ID NO.14, is a modified version of phospholipase D with the amino acid sequence SEQ ID NO.1 showing the substitution of alanine at position 59 with leucine, tryptophan at position 231 with alanine, and arginine at position 409 with asparagine. It is abbreviated as A59L / W231A / R409N. *; PLD-14, with the amino acid sequence shown in SEQ ID NO.15, is a modified version of phospholipase D shown in SEQ ID NO.1, where leucine at position 178 is replaced with tyrosine, tryptophan at position 231 is replaced with alanine, and arginine at position 409 is replaced with asparagine. It is abbreviated as L178Y / W231A / R409N. *; PLD-15, with the amino acid sequence shown in SEQ ID NO.16, is a modified version of phospholipase D with the amino acid sequence SEQ ID NO.1, where position 59 (alanine) is replaced with leucine, position 178 (leucine) with tyrosine, position 231 (tryptophan) with alanine, and position 409 (arginine) with asparagine. It is abbreviated as A59L / L178Y / W231A / R409N. *

[0029] Amino acid mutations were performed on the protein sequences encoded by each gene, and the corresponding gene sequences after mutation were cloned into the expression vector pET26 plasmid to obtain the corresponding recombinant vectors. After the recombinant vectors were verified to be correct by sequencing, they were introduced into Escherichia coli BL21(DE3) to obtain the corresponding expression strains.

[0030] Example 2 Enzyme activity assay of phospholipase D mutant Test methods The phospholipase D mutant obtained in Example 1 was subjected to enzyme activity assay and screening.

[0031] Using soybean PE (phosphatidylethanolamine, purity ≥90%) as a substrate, the content of phosphatidylserine (PS) generated by the enzymatic reaction was determined by high performance liquid chromatography (HPLC) to evaluate the catalytic activity of the mutant.

[0032] Preparation of crude enzyme solution of phospholipase D mutant: Recombinant bacteria were inoculated into TB (Terrific Broth) liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 150 r / min until the absorbance value OD reached the specified value.600 The concentration was approximately 1.5. IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.1 mM for induction at 25°C for 16 h. Then, the cells were collected by centrifugation at 10000 r / min for 10 min. One g of cells was resuspended in 10 mL of phosphate buffer (0.1 mol / L, pH 7.0), sonicated, and centrifuged at 10000 r / min for 5 min. The supernatant was collected as the recombinant crude enzyme solution.

[0033] (1) Preparation of the reaction system In a test tube (working volume 5.0 mL), add the components in the following order: Table 1 Enzyme-catalyzed reaction system

[0034] Add the above components to a test tube and place it in a constant temperature shaking incubator. React at 45 ℃ and 100 r / min for 1 h. After the reaction is complete, boil the test tube in a boiling water bath for 5 min to terminate the reaction. Transfer 100 μL of the reaction solution to a 1.5 mL centrifuge tube, add 300 μL of diluent (hexane:isopropanol:water = 3:2:1, volume ratio), vortex to mix for 1 min, and centrifuge at 4 ℃ and 12000 r / min for 10 min. Filter the upper organic phase through a 0.22 μm organic phase filter membrane into a sample vial for HPLC analysis.

[0035] Determination of enzyme activity by high performance liquid chromatography High-performance liquid chromatography (HPLC) was used for detection. The chromatographic conditions were as follows: Kromasil 100-5 C18 column (4.6 × 250 mm, 5 μm); mobile phase (volume ratio of acetonitrile:methanol:phosphoric acid = 95:5:0.5); flow rate of 1.0 mL / min; detection wavelength of 205 nm; and column temperature of 35 ℃.

[0036] Enzyme activity is defined as the amount of enzyme required to produce 1 μg PS per minute using soybean PE as a substrate at 45 ℃. This amount is denoted as U / mL.

[0037] Test results Positive mutants with significantly enhanced catalytic activity against phosphatidylethanolamine were screened by HPLC. The enzyme activity results of each phosphatase D mutant are shown in Table 1.

[0038] Among them, PLD-15, which is a phospholipase D mutant containing four mutation points A59L, L178Y, W231A and R409N, has the highest relative enzyme activity. This mutant has the ability to efficiently catalyze the synthesis of phosphatidylserine from phosphatidylethanolamine. Compared with the original phosphatidyllipase D, its relative enzyme activity in catalyzing the synthesis of phosphatidylserine from phosphatidylethanolamine increases from 4.52 U / mg to 14.38 U / mg.

[0039] Table 1. Results of increased enzyme activity in each mutant

[0040] The amino acid sequence of the PLD-15 mutant is shown in SEQ ID NO.16, and the nucleotide sequence is shown in SEQ ID NO.18.

[0041] The nucleotide sequence of the PLD-15 mutant is as follows: Example 3: Construction of recombinant engineered bacteria expressing the PLD-15 mutant The nucleotide sequence SEQ ID NO.18 of the mutant PLD-15 was cloned into the Escherichia coli-Bacillus subtilis shuttle expression vector pBE-S. Positive clones were screened and plasmids were extracted for sequencing verification, and the recombinant plasmid pBE-S-PLD with correct sequencing was obtained.

[0042] The recombinant plasmid pBE-S-PLD constructed above was transformed into Bacillus subtilis competent cells. 5 μL of recombinant plasmid DNA was added to 500 μL of Bacillus subtilis competent cells, and after electroporation, the cells were incubated at 37 °C with shaking for 30 min. Then, 300 μL of LB medium was added and incubated for another 30 min for recovery. The recovered bacterial culture was plated on LB agar plates containing kanamycin (15 μg / mL) and incubated overnight at 37 °C. Positive transformants were screened to obtain the recombinant engineered B. subtilis / pBE-S-PLD.

[0043] The successfully constructed recombinant engineered bacteria were inoculated into a liquid culture medium containing kanamycin and fermented at 37 °C and 150 r / min for 24 h. The fermentation supernatant was collected by centrifugation. The supernatant obtained by centrifugation was concentrated 5 times by ultrafiltration (molecular cutoff membrane of 20 kDa) to obtain PLD-15 crude enzyme solution.

[0044] The liquid culture medium contains the following components: glucose 10 g / L, yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, pH 7.0.

[0045] Example 4: In vitro catalytic synthesis of PS from PE using the PLD-15 mutant Test methods Preparation of PLD-15 enzyme solution: The recombinant bacteria were inoculated into a liquid culture medium containing kanamycin and fermented at 37℃ and 150 r / min for 24 h. The fermentation supernatant was collected by centrifugation. The supernatant obtained by centrifugation was concentrated 5 times by ultrafiltration (molecular cutoff membrane of 20 kDa) to obtain crude enzyme solution.

[0046] The total volume of the reaction system was 5 L, and the components of the reaction system were as follows: 150 g / L soybean PE (60% purity), 300 g / L L-serine, 20 g / L calcium chloride, 30 g / L PLD-15 (SEQ ID NO.16) enzyme solution, and the pH was adjusted to 4.5.

[0047] The reaction system was placed in a mechanically stirred apparatus preheated to 45 °C, and the reaction was stopped after 2 h.

[0048] Test results The final yield of soybean PS product was 617.63 g, with a PS content of 89.49% and a yield of 82.35%. HPLC chromatograms before and after the reaction are shown below. Figure 1 and Figure 2 .

[0049] The present invention provides a highly efficient phospholipase D mutant for the synthesis of phosphatidylserine from phosphatidylethanolamine and its application. Specifically, it relates to a highly catalytically active phospholipase D mutant, its encoded amino acid, a recombinant expression vector, an engineered bacterium, and a method for catalyzing the synthesis of a series of phosphatidylserines using the mutant.

Claims

1. A phospholipase D mutant that efficiently catalyzes the synthesis of phosphatidylserine from phosphatidylethanolamine, wherein the phospholipase D mutant is obtained by mutating any one or more of the four sites at positions 59, 178, 231 or 409, based on the amino acid sequence shown in SEQ ID NO.

1.

2. The phospholipase D mutant of claim 1, wherein the phospholipase D mutant is based on phospholipase D with an amino acid sequence as shown in SEQ ID NO. 1, and the amino acid at position 59, position 178, position 231 or position 409 is mutated to leucine, tyrosine, alanine or asparagine.

3. The phospholipase D mutant as described in claim 1, wherein the mutation is: Replace the alanine at position 59 of phospholipase D, as shown in SEQ ID NO.1, with leucine; The leucine at position 178 of phospholipase D, as shown in SEQ ID NO.1, is replaced with tyrosine. Replace tryptophan at position 231 of phospholipase D, as shown in SEQ ID NO.1, with alanine; Replace the arginine at position 409 of phospholipase D, as shown in SEQ ID NO.1, with asparagine; Replace the alanine at position 59 of phospholipase D, as shown in SEQ ID NO.1, with leucine, the leucine at position 178 with tyrosine, the tryptophan at position 231 with alanine, or the arginine at position 409 with asparagine.

4. A plasmid loaded with the gene encoding the phospholipase D mutant as described in claim 1.

5. An engineered bacterium expressing the phospholipase D mutant as described in claim 1, wherein the genome of the engineered bacterium contains the coding gene of the phospholipase D mutant, or the engineered bacterium contains a plasmid loaded with the coding gene of the phospholipase D mutant.

6. The engineered bacteria as described in claim 5, wherein the chassis strain of the engineered bacteria is yeast or bacteria.

7. The engineered bacteria as described in claim 5, wherein the chassis strain of the engineered bacteria is selected from one of Saccharomyces cerevisiae, Pichia pastoris, Yersinia lipolytica, Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis.

8. The application of the phospholipase D mutant as described in claim 1 in the biocatalytic synthesis of phosphatidylserine from phosphatidylethanolamine.

9. The application as described in claim 8, wherein the biocatalysis includes enzyme catalysis and whole-cell catalysis.

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