Phospholipase a1 mutant and use thereof

Abstract

The application discloses a phospholipase A1 mutant and application thereof, and belongs to the technical field of biology and the field of enzyme engineering. The mutant is obtained by site-directed mutagenesis on phospholipase A1-MFI, and is any one selected from W166V, W166L, A116F or L170H. Enzymatic property determination shows that the mutant has higher hydrolysis activity under the conditions of pH 8-10 and temperature 20-35 DEG C. Compared with the wild type, the specific enzyme activity, PC hydrolysis rate and LPC yield of the mutant are significantly improved. The application further discloses a nucleotide molecule encoding the mutant, a recombinant vector, a host cell and application thereof in hydrolysis of phospholipids to prepare lysophospholipids. The phospholipase A1 mutant of the application has excellent catalytic performance, and is suitable for the fields of food, medicine and feed and the like.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and enzyme engineering, and specifically relates to a phospholipase A1 mutant, a nucleotide molecule encoding the mutant, a recombinant vector containing the nucleotide molecule and a host cell, and the application of the mutant in the preparation of lysophospholipids by hydrolysis of phospholipids. Background Technology

[0002] Phospholipase A1 (PLA1, EC 3.1.1.32) is an enzyme that hydrolyzes phospholipids to produce 2-acyllysophospholipids and free fatty acids (FFA), and catalyzes the hydrolysis of phospholipids. Sn- 1 Studies have shown that it possesses relatively broad substrate specificity. Phospholipase A1 is widely distributed in nature. Comparison of known phospholipase A1 amino acid sequences reveals that most phospholipase A1s have low similarity to lipases, and the sequence similarity between phospholipase A1s from different sources is also very low. The only commonality is that all phospholipase A1s possess the lipase-identical sequence GXSXG, where serine is considered to be the site of lipase activity. Currently, the PLA1 used in industrial applications is mainly the commercial enzyme Lecitase® Ultra from Novozymes, which has an optimal pH of acidic (5.0~6.0) and an optimal temperature of relatively high (50~55℃). Furthermore, there is currently no research on its hydrolysis of phospholipids to enrich polyunsaturated fatty acids such as DHA and EPA. Therefore, exploring novel PLA1s with unique catalytic properties that can adapt to different application scenarios such as alkaline or low temperatures is of great significance for enriching the enzyme library and expanding the application boundaries of phospholipases.

[0003] Phospholipids, also known as phospholipids, are lipids containing phosphate groups. They are amphoteric molecules, with a hydrophilic nitrogen or phosphorus-containing head group at one end and a hydrophobic long hydrocarbon chain at the other. Based on the different head groups, glycerophospholipids can be classified into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylglycerol (PG), among others. Lysophospholipids (LPLs), currently the smallest and simplest phospholipids discovered, are products obtained by hydrolyzing phospholipids and losing one fatty acyl group. Due to their excellent emulsifying properties, easy absorption due to their small molecular size, and endogenous safety, lysophospholipids have been extensively developed and widely used in various industrial fields such as food processing, pharmaceuticals, cosmetics, and animal feed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a phospholipase A1 mutant with improved catalytic performance and its application, so as to further enrich the phospholipase A1 tool enzyme library and meet the needs of different application scenarios such as alkaline or low temperature.

[0005] The technical solution adopted in this invention is as follows: First, the present invention provides a phospholipase A1 mutant, which is obtained by site-directed mutagenesis of the amino acid sequence of phospholipase A1-MFI; The amino acid sequence of the phospholipase A1-MFI is shown in SEQ ID NO: 1, and its encoding nucleotide sequence is shown in SEQ ID NO: 2; The mutant is selected from any one of the following: The amino acid at position 166 was mutated from tryptophan to valine (W166V). The amino acid at position 166 was mutated from tryptophan to leucine (W166L). The amino acid at position 116 was mutated from alanine to phenylalanine (A116F). The 170th amino acid was mutated from leucine to histidine (L170H).

[0006] Preferably, the mutant is W166V.

[0007] Furthermore, the amino acid sequence of the mutant W166V is shown in SEQ ID NO: 3, and its encoding nucleotide sequence is shown in SEQ ID NO: 4.

[0008] Preferably, the mutant is W166L.

[0009] This invention also provides a method for preparing a phospholipase A1 mutant, comprising the following steps: (1) Construct a recombinant vector containing a nucleotide molecule encoding the above-mentioned phospholipase A1 mutant; (2) The recombinant vector is transformed into host cells to obtain recombinant engineered bacteria; (3) Culture the recombinant engineered bacteria and induce the expression of phospholipase A1 mutant; (4) Isolate and purify the expressed phospholipase A1 mutant.

[0010] Second, the present invention also provides a nucleotide molecule encoding the above-mentioned phospholipase A1 mutant.

[0011] Third, the present invention also provides a recombinant vector comprising the above-mentioned nucleotide molecules.

[0012] Preferably, the recombinant vector is a pET32a(+) derivative vector.

[0013] Fourth, the present invention also provides a host cell comprising the above-mentioned recombinant vector.

[0014] Preferably, the host cell is Escherichia coli .

[0015] Fifth, the present invention also provides the application of the above-mentioned phospholipase A1 mutant in the preparation of lysophospholipids.

[0016] Preferably, the lysophospholipid is lysophosphatidylcholine (LPC).

[0017] Sixth, the present invention also provides a method for hydrolyzing phospholipids, comprising the following steps: using the above-mentioned phospholipase A1 mutant, catalyzing the hydrolysis of substrate phospholipids under pH 8-10 and / or temperature 20-35℃ conditions to generate lysophospholipids.

[0018] Preferably, the substrate phospholipid includes phosphatidylcholine (PC), and the lysophospholipid is lysophosphatidylcholine (LPC).

[0019] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) High catalytic activity The phospholipase A1 mutant provided by this invention, particularly W166V, exhibits significantly higher specific enzyme activity, PC hydrolysis rate, and LPC yield than the wild-type phospholipase A1-MFI. Enzymatic property assays show that W166V has an optimal reaction temperature of 25℃, an optimal pH of 9, and good stability within a temperature range of 20–30℃ and a pH range of 8–10.

[0020] (2) High product conversion efficiency The mutant of this invention exhibits higher substrate conversion and product yield in the catalytic hydrolysis of phospholipids to prepare lysophosphatidylcholine (LPC), making it suitable for industrial production.

[0021] (3) The mechanism of action is clear Molecular docking analysis showed that the number of hydrogen bonds increased significantly after the W166V mutation, forming a network of multiple hydrogen bond interactions, resulting in stronger substrate anchoring; hydrophobic interactions were significantly enhanced, improving the matching degree between the hydrophobic pocket and the unsaturated acyl chain; electrostatic interactions were more rationally distributed, and the overall synergistic effect of the interactions was stronger, thereby greatly improving substrate affinity and catalytic performance.

[0022] (4) Enriched the phospholipase A1 tool enzyme library The novel phospholipase A1 mutant provided by this invention, especially W166V, offers a new tool enzyme for the application of phospholipase in food processing, pharmaceuticals, cosmetics, and animal feed. Attached Figure Description

[0023] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 PC hydrolysis rate, LPC yield, and LPC concentration of crude enzyme solutions of wild type and each mutant; where WT is wild type PLA1-MFI.

[0025] Figure 2 SDS-PAGE results of wild-type and mutant purified enzymes; lane M: protein molecular weight marker; lane "MFI": wild-type PLA1-MFI; the remaining lanes are A116F, W166V, W166L, L170H, and T223I.

[0026] Figure 3SDS-PAGE results of the W166V mutant nickel column affinity purification process; where lane M: protein molecular weight marker; lane "crude enzyme": crude enzyme solution after induction expression; lane "flow-through": flow-through solution after sample loading; lanes "10 mM, 20 mM, 40 mM, 80 mM, 120 mM": elution fractions of imidazole at different concentrations.

[0027] Figure 4 Comparison of SDS-PAGE results of mutant W166V before and after purification; where lane M: protein molecular weight marker; lane "crude enzyme": crude enzyme solution before purification; lane "purified enzyme": enzyme solution after purification.

[0028] Figure 5 Comparison of specific enzyme activities between wild-type and each mutant pure enzyme; where WT is wild-type PLA1-MFI.

[0029] Figure 6 PC hydrolysis rate, LPC yield, and LPC concentration of each mutant pure enzyme; where WT is wild-type PLA1-MFI.

[0030] Figure 7 The optimal temperature for the hydrolysis activity of W166V.

[0031] Figure 8 Temperature stability of W166V hydrolysis activity.

[0032] Figure 9 Effect of pH on the hydrolytic activity of W166V.

[0033] Figure 10 pH stability of W166V hydrolytic activity.

[0034] Figure 11 Molecular docking results between wild-type PLA1-MFI and substrate molecules (two-dimensional schematic diagram of the interaction).

[0035] Figure 12 Molecular docking results between mutant W166V and substrate molecules (two-dimensional schematic diagram of interaction). Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: Discovery and gene synthesis of phospholipase A1-MFI This invention discovered a phospholipase A1 derived from *Serratia sp.* (NCBI ID: WP_399669122.1) in the NCBI database, named phospholipase A1-MFI (abbreviated as PLA1-MFI). Its open reading frame is 999 bp in length, encoding 333 amino acids. The amino acid sequence of this phospholipase A1-MFI is shown in SEQ ID NO: 1, and its encoding nucleotide sequence is shown in SEQ ID NO: 2.

[0039] Based on the codon preference of the host *E. coli*, the target gene was codon optimized, and the signal peptide was deleted. The optimized target gene was obtained using whole-genome synthesis technology and inserted into the multiple cloning site of the pET32a(+) plasmid. The plasmid map was constructed using SnapGene software. Bioinformatics software predicted the protein size to be 58-60 kDa. Multiple sequence alignment of amino acid sequences closely homologous to PLA1-MFI was performed using Clustal X software. Positions 208-212 contained a conserved GXSXG pentapeptide sequence typical of lipases, and a Ser-His-Asp catalytic triplet, in which the nucleophilic Ser residue in the catalytic triplet was located within a highly conserved pentapeptide motif. BLAST analysis showed that the amino acid sequence of this protein shared only 64.58% homology with the cold-adapted basic PLA1 (NCBI ID: ABB91802.1) derived from Serratia sp. xjF1 (NCBI ID: ABB91802.1), which has been reported to have high activity, and only 56.94% homology with the PLA1 derived from Serratia sp. MK1 (NCBI ID: AAA26552.1), which has been reported to have high activity. The sequence similarity with the template sequence of PLA1 derived from Serratia sp. MK1 was 71.64%.

[0040] Example 2 Construction and transformation of recombinant plasmids 2.1 Construction of Recombinant Plasmids The target gene fragment and the linearized vector were ligated using seamless cloning technology. The reaction system consisted of 5 μL of C115 ligase, 2 μL of the target fragment, and 3 μL of the linearized vector. After mixing, the mixture was ligated in a 50°C water bath for 20 min, followed by an ice bath for 10 min.

[0041] 2.2 Plasmid Transformation The recombinant plasmid was transformed into E. coli DH5α using the heat shock method. The dry plasmid powder was simply centrifuged and dissolved in ultrapure water to make the plasmid concentration 10 ng / μL. 2 μL of plasmid was added to 50 μL of DH5α competent cells, gently pipetted to mix, and incubated on ice for 30 min. Then, it was heat-shocked in a water bath at 42℃ for 90 s, followed by another ice bath for 2 min. 700 μL of LB antibiotic-free liquid medium was added to a clean bench, and the cells were incubated in a constant temperature air shaker for 45 min (37℃, 220 rpm). After the incubation, the cells were centrifuged and concentrated. 100 μL of the concentrated medium was spread on LB agar plates containing ampicillin and incubated upside down at 37℃ for 10-12 h before observing the colonies.

[0042] 2.3 Colony Validation After overnight incubation, single colonies were picked for PCR verification. 10 μL of deionized water was placed in a PCR tube; a single colony was picked and transferred to the PCR tube, mixed thoroughly by pipetting, and 8 μL was transferred to a new PCR tube. The tube was immediately capped to prevent contamination. The remaining 2 μL was used for PCR verification. After PCR, agarose gel electrophoresis was performed, and colonies with correct positive bands were selected for sequencing. Single colonies with correct sequencing results were activated and stored at -80°C.

[0043] The activated DH5α bacterial culture was used to extract recombinant plasmids using a rapid plasmid miniprep kit. The recombinant plasmids were then transformed into E. coli BL21 strain according to the transformation steps described above. Colony verification, sequencing verification, and preservation of the bacteria were then performed. The preservation tubes were stored at -80°C for subsequent fermentation and enzyme production.

[0044] Example 3 Expression and purification of phospholipase A1-MFI 3.1 Expression of phospholipase A1-MFI Recombinant engineered bacterial strains grown on ampicillin-resistant plates were selected and inoculated into 5 mL of LB liquid medium containing 25 μg / mL ampicillin. The culture was carried out at 37 ℃ and 220 rpm for 12 hours. The inoculum was then transferred to ZYP-5052 medium containing 25 μg / mL ampicillin at a 1% inoculation rate and cultured at 20 ℃ and 200 rpm for 48 hours to induce the expression of phospholipase A1-MFI.

[0045] 3.2 Purification of phospholipase A1-MFI Take the culture medium, centrifuge at 10,000 rpm for 10 min at 4 ℃, collect the bacterial cells, resuspend them in Tris-HCl buffer (50 mM, pH 8.0), and sonicate (300 W power, 3 s working time, 2 s interval, total time 30 min). After disruption, centrifuge completely (4 ℃, 10,000 rpm, 20 min). The supernatant is the crude enzyme solution.

[0046] The crude enzyme solution was purified by affinity chromatography using a nickel column. The column was equilibrated with 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), and then weakly binding proteins were eluted with 40 mM imidazole solution (40 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). The target protein was eluted with 80 mM imidazole solution (80 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). This eluent was collected as the purified enzyme solution and analyzed by SDS-PAGE. The molecular weight of the purified protein was approximately 58 kDa, which was almost consistent with the prediction.

[0047] Example 4 Mutational modification of phospholipase A1-MFI The hydrolysis rate and substrate conversion efficiency of phospholipase A1-MFI need improvement. Therefore, this embodiment attempts to modify it using molecular biology techniques to enhance its potential for phospholipid hydrolysis. Homology modeling of PLA1-MFI was performed using the online analysis tools Swiss-Model and AlphaFold2 to obtain its three-dimensional structure. The obtained three-dimensional structures were uploaded to HotSpot Wizard 3.1, PROSS, and Fireprot to predict key amino acids. Based on the prediction results of these three tools, 22 mutation sites were selected for site-specific modification to improve the enzyme's catalytic performance.

[0048] The constructed mutants are as follows: Amino acid at position 111 of PLA1-MFI was mutated from serine to tyrosine (S111Y); amino acid at position 116 was mutated from alanine to cysteine, phenylalanine, and methionine (A116C, A116F, A116M); amino acid at position 127 was mutated from valine to glutamic acid (V127E); amino acid at position 166 was mutated from tryptophan to cysteine, phenylalanine, leucine, methionine, and valine (W166C, W166F, W166L, W166M, W166V); amino acid at position 170 was mutated from leucine to alanine, aspartic acid, and histidine (L170A, L170D, L170H); amino acid at position 176 was mutated from alanine to glycine (A176G); amino acid at position 177 was mutated from leucine ...77 was mutated from leucine to glutamic acid (V127E); amino acid The amino acids at positions 1-177 were mutated from threonine to phenylalanine, leucine, and methionine (T177F, T177L, T177M); valine at position 182 was mutated from valine to proline (V182P); histidine at position 186 was mutated from histidine to glutamine (H186Q); serine at position 193 was mutated from serine to isoleucine (S193I); alanine at position 196 was mutated from alanine to lysine and valine (A196K, A196V); arginine at position 201 was mutated from arginine to aspartic acid and alanine (R201D, R201A); isoleucine at position 204 was mutated from isoleucine to phenylalanine and valine (I204F, I204V); serine at position 220 was mutated from serine to alanine (S220A); and threonine at position 223 was mutated from threonine to isoleucine (T223I). The amino acids at positions 244 and 262 were mutated from valine to serine and proline, respectively (V224S, V224P); from asparagine to aspartic acid (N244D); from asparagine to leucine (N261L); from glutamic acid to valine (E262V); from threonine to leucine (T270L); from serine to lysine (S284K); and from threonine to glutamine (T310Q).

[0049] A total of 36 mutants were obtained, and their names are shown above. Accordingly, the codons at the mutation sites on the coding genes were mutated to the codons of the corresponding amino acids.

[0050] Using Snapgene software, primers were designed with pET-32a-PLA1-MFI as a template to construct recombinant plasmids of the above mutants. Engineered bacteria were constructed using conventional molecular biology methods. After conventional culture, induced expression, and crude enzyme extraction, crude enzyme solutions of each mutant were obtained.

[0051] Example 5: Application of phospholipase A1-MFI and its mutants in the hydrolysis preparation of lysophosphatidylcholine 5.1 Method for determining hydrolytic activity Soybean lecithin (PC) was used as a substrate, and its hydrolytic activity was determined using a classic biphasic reaction system. The substrate solution was 20 mg / mL soybean lecithin (dissolved in cyclopentyl methyl ether). The reaction system consisted of 0.5 mL substrate solution, 0.001 mL 0.2 M Ca2+, 0.1 mL crude enzyme solution, and 0.39 mL pH 9.0 buffer solution. The reaction was carried out at 20℃ and 220 rpm in a water bath for 10 min. After the reaction was completed, 2 mL of anhydrous ethanol was immediately added and mixed to inactivate the enzyme. The mixture was then filtered through a 0.22 μm nylon membrane and analyzed by HPLC-ELSD. The enzyme activity unit (U) was defined as the amount of enzyme required per minute to hydrolyze the substrate to produce 1 μmol of product LPC.

[0052] 5.2 Plotting the Standard Curve Substrate PC, product LPC, and GPC standards were dissolved in anhydrous ethanol to prepare standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. After filtration through a 0.22 μm nylon membrane, the solutions were analyzed by HPLC-ELSD. Standard curves were plotted with the logarithm of concentration on the x-axis and the logarithm of the ELSD peak area on the y-axis.

[0053] HPLC-ELSD detection conditions: The chromatographic column was a Restek Silica (5 μm, 250 mm × 4.6 mm) silica gel column, the column temperature was 40℃, mobile phase A was methanol, mobile phase B was water, the flow rate was 0.8 mL / min, the injection volume was 5 μL, and the gradient elution program was shown in Table 1. Detector: N2 flow rate was 1.6 L / min, and the drift tube temperature was 40℃.

[0054] Table 1. HPLC-ELSD binary elution program 0 92 8 4.0 92 8 8.0 91 9 8.4 90 10 9.0 87 13 15.0 87 13 20.0 92 8 5.3 Calculation of hydrolysis rate and yield The product content was calculated based on the PC, LPC, and GPC standard curves. The hydrolysis rate was calculated using the following formula: ; In the formula, PC 初PC 末 The values ​​represent the PC content in the system before and after hydrolysis (calculated based on the standard curve).

[0055] The formula for calculating LPC yield is as follows: ; In the formula, m PC m LPC m GPC The contents of PC, LPC, and GPC in the hydrolysis system are respectively (calculated based on the standard curve), M PC M LPC M GPC These are the average relative molecular masses of PC, LPC, and GPC, respectively.

[0056] 5.4 Initial screening of crude enzyme solution The crude enzyme solutions of each mutant were initially screened. The reaction system consisted of 0.5 mL substrate solution, 0.001 mL 0.2 M Ca2+, 0.1 mL crude enzyme solution, and 0.39 mL pH=9.0 buffer solution. The mixture was shaken in a water bath at 20℃ and 220 rpm for 10 min. Immediately after shaking, 2 mL of anhydrous ethanol was added to inactivate the enzyme. After enzyme inactivation, the mixture was filtered through a 0.22 μm nylon membrane and analyzed by HPLC-ELSD. Mutants with improved catalytic activity compared to the wild type were screened for subsequent purification.

[0057] 5.5 Crude enzyme PC hydrolysis rate, LPC conversion rate, and LPC concentration The LPC conversion rate and LPC concentration of the crude enzyme solutions of each mutant were determined, and the results are as follows: Figure 1 As shown.

[0058] Depend on Figure 1It can be seen that the PC hydrolysis rate of wild-type PLA1-MFI is 19.83%, the LPC yield is 15.55 mol%, and the LPC concentration is 1.04 mg / mL. The LPC yield and concentration of each mutant were significantly higher than those of the wild type. Mutant W166V exhibited the highest PC hydrolysis rate (57.09%), LPC yield (46.63 mol%), and LPC concentration (3.12 mg / mL), followed by mutant L170H with a PC hydrolysis rate of 50.35%, an LPC yield of 42.08 mol%, and an LPC concentration of 2.82 mg / mL. Mutants W166L, A116F, and T223I showed PC hydrolysis rates of 40.11%, 28.47%, and 25.26%, respectively, with LPC yields of 34.51 mol%, 28.78 mol%, and 21.54 mol%, and LPC concentrations of 2.31 mg / mL, 1.92 mg / mL, and 1.44 mg / mL, respectively. These results indicate that the mutants constructed in this example, particularly W166V and L170H, demonstrate significantly superior catalytic performance compared to the wild type, even at the crude enzyme level.

[0059] Example 6 Purification and SDS-PAGE detection of mutant enzyme 6.1 Purification by Nickel Column Affinity Chromatography (1) Column mounting Place a gasket at the bottom of the column, mix the nickel column packing material thoroughly, and slowly fill the column. Let the column stand vertically, allowing the liquid in the packing material to flow out naturally and the packing material to gradually settle to the bottom. After confirming that the packing material has completely settled, add a gasket on top of the packing material and add 20% ethanol to seal the column. The completed nickel column must be stored at 4°C. All liquid entering the column must be filtered through a 0.45μm polyethersulfone membrane.

[0060] (2) Equilibrium nickel column After the nickel column was packed, the column was rinsed sequentially with 20% ethanol filtered through a 0.45 μm polyethersulfone membrane and sterile water for 3 column volumes each. Then, 6 volumes of 10 mM imidazole buffer (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl) were added to equilibrate the nickel column.

[0061] (3) Sample loading After the crude enzyme solution was filtered through a 0.45 μm polyethersulfone filter membrane, it was loaded onto the column. After the crude enzyme solution had bound to the nickel column once, 10 mM imidazole buffer was added to elute the poorly bound proteins. After no protein was detected, the crude enzyme solution was loaded again. The column was loaded alternately a total of 3 times to ensure that the target protein was fully bound to the nickel column.

[0062] (4) Gradient elution After loading the crude enzyme solution, perform a gradient wash with imidazole elution buffer at concentrations of 10 mM, 20 mM, 40 mM, 80 mM, 120 mM, 200 mM, and 500 mM. Low concentrations of imidazole elution buffer can elute non-specifically bound proteins. During elution, use 1× Coomassie Brilliant Blue (G250) solution for rough protein determination (100 μL elution buffer + 100 μL G250 solution). Before changing to the next gradient elution buffer, ensure that the current gradient has completely eluted the proteins. Collect the elution buffer from each gradient and store it on ice. Finally, wash the column with 500 mM imidazole elution buffer to remove strongly bound proteins.

[0063] (5) Rinse the nickel column First, rinse the column with about 6 column volumes of sterile water, then rinse the column with about 3 column volumes of 20% ethanol. When the ethanol has flowed to the same volume as the packing material, seal the column and store the nickel column in a 4°C refrigerator.

[0064] 6.2 Ultrafiltration Concentration The collected eluent containing the target protein was placed on ice and concentrated using a 10 kDa ultrafiltration tube at 4°C and 4000 rpm. After concentration, the imidazole was replaced with Buffer A to prevent high concentrations of imidazole from affecting enzyme activity. The resulting pure enzyme solution was aliquoted and stored at 4°C. The ultrafiltration tubes were washed and sealed with 20% ethanol and stored at 4°C for later use.

[0065] 6.3 Mutant Purification The crude enzyme solutions of the five mutants (A116F, W166V, W166L, L170H, and T223I) obtained from the initial screening were purified according to the above method to obtain pure enzyme solutions of the five mutant enzymes.

[0066] Example 7: SDS-PAGE detection and comparison of specific enzyme activities of wild-type and mutant enzymes 7.1 SDS-PAGE Detection (1) Making adhesive After installing the protein electrophoresis gel preparation plate, add an appropriate amount of water to check for leaks (let it stand for 10 minutes, ensuring the liquid level does not drop), confirming that the glass plate is well sealed. Follow the instructions of the protein electrophoresis gel preparation kit: first, prepare the lower separating gel, pour it to the specified height, then add anhydrous ethanol to the upper layer to flatten the gel surface, let it stand for 30 minutes to solidify, and then pour off the anhydrous ethanol; then prepare the upper stacking gel, insert the comb, and let it stand for 30 minutes until the gel is completely solidified.

[0067] (2) Sample preparation Mix the corresponding gradient protein elution buffer with 5× protein loading buffer at a volume ratio of 1:4 and incubate in a boiling water bath for 10 min.

[0068] (3) Sample loading Take appropriate amounts (20 μL macroporous gel, 10 μL microporous gel) of sample and 10 μL of 180 kDa protein molecular weight marker and add them into the pores in the set order.

[0069] (4) Running glue Follow the instructions in the kit to set the gel running program until electrophoresis is complete (120 V, 75 min or 80 V, 120 min).

[0070] (5) Staining Remove the electrophoresis tank, carefully peel the protein electrophoresis gel into a petri dish, rinse with water, pour in the prepared protein staining solution to submerge the electrophoresis gel, and stain on a shaker for 1.5 h.

[0071] (6) Decolorization Recover the staining solution and wash with water. After thorough washing, add the prepared destaining solution, immerse the electrophoresis gel, and destain on a shaker until the protein gel background is clean and the bands are clearly visible.

[0072] Test results: SDS-PAGE results of wild-type and mutant purified enzymes are as follows: Figure 2 As shown, by Figure 2 It can be seen that, using the pure enzyme solution of wild-type phospholipase A1-MFI as a control, the protein molecular weight of wild-type phospholipase A1-MFI and each mutant enzyme is consistent, all about 58kDa, which is close to the theoretical molecular weight. Moreover, the bands are single and there are no significant impurities, indicating that the protein purity is high.

[0073] The nickel column affinity purification process of mutant W166V is as follows: Figure 3 As shown. By Figure 3 It can be seen that the target protein (approximately 58 kDa) was mainly eluted at a concentration of 40-80 mM imidazole, and the purification effect was good.

[0074] The SDS-PAGE comparison results of mutant W166V before and after purification are as follows: Figure 4 As shown. By Figure 4 It can be seen that the purified enzyme solution has a single band with a molecular weight of approximately 58 kDa, indicating that impurities and proteins were effectively removed compared to the crude enzyme solution.

[0075] 7.2 Enzyme activity assay The specific enzyme activity is calculated based on the mass of pure enzyme added. The formula for calculating the specific enzyme activity is as follows: Enzyme activity (U / mg) = A / M; In the formula, A represents the hydrolytic enzyme activity (U); M represents the pure enzyme mass (mg), and the unit of specific enzyme activity is U / mg.

[0076] The measurement results are as follows Figure 5 As shown, the specific activity of wild-type phospholipase A1-MFI is 648.69 U / mg, the specific activity of mutant A116F is 741.95 U / mg, the specific activity of mutant W166V is 833.96 U / mg, the specific activity of mutant W166L is 788.63 U / mg, and the specific activity of mutant L170H is 752.00 U / mg. Figure 5 It can be seen that the specific enzyme activity of each mutant is higher than that of the wild type, among which the specific enzyme activity of mutant W166V is the highest, which is about 28.6% higher than that of wild type.

[0077] 7.3 Hydrolysis rate of pure enzyme PC and conversion rate of LPC The purified mutant enzymes were subjected to PC hydrolysis rate and LPC conversion rate determination, and the results are as follows: Figure 6 As shown.

[0078] Depend on Figure 6 It was found that the PC hydrolysis rate of wild-type PLA1-MFI was 68.37%, the LPC yield was 66.29 mol%, and the LPC concentration was 4.45 mg / mL. The catalytic performance of each mutant was significantly better than that of the wild type, among which: The PC hydrolysis rate of mutant W166V was 92.43%, the LPC yield was 85.22 mol%, and the LPC concentration was 5.71 mg / mL. The PC hydrolysis rate of mutant W166L was 92.66%, the LPC yield was 80.59 mol%, and the LPC concentration was 5.40 mg / mL. The PC hydrolysis rate of mutant L170H was 83.02%, the LPC yield was 76.85 mol%, and the LPC concentration was 5.15 mg / mL; The PC hydrolysis rate of mutant A116F was 89.65%, the LPC yield was 75.82 mol%, and the LPC concentration was 5.08 mg / mL.

[0079] The results indicate that each mutant, especially W166V and W166L, exhibits significantly superior PC hydrolysis capacity and LPC conversion efficiency compared to the wild type at the pure enzyme level.

[0080] 7.4 Mutant Screening Instructions During the mutant screening process, mutant T223I was discarded because its protein expression level was too low and insufficient pure enzyme could not be obtained. Therefore, it was not included in the subsequent purification comparison.

[0081] 7.5 Confirmation of the construction of mutant W166V: The 166th amino acid of PLA1-MFI was site-directedly mutated from tryptophan (Trp) to valine (Val), and the codon corresponding to the 166th amino acid in the coding gene was changed from the tryptophan codon "TGG" to the valine codon "GTA". The amino acid sequence of the mutant W166V is shown in SEQ ID NO: 3, and its coding nucleotide sequence is shown in SEQ ID NO: 4. Example 8: Investigation of the enzymatic properties of mutant W166V 8.1 Optimal temperature for hydrolysis activity To investigate the effect of temperature on the hydrolytic activity of W166V, pure W166V enzyme solution was incubated in water baths at 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃ for 2–3 min. Substrate PC was added to catalyze hydrolysis, and anhydrous ethanol was added to terminate the reaction after 10 min. After centrifugation, the supernatant was filtered through a 0.22 μm nylon membrane, and the hydrolytic enzyme activity was determined by HPLC-ELSD. The residual enzyme activity was calculated. Results are as follows: Figure 7 As shown.

[0082] Depend on Figure 7 It was found that the hydrolytic activity of W166V increased with increasing temperature within the range of 15-30℃, reaching its highest relative enzyme activity (100%) at 25℃. When the temperature exceeded 30℃, the enzyme activity gradually decreased, with a relative enzyme activity of approximately 96% at 40℃ and dropping to 18% at 50℃. These results indicate that the optimal reaction temperature for W166V is 25℃, and it maintains high activity (relative enzyme activity ≥95%) within the range of 20-35℃.

[0083] 8.2 Hydrolysis activity and temperature stability To investigate the temperature stability of the hydrolytic activity of W166V, pure W166V enzyme solution was incubated in water baths at 20℃, 25℃, 30℃, 35℃, and 40℃ for 0, 4, 8, 12, 16, 20, and 24 h, respectively. After incubation, substrate PC was added and mixed. The hydrolytic enzyme activity was then measured at the optimal temperature, and the residual enzyme activity was calculated. The results are as follows: Figure 8 As shown.

[0084] Depend on Figure 8 It can be seen that W166V exhibits different stability characteristics at different temperatures: At 20℃: after incubation for 24 h, the residual enzyme activity remained at 73.35%, showing good stability.

[0085] At 25℃: after incubation for 24 h, the residual enzyme activity was approximately 60.91%, indicating good stability.

[0086] At 30℃: after incubation for 24 h, the residual enzyme activity decreased to 67.94%, and the stability decreased significantly.

[0087] At 35℃: after 24 h of incubation, the residual enzyme activity was only 26.68%, indicating rapid enzyme inactivation.

[0088] The above results indicate that W166V has good temperature stability in the range of 20-30℃, making it suitable for storage and use at room temperature or low temperature; however, long-term incubation at temperatures above 35℃ will lead to a significant loss of enzyme activity.

[0089] 8.3 Optimal pH for hydrolytic activity To investigate the effect of pH on the hydrolytic activity of W166V, the type of buffer solution and the corresponding pH value in the hydrolase activity assay were changed: 0.1 M citrate-sodium citrate buffer at pH 5.0 and 6.0; 0.1 M phosphate buffer at pH 6.0, 7.0, and 8.0; 0.1 M Tris-HCl buffer at pH 8.0 and 9.0; and 0.1 M Gly-NaOH buffer at pH 9.0, 10.0, and 11.0. After the reaction, the hydrolase activity was measured, and the residual enzyme activity was calculated. The results are as follows: Figure 9 As shown.

[0090] Depend on Figure 9 It was found that W166V exhibits high hydrolytic activity within the pH range of 9 to 10. Its optimal reaction pH is 9, where it reaches its highest relative enzyme activity (100%). Enzyme activity significantly decreases when the pH is below 8 or above 11. These results indicate that W166V is an alkaline phospholipase A1 with good catalytic activity within the pH range of 8-10.

[0091] 8.4 Hydrolysis activity and pH stability To investigate the pH stability of W166V hydrolytic activity, pure W166V enzyme solution was placed in different pH buffer environments and incubated at 4°C for 0, 12, 36, 60, 84, and 108 hours. After incubation, the hydrolytic enzyme activity was measured, and the residual enzyme activity was calculated. The results are as follows: Figure 10 As shown.

[0092] Depend on Figure 10 It can be seen that W166V exhibits different stability characteristics under different pH conditions: At pH 8.0, the residual enzyme activity remained at 77.62% after 108 h of incubation, indicating good stability.

[0093] At pH 9.0, the residual enzyme activity was approximately 80.77% after 108 h of incubation, demonstrating excellent stability.

[0094] Under pH 10.0 conditions: after incubation for 108 h, the residual enzyme activity was approximately 64.91%, indicating good stability.

[0095] At pH 11.0: after incubation for 108 h, the residual enzyme activity decreased to 56.12%, indicating relatively poor stability.

[0096] The above results indicate that W166V has good pH stability in the pH range of 8.0-10.0, especially at pH 9.0, and is suitable for storage and use under weakly alkaline to alkaline conditions.

[0097] Example 9 Molecular docking analysis To elucidate the mechanism of the enhanced hydrolytic activity of the mutant W166V, molecular docking was performed between wild-type PLA1-MFI and the mutant W166V, and the structure with the lowest binding free energy was selected for analysis.

[0098] The docking results of wild-type PLA1-MFI are shown in Figure 11. The interaction between the wild-type enzyme and the substrate mainly depends on residues such as SER-208, THR-163, HIS-207, TYR-110, ILE-314, ASN-164, and LEU-276. The interaction types include van der Waals forces, π-cation interactions, conventional hydrogen bonds, and C-H bonds. The docking results of the mutant W166V are shown in Figure 12. After mutation, the amino acid at position 166 is changed from tryptophan (TRP) to valine (VAL), and the interaction network changes significantly, involving residues such as THR-163, SER-208, LEU-209, ASN-164, HIS-312, LEU-276, and ILE-314. The interaction types include van der Waals forces, conventional hydrogen bonds, C-H bonds, and alkyl interactions.

[0099] Docking results showed that, compared to the wild-type PLA1-MFI, the mutant W166V exhibited superior substrate binding ability and catalytic advantages. Firstly, the mutant fully retained the key hydrogen bonds between the catalytic core Ser208 and Thr163 and the substrate ester carbonyl group, maintaining the fundamental mechanism of the catalytic reaction. Secondly, the hydrogen bond network between the enzyme and substrate was further strengthened, with Thr163 becoming the core site for hydrogen bonding, while the newly added Leu209 participated in hydrogen bond formation, further stabilizing the spatial position of the substrate reaction region. Thirdly, after Trp166 mutated to Val, the steric hindrance of the binding pocket was significantly reduced, the original π-cation interaction disappeared, and alkyl interactions appeared in the system, significantly enhancing the binding ability of the enzyme to the hydrophobic long chain of the substrate. Furthermore, both mutants retained van der Waals forces and C-H bonds, resulting in a richer variety of intermolecular forces and a more compact and stable overall binding network. In summary, the W166V mutation improves the binding state of the enzyme and substrate from multiple dimensions by reconstructing the hydrogen bond system, optimizing the hydrophobic pocket microenvironment, and adding alkyl interactions, thus explaining the intrinsic mechanism of the enhanced hydrolytic activity of this mutant at the molecular structure level.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A phospholipase A1 mutant, characterized in that, The mutant is derived from Serratia sp The amino acid sequence of phospholipase A1-MFI was obtained by site-directed mutagenesis, and the amino acid sequence of phospholipase A1-MFI is shown in SEQ ID NO:

1. The mutant is selected from any one of the following: (a) The amino acid at position 166 is mutated from tryptophan to valine W166V; (b) The amino acid at position 166 is mutated from tryptophan to leucine W166L.

2. The phospholipase A1 mutant according to claim 1, characterized in that, The amino acid sequence of the mutant W166V is shown in SEQ ID NO:

3.

3. The application of the phospholipase A1 mutant according to claim 1 or 2 in the preparation of lysophospholipids, wherein the lysophospholipid is lysophosphatidylcholine (LPC).

4. A nucleotide molecule encoding a phospholipase A1 mutant as described in claim 1 or 2.

5. A recombinant vector, characterized in that, It comprises the nucleotide molecule of claim 4.

6. The recombinant vector according to claim 5, characterized in that, The recombinant vector is a pET32a(+) derivative vector.

7. A host cell, characterized in that, It includes the recombinant vector as described in claim 5 or 6.

8. A method for hydrolyzing phospholipids, characterized in that, Using the phospholipase A1 mutant as described in claim 1 or 2, the substrate phospholipid is hydrolyzed under pH 8-10 and / or temperature 20-35°C to generate lysophospholipids. The substrate phospholipid is phosphatidylcholine PC, and the lysophospholipid is lysophosphatidylcholine LPC.

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