Lipase PAL mutant and application thereof

Abstract

The invention discloses a lipase PAL mutant and application thereof. Based on the amino acid sequence of wild type lipase PAL, the amino acid sequence of the mutant comprises substitution of L146S and G230N, and the amino acid sequence of the wild type lipase PAL comprises a sequence as shown in SEQ ID No.1. The enzyme activity of the lipase PAL mutant disclosed by the invention is obviously superior to that of wild type lipase PAL; the lipase PAL mutant can efficiently catalyze hydrolysis of 2-phenoxy methyl propionate to obtain R-2-phenoxy methyl propionate, and the catalysis time is short, so that the lipase PAL mutant has wide application prospects in the fields of food, pharmacy, fine chemical engineering and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis and enzyme engineering, and particularly relates to a lipase PAL mutant and application thereof. Background Art

[0002] As the world's second largest herbicide category (second only to glyphosate), phenoxypropionic acid herbicides are an important tool for controlling grass weeds in modern agriculture. Its development can be traced back to the 1960s, when Germany's Hoechst Company first developed diclofop-butyl. Subsequently, active groups such as heterocycles and fluorine atoms were introduced through structural modification to form high-efficiency varieties such as clodinafop-butyl and cyhalofop-butyl. Global sales reached US$667 million in 2019. This type of compound inhibits acetyl-CoA carboxylase (ACCase), blocks fatty acid synthesis, and causes damage to the membrane structure of weeds. It has the characteristics of high efficiency, low toxicity and strong selectivity. At present, more than 5,000 derivatives have been developed, and nearly 140 patents have been laid out, forming a product matrix covering crops such as rice, wheat, and corn. Among them, R-2-phenoxypropionic acid methyl ester is a key chiral intermediate, and its synthesis efficiency directly affects the industrialization process of downstream herbicides (such as hydrochloride).

[0003] The industrial synthesis of methyl R-2-phenoxypropionate relies primarily on three methods: 1) Chiral source synthesis (e.g., Williamson ether synthesis) achieves configuration inversion through an SN2 reaction. While yields reach 73%, enantioselectivity is only 75% ee, requiring high-temperature reflux, solvent distillation, and multiple purification steps, resulting in complex and energy-intensive processes. 2) Asymmetric catalysis (e.g., copper-catalyzed alkylation of ethyl diazopropionate) can achieve high selectivity (99% ee), but relies on precious metal catalysts (CuCl / NaBARF) and toxic solvents (dichloromethane), resulting in high costs and environmental pollution. 3) Racemic bioresolution (e.g., Brevibacterium imperialis B222 hydrolase-catalyzed process) operates under mild conditions but suffers from poor chiral recognition (42% ee), low conversion efficiency (reaction termination requires 50% conversion), and cumbersome product purification steps. None of these methods offer the necessary balance between environmentally friendly production, high selectivity, and economies of scale. Therefore, there is an urgent need to develop a new bio-enzyme catalytic system to break through the efficiency bottleneck of the hydrolytic kinetic resolution of racemic forms by targeted optimization of the chiral recognition ability and catalytic activity of lipase, and to achieve the sustainable preparation of high-purity R-configuration products.

[0004] None of the above existing technologies can achieve high selectivity, low cost, and green process requirements. Therefore, there is an urgent need to develop a new catalytic system based on enzyme-directed optimization. By improving the chiral recognition ability and reactivity of lipase, it can break through the selectivity bottleneck of the hydrolytic kinetic resolution of the racemate and achieve the efficient and green preparation of R-2-phenoxypropionic acid methyl ester.

[0005] The lipase catalytic system has become the preferred solution for the synthesis of chiral intermediates due to its strong stereoselectivity and mild reaction conditions. The catalytic splitting of pregabalin intermediates achieved an ee value of 98% and 10 recycling times. However, there are still gaps in the enzymatic research on methyl 2-phenoxypropionate: Miyazawa's team used Aspergillus niger lipase to catalyze the vinyl ester conversion, but the ee value was only 83%; Wakana et al. achieved a 92% ee value through papaya lipase transesterification, but the conversion rate was less than 46%.

[0006] Our team previously discovered that lipase (PAL) from Pseudomonas alcaligenes exhibits excellent selectivity in the resolution of menthol and holds great promise. However, the enzyme's long catalytic reaction time and low efficiency warrant further modification and improvement.

[0007] Therefore, there is an urgent need for a lipase that is efficient, highly stereoselective, and can resolve racemic 2-phenoxypropionic acid methyl ester. Summary of the Invention

[0008] To address the problems of severe pollution and high energy consumption in the chemical synthesis of R-2-phenoxypropionic acid methyl ester, and the technical bottleneck of low substrate selectivity in traditional biocatalysis, the present invention provides a lipase PAL mutant and its application. The mutant can efficiently resolve the racemic 2-phenoxypropionic acid methyl ester, thereby improving conversion efficiency and selectivity.

[0009] The present invention is achieved through the following technical ideas: using the wild-type lipase PAL gene from Pseudomonas aeruginosa as a template, a mutant library is obtained by molecular docking with sites near the substrate and the protein channel, primers are designed for site-directed saturation mutagenesis, and a recombinant plasmid library is then constructed using pET30a as a backbone. The recombinant plasmid library is transformed and introduced into Escherichia coli BL21 (DE3), and a high-throughput screening system for the mutant library is established. Through primary screening in 96-well plates and secondary screening in test tubes and shake flasks, lipase PAL mutants with enhanced enzyme activity are finally screened.

[0010] The first aspect of the present invention protects a lipase PAL mutant, based on the amino acid sequence of the wild-type lipase PAL, the amino acid sequence of the mutant includes substitutions L146S and G230N, and the amino acid sequence of the wild-type lipase PAL includes the sequence shown in SEQ ID No. 1.

[0011] In certain embodiments, the wild-type lipase PAL is derived from Pseudomonas aeruginosa.

[0012] In certain embodiments, the amino acid sequence of the mutant includes the sequence shown in SEQ ID No. 2 (L146S) or the sequence shown in SEQ ID No. 3 (L146S / G230N).

[0013] In certain embodiments, the enzyme activity of the mutant is increased by at least 14.83 times compared to the wild-type lipase PAL.

[0014] In certain embodiments, the enantioselectivity (ee value) of the mutant is increased by at least 58.1% compared to the wild-type lipase PAL.

[0015] The present invention uses a semi-rational design strategy to modify the key sites of the lipase PAL derived from Pseudomonas aeruginosa, screens and obtains the single mutant L146S, and obtains the double-point mutant L146S / G230N based on the single mutant. The enzyme activity of the double mutant reaches 40.36 U / mg, which is 14.83 times higher than that of the wild type; compared with the wild-type PAL, the enantioselectivity (ee value) of the double mutant for the substrate is increased from 57% to 90.1%, the reaction time is shortened from more than 20 hours to 60 minutes, and it exhibits excellent substrate selectivity. The mutant can efficiently catalyze the synthesis of the chiral intermediate R-2-phenoxypropionic acid methyl ester of phenoxy herbicide under mild conditions of pH 9.0 and 37°C. The lipase mutant of the present invention can be used for the green synthesis of chiral intermediates of phenoxy herbicides, has industrial production potential, and provides a new strategy for the semi-rational design of lipases and the synthesis of chiral drugs.

[0016] Another aspect of the present invention is to protect a method for constructing a mutant as described above, comprising the following steps:

[0017] (a) Molecular docking and active pocket analysis were used to screen 62 potential mutation sites in wild-type lipase PAL, including L146;

[0018] (b) performing a first round of saturation mutagenesis at the potential mutation sites and constructing a single-point mutant library, screening L146S as the optimal single-point mutant;

[0019] (c) Using L146S as a template, a second round of iterative saturation mutagenesis was performed based on the overlapping sites of the substrate channel and the active center, and the double-point mutant L146S / G230N was screened and obtained.

[0020] In certain embodiments, molecular dynamics simulation is also included to verify the reconstruction of the hydrogen bond network of the mutant substrate binding pocket and the improvement of the conformational stability of key residues.

[0021] Another aspect of the present invention protects an isolated polynucleotide encoding a mutant as described above.

[0022] Another aspect of the present invention provides a nucleic acid construct comprising the polynucleotide described above, including but not limited to a prokaryotic expression vector pET30a and a cloning vector.

[0023] Another aspect of the present invention provides a host cell comprising the nucleic acid construct described above or a polynucleotide described above integrated into its genome. The host cell is Escherichia coli BL21 (DE3).

[0024] Another aspect of the present invention protects the use of the mutant described above, the polynucleotide described above, the nucleic acid construct described above, or the host described above in the preparation of R-2-phenoxypropionic acid methyl ester.

[0025] Another aspect of the present invention is a method for preparing methyl R-2-phenoxypropionate, comprising the steps of:

[0026] The mutant as described above, or the polynucleotide as described above, or the nucleic acid construct as described above, or the host as described above and the substrate carry out a catalytic reaction.

[0027] In certain embodiments, the substrate comprises methyl 2-phenoxypropionate, which is a methyl phenoxypropionate racemate. The methyl phenoxypropionate racemate refers to a mixture of equal amounts of methyl R-2-phenoxypropionate and methyl S-2-phenoxypropionate.

[0028] In certain specific embodiments, based on the total volume of the reaction system, the concentration of the substrate is 40-60 mM, preferably 50 mM.

[0029] In certain embodiments, the temperature of the catalytic reaction is 30-40°C, preferably 37°C.

[0030] In certain embodiments, the catalytic reaction time is less than 1 hour.

[0031] In certain specific embodiments, the crude enzyme solution obtained by ultrasonically disrupting the wet bacteria of the host described above is used as a catalyst to catalyze the reaction of the substrate methyl 2-phenoxypropionate.

[0032] In certain more specific embodiments, the catalytic reaction medium is 50 mM glycine buffer at pH 9.0.

[0033] In certain more specific embodiments, the concentration of the substrate is 50 mM based on the total volume of the reaction system.

[0034] In certain more specific embodiments, based on the total volume of the reaction system, the amount of the crude enzyme added is 0.11 mg / mL and the enzyme activity is 30.02 U / mL.

[0035] Furthermore, the wet bacteria were prepared as follows: the host described above was inoculated into LB liquid culture medium, cultured at 37°C for 6-8 hours, inoculated into LB liquid culture medium at a volume concentration of 1%, and cultured at 37°C until the culture OD 600 The pH value was between 0.6 and 0.8, and a final concentration of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added. The cells were induced at 16°C for 16-18 hours, centrifuged, and the cells were harvested and washed twice with physiological saline to obtain wet cells. The wet cells were ultrasonically disrupted (ice bath, power 300 W, 3 s operation, 5 s interval, total time 30 min) and centrifuged (12,000 × g, 4°C, 30 min) to obtain a crude enzyme solution.

[0036] Furthermore, the method further includes a step of purifying the wet cells, wherein the purification is a Ni-NTA affinity chromatography method, specifically comprising the following steps:

[0037] Equilibration: Remove the stored ethanol solution and rinse the Ni-NTA affinity column with approximately 10 column volumes of deionized water. Then, equilibrate the Ni-NTA affinity column with 10 column volumes of 50mM imidazole to restore matrix activity. Loading: The lysate containing the 6×His-tagged mutant protein (filtered through a 0.45μm filter) was loaded five times by gravity flow (0.5-1mL / min). The binding efficiency was improved by extending the contact time. Purification: The Ni-NTA affinity column was rinsed with approximately 15-20 column volumes of 50mM imidazole to selectively elute non-specifically adsorbed host proteins. The Ni-NTA affinity column was rinsed with approximately 10-15 column volumes of 250mM imidazole to elute the target protein and collect the eluate on ice to maintain its stability. Further treatment of the target protein: The purified target protein eluate was concentrated using a 30 kDa molecular weight cutoff ultrafiltration centrifuge tube (4°C, 6000 × g). After replacement with a pH 9.0 Gly-NaOH buffer system, the concentrated product was aliquoted into sterile cryovials. The protein concentration was quantified using the BCA assay, and the pure enzyme solution was finally stored at -80°C for a long term. The binding buffer was a pH 9.0, 50 mM Gly-NaOH buffer containing a final concentration of 500 mM NaCl. The different concentration imidazole buffers were pH 9.0, 50 mM Gly-NaOH buffers containing a final concentration of 500 mM NaCl and different concentrations of imidazole.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1) The present invention uses semi-rational design to molecularly modify lipase to obtain a lipase PAL double mutant (L146S / G230N), whose enzyme activity is increased by 14.83 times compared with the wild-type PAL.

[0040] 2) The lipase PAL mutant of the present invention was used to hydrolyze methyl 2-phenoxypropionate at room temperature (37° C.), and the reaction time was shortened from more than 20 hours (wild-type lipase PAL) to 1 hour; the enantioselectivity (ee value) for methyl 2-phenoxypropionate was increased from 57% (wild-type lipase PAL) to 90.1%, an increase of 58.1%.

[0041] 3) The lipase PAL mutant of the present invention has good application prospects in efficiently catalyzing the hydrolysis of 2-phenoxypropionic acid methyl ester to obtain R-2-phenoxypropionic acid methyl ester. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a molecular docking diagram of the wild-type lipase PAL and the substrate S-2-phenoxypropionic acid methyl ester in Example 1.

[0043] Figure 2 This is a diagram of the substrate channel in Example 1.

[0044] Figure 3 This is the result diagram of the single point mutation in Example 2.

[0045] Figure 4 This is the result diagram of the double-point mutation in Example 2.

[0046] Figure 5 1 is a flow chart of the synthesis reaction of methyl R-2-phenoxypropionate in Example 7.

[0047] Figure 6 This is a graph showing the changes in conversion rate and selectivity of wild-type lipase PAL as the reaction proceeds in Example 7.

[0048] Figure 7 This is a graph showing the changes in conversion rate and selectivity of the lipase PAL mutant (L146S / G230N) as the reaction proceeds in Example 7. DETAILED DESCRIPTION

[0049] The present invention will be further described below in conjunction with specific embodiments. The following examples are only specific embodiments of the present invention, but protection scope of the present invention is not limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were performed using three replicates, and the results were averaged.

[0050] The composition of the LB liquid culture medium in the following examples is: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, the solvent is purified water, and the pH value is natural.

[0051] The composition of the LB solid medium in the following examples is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar, the solvent is water, and the pH value is natural.

[0052] Example 1 Mutation site selection and screening

[0053] This example uses single-site saturation mutagenesis to mutate the lipase PAL from Pseudomonas aeruginosa. Specifically:

[0054] 1.1 Molecular docking analysis

[0055] Using 8YZN in the PDB database as a template, the FlexibleDocking module in the Discovery Studio 2019 software package was used to perform molecular docking of wild-type lipase PAL (its amino acid sequence is shown in SEQ ID No. 1) and substrate S-2-phenoxypropionic acid methyl ester. The docking parameters were set as: CHARMm force field, energy optimization iteration step size 1000 times, and weighted coefficients of van der Waals force and electrostatic potential of 0.1 and 0.2, respectively. Complex conformations with binding free energy lower than -8 kcal / mol were extracted, and the active pocket was visualized and analyzed using PyMOL 2.5. 36 key residues were selected (see Table 1 ) to evaluate the effects of hydrogen bonding, hydrophobic interactions, and π-π interactions on stereoselectivity.

[0056] Molecular docking of wild-type lipase PAL and substrate S-2-phenoxypropionate methyl ester Figure 1 .

[0057] 1.2 Substrate channel analysis

[0058] CavityPlus 2.0 software was used to probe the substrate channel of wild-type lipase PAL, identify potential channels and annotate 58 key residues (see Table 1).

[0059] 1.3 Mutation site design

[0060] Based on the results of molecular docking and substrate channel analysis, mutation sites were designed for key residues in the active pocket and substrate channel. These mutation sites were selected based on their critical roles in the active pocket and substrate channel, as well as their potential impact on substrate binding and enzyme catalysis. For each mutation site, corresponding primers were designed for subsequent single-site saturation mutagenesis experiments. The mutation sites are listed in Table 1.

[0061] The overlapping residues are near the active center The residues within the range are the residues shared by the protein channel. The first round of mutation points in Example 2 are all the mutation points of the above two. The second round of saturation mutation in Example 4 uses overlapping residue mutations based on L146S.

[0062]

[0063] Primer sequences are shown in Table 2.

[0064] Table 2 Primer design table

[0065]

[0066]

[0067] Example 2 Construction of mutation library

[0068] Using pET30a-PAL as a template, PCR amplification was performed using the primers designed in Example 1 to construct a site-saturation mutation library (i.e., the first round of mutation). The specific steps are as follows: The PCR reaction system is shown in Table 3.

[0069] Table 3

[0070]

[0071] The amplification program was set as follows: initial denaturation at 98°C for 3 minutes to initiate template melting, followed by 30 amplification cycles (each cycle consisting of denaturation at 98°C for 15 seconds, gradient annealing at 55-65°C for 15 seconds (temperature optimized based on primer Tm values), and extension at 72°C for 2 minutes). After the cycle was completed, a final extension at 72°C for 10 minutes was performed to ensure product integrity. Finally, the amplified product was stored at 4°C for future use.

[0072] The amplified PCR product was digested with DpnI (37°C, 2 h) to remove the template, transformed into E. coli BL21 competent cells, evenly spread on LB agar plates (LB solid medium) containing kanamycin (final concentration 50 μg / mL), and cultured in an inverted manner at 37°C overnight to obtain a single-point saturation mutation library.

[0073] Cloning and verification: Three single clones were randomly selected from each library for sequencing to verify the mutation site. The plates with mutation sites confirmed by sequencing were stored at 4°C for subsequent functional screening.

[0074] Example 3 Strain cultivation and preservation

[0075] The monoclonal clone obtained in Example 2 and 12 wells of the wild-type control strain were inoculated into 200 μL of LB liquid medium (containing 34 mg / mL chloramphenicol and 100 mg / mL ampicillin) in a 96-well plate and cultured with shaking at 37°C and 250 rpm for 12-16 hours to obtain an overnight bacterial solution. 50 μL of the bacterial solution was transferred using a 12-channel pipette to a daughter plate containing 150 μL of antibiotic medium at the same concentration. The mother plate was supplemented with a final concentration of 20% glycerol and then frozen at -80°C.

[0076] Induction of expression and bacterial cell acquisition: After the daughter plate is cultured at 37°C and 250 rpm until the OD600 reaches 0.6-0.8, 0.1 mM IPTG is added and low-temperature induction is carried out at 25°C and 250 rpm for 16 hours; centrifuge at 4°C and 5000×g for 20 minutes, discard the supernatant, obtain the bacterial cells, and freeze at -80°C until use.

[0077] Preparation of crude enzyme solution: Remove frozen bacterial cells and add 200 μL of Gly-NaOH buffer (5 mM, pH 9.0) containing 0.1 g / L lysozyme to each well. Lyse the cells at 25°C and 220 rpm for 2 hours. Centrifuge at 4°C and 12,000 × g for 10 minutes. Collect the supernatant as the soluble lipase source.

[0078] Example 4 High-throughput screening method

[0079] In this example, ester hydrolysis reaction and pH indicator Bromothymol Blue (BTB) were used to screen for highly active mutants in the lipase mutant library. These include:

[0080] Principle: Ester hydrolysis generates acid, causing a decrease in the reaction system's pH, which can be monitored by the color change of the pH indicator bromothymol blue (yellow at pH 6.0, blue at pH 7.6). Absorbance changes before and after the reaction are measured using a microplate reader. The activity of the mutant strain is compared with that of the wild type to identify highly active mutants.

[0081] The entire reaction system was 200 μL: 50 μL of the crude enzyme solution obtained in Example 3, 50 μL of indicator (bromothymol blue dissolved in 20% ethanol aqueous solution, concentration was 1 mM), and 100 μL of 50 mM R-2-phenoxypropionic acid methyl ester substrate solution (dissolved in DMSO).

[0082] Experimental steps: 1) Add crude enzyme solution to a 96-well reaction plate, then add indicator. After mixing, a dark blue color will appear. Then, add substrate solution and measure the initial OD value of each reaction well at 630nm. 2) After reacting at 37℃ for 30 minutes, a color change is observed and the OD value at the end of the reaction is measured using a microplate reader. 3) Based on the absorbance activity data of the mutant library, the screening threshold is set using the wild-type strain as a control (the absorbance difference is ≥50% higher than the wild-type average value), and the target clone is locked through data normalization analysis. 4) After locating the corresponding well position of the mother plate, 5μL of bacterial solution is inoculated into LB liquid medium containing kanamycin and amplified overnight at 37℃. Subsequently, the plasmid is extracted using a plasmid kit and sequenced by Beijing Qingke Co., Ltd. The resulting sequence is aligned with the original gene using snapgene software to accurately identify the amino acid substitution site and rescreened using HPLC liquid chromatography. The wild-type and sequenced improved sites are re-cultured in 50mL shake flasks to measure the crude enzyme activity, providing data support for the improvement of lipase activity.

[0083] Enzyme activity results are shown in Figure 3 .

[0084] The method for enzyme activity determination is as follows:

[0085] The reaction system included 50 μL of 2M methyl 2-phenoxypropionate and 450 μL of crude enzyme solution, totaling 0.5 mL of reaction system. The mixture was shaken at 37° C. for 10 min to measure the enzyme activity.

[0086] The reaction solution obtained after the reaction was diluted by adding 500 μL of acetonitrile solution containing 0.5% phosphoric acid, filtered through a 0.25 μm organic filter membrane, and analyzed by HPLC.

[0087] Enzyme activity assay: The HPLC instrument used a C-18 column, 4.6 mm × 250 mm. The mobile phase consisted of acetonitrile / 0.1% aqueous phosphoric acid (50 / 50). The detection wavelength was 254 nm, the column temperature was 30°C, the mobile phase flow rate was 1 mL / min, and the injection volume was 20 μL. The peak elution time of the product acid was approximately 5.3 min, and the peak elution time of the substrate was approximately 8.6 min.

[0088] Enzyme activity definition: One enzyme activity unit (1U) is the amount of enzyme required to catalyze the production of 1 μmol of 2-phenoxypropionic acid per minute at 37°C and pH 9.0.

[0089] from Figure 3It can be seen that the mutations with improved crude enzyme activity compared with the wild type include L146M, L146C, L146S, L226I, I157Q and P370G, among which the L146S mutant has the highest crude enzyme activity, followed by L146C, I157Q and L146M. The amino acid sequence of the L146S mutant is shown in SEQ ID No. 2.

[0090] Furthermore, using L146S as a template, a second round of iterative saturation mutagenesis was performed at the overlapping sites of the substrate channel and the active center, resulting in six double mutants, including: L146S / G230N, L146S / L476P, L146S / L476A, L146S / L476H, L146S / A154K, and L146S / L226M. The crude enzyme activity results of the double mutants are shown in Figure 4 Among them, L146S is marked as 1M.

[0091] from Figure 4 It can be seen that the double mutant L146S / G230N has the highest enzyme activity, followed by L146S / L476P, L146S / A154K and L146S / L226M. The amino acid sequence of the double point mutant L146S / G230N is shown in SEQ ID No.3.

[0092] Example 5 Expression and purification of lipase mutants

[0093] In this example, the mutant strain screened in Example 4 was expressed and purified, including the following steps:

[0094] 5.1. Strain culture and induced expression

[0095] The mutant strain was inoculated into LB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37°C until OD 600 =0.6-0.8, add 0.5mM IPTG, and induce expression at 16℃ for 18h.

[0096] 5.2 Cell disruption

[0097] The culture obtained in step 5.1 was centrifuged to collect the wet cells, which were then disrupted by ultrasonication in an ice bath (power 300 W, working 3 s, interval 5 s, total time 30 min), and then centrifuged at 4°C and 12,000×g for 30 min to obtain the supernatant crude enzyme solution.

[0098] 5.3 Ni-NTA affinity chromatography purification

[0099] Experimental reagents: ethanol solution, i.e., 20% ethanol aqueous solution by volume, gradient imidazole buffer solutions of different concentrations (50 mM, 250 mM), i.e., 500 mM NaCl, corresponding concentrations of imidazole, 50 mM Gly-NaOH buffer, adjusted to pH 9.0.

[0100] The mutant protein (pET30a-PaL) with a 6×His tag fused to the N-terminus was purified using a gradient imidazole buffer system.

[0101] 1) Equilibration: Remove the stored ethanol solution and wash the Ni-NTA affinity column with approximately 10 column volumes of deionized water. Then, equilibrate the Ni-NTA affinity column with 10 column volumes of 50 mM imidazole to restore matrix activity.

[0102] 2) Loading: The lysate containing the 6×His-tagged mutant protein (filtered through a 0.45 μm filter) was loaded five times by gravity flow (0.5-1 mL / min). The binding efficiency was improved by extending the contact time. The liquid flowing out of the chromatography column after loading was the flow-through.

[0103] 3) Purification: Wash the Ni-NTA affinity column with approximately 15-20 column volumes of 50 mM imidazole to selectively elute non-specifically adsorbed host proteins. Wash the Ni-NTA affinity column with approximately 10-15 column volumes of 250 mM imidazole to elute the target protein and collect the eluate on ice to maintain stability.

[0104] 4) Further processing of the target protein: The purified target protein eluate was concentrated using a 30 kDa molecular weight cutoff ultrafiltration centrifuge tube (4°C, 6000 × g). After replacement with a pH 9.0 Gly-NaOH buffer system, the concentrated product was aliquoted into sterile cryovials. The protein concentration was quantified using the BCA assay and the product was stored at -80°C for long-term storage.

[0105] Protein concentration was determined using the BCA assay with bovine serum albumin as the standard. Furthermore, enzyme activity was determined as described in Example 4, except that a concentrated product was used instead of a crude enzyme solution.

[0106] The protein concentration and enzyme activity after purification are shown in Table 4.

[0107] Table 4

[0108] mutant Protein content (mg / mL) Enzyme activity (U / mg) Wild-type PAL 9.82 2.55 L146M mutant 13.20 4.50 L146C mutant 11.04 18.89 L146S mutant 9.22 20.34 L146S / A154K mutant 11.22 31.24 L146S / L226M mutant 9.96 30.06 L146S / G230N mutant 9.88 40.36 L146S / L476A mutant 12.08 22.06 L146S / L476P mutant 13.10 29.22 L146S / L476H mutant 6.20 29.18

[0109] As shown in Table 4, the enzyme activity of the lipase PAL double mutant (L146S / G230N) was increased by 14.83 times compared with the wild-type PAL ((double mutant enzyme activity-wild-type PAL enzyme activity) / wild-type PAL enzyme activity) (p < 0.05).

[0110] Example 6 Determination of kinetic parameters of PAL lipase and its mutants

[0111] The kinetic parameters of the protein purified in Example 3 were determined in the following steps:

[0112] Methyl 2-phenoxypropionate was used as substrate, and pure enzyme solutions of wild-type PAL, L146S mutant, and L146S / G230N mutant were used as catalysts.

[0113] Enzyme activity was measured using methyl 2-phenoxypropionate as a substrate, with substrate solutions ranging from 5 to 20 mmol / L. The reaction was performed at 37°C, a pH of 9, and a duration of 1 hour. The Lineweaver-Burk double reciprocal method was used to plot the kinetic constants of the enzyme reaction. The results are shown in Tables 5 and 6. The method for enzyme activity measurement is described in Example 4.

[0114] Table 5

[0115]

[0116] As shown in Table 5 , the maximum reaction rate, Michaelis constant, catalytic constant and catalytic efficiency of the L146S / G230N double mutant were higher than those of the wild-type PAL and the single mutant containing only L146S.

[0117] Table 6

[0118]

[0119]

[0120] As shown in Table 6, the L146S / G230N double mutant has the highest maximum reaction rate, Michaelis constant, catalytic constant and catalytic efficiency.

[0121] Example 7 Reaction Progress of Mutant-Catalyzed Selective Hydrolysis of Methyl 2-Phenoxypropionate

[0122] In this example, the selective synthesis of R-2-phenoxypropionate methyl ester by wild-type PAL and PAL double mutant (L146S / G230N) from substrate 2-phenoxypropionate methyl ester was studied. The synthesis process is shown in Figure 5 . Including the following:

[0123] The reaction conditions were as follows: 85 mL of 50 mM Gly-NaOH buffer, 5 mL of crude enzyme solution (wild-type PAL and L146S / G230N), and 10 mL of 500 mM methyl 2-phenoxypropionate were added to a 250 mL three-necked flask, with DMSO as a cosolvent. The pH of the reaction system was monitored in real time using a pH meter, and 1 M NaOH was automatically added dropwise to maintain the pH of the system at 9.0. The enzyme selectivity for the substrate was determined using a high-performance liquid chromatograph, as follows:

[0124] Selective Detection: The HPLC column used was a CHIRALPAK IB N-5 column (4.6 mm × 250 mm). The mobile phase consisted of n-hexane / isopropanol (90 / 10), with a detection wavelength of 254 nm, a column temperature of 30°C, a mobile phase flow rate of 1 mL / min, and a 10 μL injection volume. The peak elution time for the S-configuration substrate was 6.8 min, and the peak elution time for the R-configuration substrate was 13.4 min.

[0125] Calculation of ee value: ee = [(RS) / (R+S)] × 100%.

[0126] Enantiomeric excess (ee) refers to the percentage by which the amount of one enantiomer exceeds the amount of the other enantiomer in a mixture of a pair of enantiomers.

[0127] The conversion rate and ee value of wild-type lipase PAL for substrate are shown in Figure 6 Conversion rate = substrate consumption / initial substrate amount * 100%

[0128] from Figure 6 It can be seen that the conversion rate of wild-type PAL quickly increased to 30% within the first 20 h (finally stabilized at 60%), but the growth rate slowed down due to substrate consumption or product inhibition, and its enantiomeric excess (ee) reached 57% and then stagnated.

[0129] The conversion rate and ee value of the PAL double mutant (L146S / G230N) for the substrate are shown in Figure 7 .

[0130] from Figure 7 The mutant (L146S / G230N) exhibited significantly improved catalytic performance through site-specific mutation optimization, with conversion increasing linearly to 50% within 60 minutes (reaching 65% in 300 minutes). The ee value remained above 90% throughout this period (with an initial peak of 90.1%), demonstrating the mutant's precise recognition of the target enantiomer. A slight decrease in the ee value during the latter stages was attributed to competitive hydrolysis caused by limited mass transfer of the S-configuration substrate. In contrast, the conversion of wild-type PAL rapidly increased to 30% within 20 hours (ultimately stabilizing at 60%), indicating that the catalytic energy of the mutant for substrate conversion was shortened from 20 hours to 1 hour.

[0131] In summary, wild-type PAL and PAL mutants show significant differences in conversion rate and selectivity. Through the modification of the active center, the mutant achieves efficient and stable stereoselective catalysis, which can effectively break through the industrial bottleneck of insufficient activity and selectivity attenuation of traditional enzymatic methods, and provide technical support for the green synthesis of chiral propionate compounds.

[0132] The present invention is not limited by the above specific description. Various changes can be made to the present invention within the scope outlined by the claims, and these changes are all within the scope of the present invention.

Claims

1. A lipase PAL mutant, characterized in that: Based on the amino acid sequence of the wild-type lipase PAL, the amino acid sequence of the mutant includes substitutions of L146S and G230N. The amino acid sequence of the wild-type lipase PAL includes the sequence shown in SEQ ID No.

1.

2. The mutant according to claim 1, characterized in that Compared with the wild-type lipase PAL, the enzyme activity of the mutant is increased by at least 14.83 times.

3. An isolated polynucleotide, characterized in that Encoding the mutant according to claim 1 or 2.

4. A nucleic acid construct, characterized in that Comprising the polynucleotide according to claim 3.

5. A host, characterized in that The nucleic acid construct according to claim 4 is included, or the polynucleotide according to claim 3 is integrated into the genome.

6. Use of the mutant according to claim 1, the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, or the host according to claim 5 as a catalyst in the preparation of R-2-phenoxypropionic acid methyl ester.

7. A method for preparing methyl R-2-phenoxypropionate, characterized in that: The steps include: The mutant according to claim 1, the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, or the host according to claim 5 and the substrate carry out a catalytic reaction.

8. The method according to claim 7, wherein: The substrate includes methyl 2-phenoxypropionate.

9. The method according to claim 7, wherein: The temperature of the catalytic reaction is 30-40°C; And / or, the catalytic reaction time is less than 1 hour.

10. The method according to claim 8, wherein: Based on the total volume of the reaction system, the concentration of the substrate is 40-60 mM.

Images