A lipase mutant and its application in the preparation of epoxides

Abstract

The present invention relates to a kind of lipase mutant and its application in preparing epoxide, specifically to the lipase mutant with significantly improved peroxidation activity derived from Indian tilletia (Tilletia indica), its encoding nucleic acid, recombinant expression vector and recombinant expression transformant containing the nucleic acid sequence, and using the recombinant lipase or recombinant expression transformant as catalyst to prepare peroxy acid, and then the application of peroxy acid and olefin reaction to generate epoxide. Compared with other methods (including chemical method or biological method etc.) for preparing α-epoxy pinane, application enzyme disclosed in the present invention and its technical method, there are the advantages such as mild reaction conditions, environmental friendliness, lower cost, simple and easy to operate, easy to amplify, and have good application prospect in the field of producing epoxy products with carbon-carbon double bond as substrate.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology and relates to a lipase mutant with significantly improved peroxidation activity, a nucleic acid encoding the lipase mutant, a recombinant expression vector and a recombinant expression transformant containing the nucleic acid sequence, and the use of the recombinant lipase mutant or the recombinant expression transformant as a catalyst for preparing peroxyacids, and further reacting the peroxyacids with alkenes to produce epoxides. Background Art

[0002] Epoxidation reaction is an extremely important type of oxygenation reaction. Its product, epoxide, contains an active three-membered oxygen ring structure and is prone to ring-opening reaction under various conditions. It can react with various functional groups such as amines and alcohols to produce high-value-added fine chemical products and intermediates. It is a widely used and extremely important chemical raw material, widely used in various chemical production fields such as organic synthesis and fine chemicals.

[0003] Olefin epoxidation is an important method for preparing epoxides, including chemical, biological and chemical-enzymatic methods. Among them, the chemical Prilezhaev epoxidation method uses hydrogen peroxide as an oxidant and a strong acid as a catalyst to catalyze the epoxidation of olefins. This method has a fast reaction speed, but has many side reactions, is difficult to recycle waste, is not environmentally friendly, and the acid can easily cause equipment corrosion. The biological method uses monooxygenases, such as cytochrome P450 and glucose monooxygenase, as catalysts and oxygen as an oxidant to catalyze the epoxidation of olefins. The biological method has mild reaction conditions and high selectivity, but often has problems such as poor enzyme stability, low catalytic efficiency, low substrate concentration, high product separation cost, and the need for expensive coenzymes. The chemical-enzymatic method uses hydrogen peroxide as an oxidant and a relatively stable lipase as a catalyst. It does not require a coenzyme and is green and environmentally friendly, combining the advantages of both chemical and biological methods.

[0004] The chemical-enzymatic epoxidation reaction is derived from the Prilezhaev epoxidation reaction and is divided into two steps: first, in the presence of hydrogen peroxide, lipase catalyzes the peroxidation reaction of carboxylic acid to produce the corresponding peroxycarboxylic acid; then, the generated peroxycarboxylic acid transfers the active oxygen to the carbon-carbon double bond of the olefin to generate the corresponding epoxide, and at the same time, the peroxyacid is reduced to carboxylic acid to participate in the next round of the cycle.

[0005] In 1990, Screening revealed that lipases from various sources can catalyze olefin epoxidation, but only immobilized Candida antarctica lipase B (Novozym 435) has been widely studied. Researchers used Novozym 435 to catalyze the epoxidation of unsaturated fatty acids. Using 4.0 wt% Novozym 435 to catalyze the epoxidation of 8.0 wt% oleic acid at 35°C for 24 hours, the yield of epoxidized fatty acids approached 100% (ACS Sustain. Chem. Eng., 2018, 6:8578-8583). Ankudey et al. used Novozym 435 to catalyze the epoxidation of phenylcyclohexene in the presence of 50% aq hydrogen peroxide and octanoic acid. After each 5-hour reaction, Novozym 435 was washed with ethyl acetate and found to completely lose activity after the third use (Green Chem., 2006, 8:923-926). Studies on epoxidation reactions using lipases from other sources are less common. Madalina et al. screened a number of known lipases and identified an Aspergillus niger lipase-mediated epoxidation of α-pinene. Using 1 M α-pinene and oxidant concentrations and 1 g / L free or immobilized enzyme, the reaction achieved a conversion of 65.4% at room temperature for 24 hours (J. Mol. Catal. B-Enzym., 2016, 134:9-15). Silva et al. used methyl oleate as a substrate, an ionic liquid as a solvent, and 30% (v / v) H₂O₂ as an oxygen carrier, selecting lipases from Candida rugosa, Pseudomonas cepacian, and Pseudomonas fluorescens to mediate epoxidation. The yields were 69%, 62%, and 54%, respectively, at 30°C for 1 hour (J. Mol. Catal. B-Enzym., 2011, 68:98-103).

[0006] Currently, the availability of lipases for chemo-enzymatic epoxidation is limited, significantly restricting its application in the production of chemical intermediates. Exploring novel lipases with superior performance beyond Candida antarctica lipase B is crucial for expanding the epoxidation lipase resource and developing highly efficient industrial enzymes.

[0007] Previous work, through extensive gene mining and screening, identified the lipase TiL (CN116949009) from the Indian fungus Tilletia indica. This enzyme exhibits the highest activity towards n-heptane, with a crude enzyme loading of 8 g / L and a reaction temperature of 50°C. It can completely epoxidize 100 mM α-pinene to the corresponding α-epoxypinene within 12 hours. While this enzyme exhibits relatively high stability, its activity remains relatively low, limiting its large-scale application. It is necessary to modify the activity of this enzyme to enhance its catalytic activity. Summary of the Invention

[0008] The present invention addresses the problems of limited sources and low activity of lipases for C=C double bond epoxidation reactions using chemical-enzymatic methods. A lipase derived from Tilletia indica obtained in previous work is modified for peroxidation activity to provide a lipase mutant with significantly improved peroxidation activity, a gene thereof, a recombinant expression vector containing the gene, and a recombinant expression transformant, as well as a method for efficiently catalyzing the synthesis of epoxides using the recombinant lipase.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The first aspect of the present invention provides a lipase mutant with improved activity.

[0011] The present invention has molecularly modified the amino acid sequence of the lipase TiL to provide a variety of lipase mutants. The lipase mutants are derived proteins with enhanced activity obtained by replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 2. Proteins corresponding to the following amino acid sequences are selected:

[0012] (1) replacing asparagine at position 72 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0013] (2) replacing asparagine at position 72 of the amino acid sequence shown in SEQ ID No. 2 with aspartic acid;

[0014] (3) replacing asparagine at position 72 of the amino acid sequence shown in SEQ ID No. 2 with serine;

[0015] (4) replacing the leucine at position 169 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0016] (5) replacing the leucine at position 169 of the amino acid sequence shown in SEQ ID No. 2 with aspartic acid;

[0017] (6) replacing the leucine at position 169 of the amino acid sequence shown in SEQ ID No. 2 with glutamic acid;

[0018] (7) replacing the leucine at position 169 of the amino acid sequence shown in SEQ ID No. 2 with glycine;

[0019] (8) replacing glutamic acid at position 218 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0020] (9) replacing glutamic acid at position 218 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0021] (10) replacing glutamic acid at position 218 of the amino acid sequence shown in SEQ ID No. 2 with lysine;

[0022] (11) replacing glutamic acid at position 218 of the amino acid sequence shown in SEQ ID No. 2 with leucine;

[0023] (12) replacing the isoleucine at position 219 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0024] (13) replacing the isoleucine at position 219 of the amino acid sequence shown in SEQ ID No. 2 with phenylalanine;

[0025] (14) replacing the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0026] (15) replacing the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 with phenylalanine;

[0027] (16) replacing the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 with leucine;

[0028] (17) The isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the glutamic acid at position 218 is replaced by isoleucine;

[0029] (18) The isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the isoleucine at position 219 is replaced by leucine;

[0030] (19) The isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by methionine, and the glutamic acid at position 218 is replaced by isoleucine;

[0031] (20) The isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by methionine, and the isoleucine at position 219 is replaced by leucine;

[0032] (21) The asparagine at position 72 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the glutamic acid at position 218 is replaced by isoleucine.

[0033] Preferably, the lipase mutant is selected from (2), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21).

[0034] Homologs of SEQ ID No. 1 are also referred to as promoter mutants. The promoter of the lipase gene can be altered by substitution, insertion, or deletion of one or more nucleotides. By changing the promoter sequence or using a more efficient promoter from a different source, the expression level of the lipase can be increased, but these changes do not negatively affect the function of the enzyme.

[0035] The second aspect of the present invention further provides an isolated nucleic acid, wherein the nucleic acid is a nucleic acid molecule encoding the lipase mutant.

[0036] The nucleotide sequence of the full-length lipase TiL gene is shown in SEQ ID No. 1, which is 1170 nucleotides long. Its coding sequence (CDS) begins at base 1 and ends at base 1170, with an ATG initiator and a TAA terminator, and no introns. The amino acid sequence of the protein encoded by this gene is shown in SEQ ID No. 2 in the sequence listing.

[0037] The third aspect of the present invention also provides a recombinant expression vector comprising the nucleic acid sequence of the lipase mutant.

[0038] The recombinant expression vector is obtained by cloning the mutant nucleic acid of the lipase TiL into various expression vectors using conventional methods in the art. The expression vector includes various conventional vectors in the art, such as commercially available plasmids, phages, or viral vectors, and the preferred vector is plasmid pET28a.

[0039] Preferably, the recombinant expression vector can be obtained by the following exemplary method: the empty plasmid pET28a and the DNA fragment of the TiL or its mutant gene obtained by PCR amplification are double-digested with restriction endonucleases EcoR I and Hind III, respectively, the lipase fragment and the empty plasmid after the above enzyme digestion are recovered, and they are ligated using T4 DNA ligase to construct a recombinant expression vector containing the lipase or its mutant gene for expression in Escherichia coli.

[0040] The fourth aspect of the present invention further provides a recombinant expression transformant comprising the lipase mutant gene or its recombinant expression vector.

[0041] The recombinant expression transformant can be prepared by transforming the above-mentioned recombinant expression vector into a host cell.

[0042] The host cell is any conventional host cell in the art, as long as the recombinant expression vector can stably replicate and the lipase mutant gene carried by the recombinant expression vector can be effectively expressed. The host cell is preferably Escherichia coli, more preferably E. coli BL21 (DE3). The preferred recombinant expression transformant of the present invention can be obtained by transforming the recombinant expression vector into E. coli BL21 (DE3).

[0043] The fifth aspect of the present invention also provides a method for preparing the recombinant lipase mutant.

[0044] The method for preparing the recombinant lipase mutant of the present invention is preferably as follows: culturing the recombinant expression transformant as described above, and isolating and obtaining the recombinantly expressed lipase. The culture medium used for culturing the recombinant expression transformant is any culture medium known in the art that can grow the transformant and produce the recombinant lipase of the present invention. The culture medium is preferably LB culture medium, having the following formula: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0. The culture method and culture conditions are not particularly limited and can be appropriately selected according to conventional knowledge in the art, as long as the transformant can grow and produce the recombinant lipase.

[0045] The specific operation of culturing the recombinant expression transformant can be carried out according to conventional operations in the art. Preferably, the recombinant E. coli of the present invention is inoculated into LB medium containing kanamycin and cultured at 37°C. When the OD 600 When the pH reaches 0.5 to 1.0 (preferably 0.6), isopropyl-β-D-thiogalactopyranoside (IPTG) is added at a final concentration of 0.1 to 1.0 mmol / L (preferably 0.2 mmol / L) to induce enzyme production, and the culture is continued at 16° C. for 24 hours to efficiently express the lipase of the present invention.

[0046] Enzyme hydrolysis specific activity assay: The change in absorbance of p-nitrophenyl ester at 405 nm was measured using a UV spectrophotometer. The standard assay system consisted of 1 mL of reaction solution containing 490 μL of potassium phosphate buffer (KPB, 100 mmol / L, pH 7.0), 10 μL of the substrate p-nitrophenyl butyrate (100 mmol / L, dissolved in dimethyl sulfoxide), and 500 μL of the incubated enzyme solution. The reaction temperature was 30°C. Enzyme activity was calculated according to the following formula:

[0047] Enzyme activity (U / g wet cell) = EW × 10 2 / (18300×l×V)

[0048] Where EW is the change in absorbance at 405 nm over 1 minute; V is the volume of the enzyme solution, in mL; 18300 is the molar extinction coefficient of p-nitrophenol, in L / (mol·cm); and l is the optical path length, in cm. One unit (U) of enzyme activity corresponds to the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol per minute under the above conditions.

[0049] In a sixth aspect, the present invention further provides a lipase catalyst, which is in any one of the following forms:

[0050] (1) culturing the recombinant expression transformant and isolating resting cells containing the lipase mutant;

[0051] (2) freeze-dried cells obtained by freeze-drying the resting cells described in form (1);

[0052] (3) disrupting the resting cells described in form (1) to obtain a cell disrupted liquid containing the lipase mutant;

[0053] (4) Freeze-dried enzyme powder obtained by freeze-drying the cell disrupted liquid described in form (3).

[0054] After the cell culture is completed, the precipitated bacterial cells are collected by centrifugation to obtain the resting cells of the recombinant expression transformant. The resulting cells are suspended in potassium phosphate buffer, disrupted by ultrasonication, and the disrupted liquid is centrifuged. The supernatant is collected to obtain a cell disrupted liquid containing the recombinant lipase. The resting cells and the cell disrupted liquid are then freeze-dried to obtain freeze-dried cells and freeze-dried enzyme powder.

[0055] In a seventh aspect, the present invention further provides the use of the above-mentioned lipase catalyst to catalyze the reaction of carboxylic acid with hydrogen peroxide to generate peroxycarboxylic acid, which then reacts with olefins in situ to prepare epoxides.

[0056] The carboxylic acid is one of acetic acid, propionic acid, n-butyric acid, n-pentanoic acid, n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-decanoic acid, lauric acid, myristic acid, palmitic acid or stearic acid.

[0057] Among them, the preferred carboxylic acid is n-heptanoic acid.

[0058] Preferably, there is provided the use of the above lipase catalyst in mediating the synthesis of α-epoxypinene from α-pinene.

[0059] The synthesis route is illustrated by taking the recombinant lipase mutant as an example, wherein the substrate n-heptanoic acid is catalyzed to prepare peroxy-n-heptanoic acid, and then the peroxy-n-heptanoic acid undergoes epoxidation with α-pinene to form α-epoxypinene, as shown below:

[0060]

[0061] The lipase catalyst of the present invention catalyzes the peroxidation reaction of the carboxylic acid, which can be carried out according to the following exemplary method:

[0062] In the presence of hydrogen peroxide, the lipase catalyst catalyzes the peroxidation of the carboxylic acid to produce the corresponding peroxycarboxylic acid, while simultaneously converting the hydrogen peroxide into water. The peroxycarboxylic acid then reacts with the olefin to produce an epoxide and a carboxylic acid, which then acts as an oxygen carrier to participate in the enzyme-catalyzed peroxidation reaction.

[0063] Preferably, the reaction conditions are as follows: the reaction is carried out in a buffered saline solution with a pH of 5.0-7.0, the concentration of the substrate carboxylic acid is 100-500 mmol / L, the molar ratio of carboxylic acid, olefin, and hydrogen peroxide is 1:1:6, and the reaction is carried out in a toluene-water two-phase system with a volume ratio of the organic phase to the aqueous phase of 5:2. The lipase activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of the corresponding substrate to product per minute. Depending on the reaction system used, the lipase dosage is 0.1-1 U / mmol of carboxylic acid compound. The phosphate buffer can be any phosphate buffer commonly used in the art, such as phosphate-sodium (potassium) phosphate buffer, as long as its pH range is 5.0-7.0. The buffer concentration can be 0.05-0.2 mol / L, preferably 0.1 mol / L. The reduction reaction temperature can be 30-60°C, preferably 50°C. During the reaction, samples are intermittently taken to determine the reaction conversion rate. The reaction time is determined by the time when the substrate is completely converted or the reaction conversion rate stops increasing, and is generally 1-12 hours. The reaction conversion was analyzed by gas chromatography.

[0064] After the reaction is completed, the organic phase is separated from the aqueous phase, and the organic phase is extracted with an equal amount of a water-insoluble organic solvent conventional in the art, such as ethyl acetate, butyl acetate, toluene, dichloromethane, isopropyl ether or methyl tert-butyl ether, etc. The organic phases are combined, the organic solvent is removed by rotary evaporation, and the product is subjected to silica gel column chromatography to obtain a high-purity epoxy product.

[0065] Compared with the prior art, the present invention has significant advantages:

[0066] Compared to the parent lipase TiL, the recombinant lipase mutant of the present invention exhibits significant advantages, including high peroxidation activity, high substrate concentration, and reduced catalyst usage. Compared to other methods for preparing α-epoxypinene, including chemical or biological methods, the enzyme and its technical methods disclosed in this invention offer advantages such as mild reaction conditions, environmental friendliness, low cost, simple operation, and ease of scale-up. These advantages hold great promise for the production of epoxides using olefins as substrates. DETAILED DESCRIPTION

[0067] The various reaction or detection conditions described in the present invention may be combined or modified according to common knowledge in the art and may be verified by experiments. The technical solutions and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, the scope of protection of the present invention is not limited to these embodiments, and any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0068] The sources of materials in the following examples are:

[0069] The expression plasmid pET28a was purchased from Novagen.

[0070] The DNA sequence optimization and synthesis of Tilletia indica lipase were completed by GenScript Biotech Co., Ltd., and the expression plasmid pET28a-TiL has been constructed.

[0071] Escherichia coli BL21 (DE3) competent cells, 2× Taq PCR MasterMix, and agarose gel DNA recovery kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0072] Restriction enzymes EcoR I, Hind III, and Dpn I are all commercially available products from New England Biolabs (NEB).

[0073] Unless otherwise stated, the specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the commercial instructions of the kits.

[0074] Example 1 Construction and expression of lipase mutants

[0075] The mutation library of lipase TiL was constructed by single-point saturation mutagenesis: the active center of TiL was molecularly docked with the substrate n-heptanoic acid to obtain the active center The sites within the range were single-point saturation mutagenesis. The corresponding mutant primers were designed and PCR amplified using the plasmid pET28a-TiL as a template and the high-fidelity polymerase PrimeSTAR. The PCR reaction conditions were as follows: 0.5-20 ng of template, 10 μL 2× PrimeSTAR (Premix), 0.4 μL of each pair of mutant primers (10 μM) were added to a 20 μL PCR reaction system, and sterile distilled water was added to 20 μL. The PCR reaction procedure was as follows: (1) denaturation at 98°C for 10 seconds, (2) annealing at 55°C for 5 seconds, (3) extension at 72°C for 90 seconds, and steps (1) to (3) were repeated 30 times. The PCR product was stored at 4°C. The PCR product was verified by agarose gel electrophoresis and then digested with the restriction endonuclease Dpn I at 37°C for 2 hours. The digested product was transformed into competent E. coli BL21(DE3) cells and plated onto LB solid medium plates containing 50 μg / mL kanamycin. The plates were then incubated in a 37°C incubator for approximately 12 hours. The resulting monoclonal colonies were transferred to LB tubes containing 50 μg / mL kanamycin and cultured with shaking at 37°C for 12 hours. The cells were then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis and transferred to 100 ml shake flasks for amplification.

[0076] Example 2 Induced expression and purification of lipase mutants

[0077] The seed solution was inoculated into a 500 mL Erlenmeyer flask containing 100 mL LB medium (containing 50 μg / mL kanamycin) at a 1% (v / v) inoculum volume and cultured at 37°C and 180 rpm. 600 When the pH value reached 0.6, IPTG was added to a final concentration of 0.2 mmoL / L for induction. After induction at 16°C for 24 hours, the culture medium was centrifuged at 8000 rpm, the cell pellet was collected, and washed with physiological saline to obtain resting cells, which were freeze-dried to prepare lyophilized cells.

[0078] The resting cells were suspended in 100 mL of potassium phosphate buffer (100 mmol / L, pH 7.0), disrupted by ultrasound in an ice-water bath, and centrifuged to collect the supernatant, which was the crude recombinant lipase solution. The resulting crude enzyme solution was freeze-dried to produce crude recombinant lipase powder.

[0079] All purification experiments were performed using nickel affinity self-packed columns. The following buffers were used during purification: Solution A: 25 mmol / L KPB, 500 mmol / L NaCl, 10 mmol / L imidazole, 1 mmol / L β-mercaptoethanol, pH 7.0; Solution B: 25 mmol / L KPB, 500 mmol / L NaCl, 500 mmol / L imidazole, 1 mmol / L β-mercaptoethanol, pH 7.0; Solution C: 25 mmol / L KPB, 500 mmol / L NaCl, 1 mmol / L DTT, 5% glycerol, pH 7.0. The purification method is as follows:

[0080] 1. Resuspend the resting cells obtained above in solution A and then disrupt them by ultrasonication. Centrifuge the crude enzyme solution at 4°C in a low-temperature high-speed centrifuge at 12,000 rpm for 30 minutes. Temporarily store the supernatant after centrifugation in a 4°C refrigerator or cold storage.

[0081] 2. Pre-equilibrate the Ni column (bed volume 2 ml) with 5 to 10 column volumes of solution A;

[0082] 3. The stored supernatant was filtered through a membrane and then loaded with sample (sample volume 10 ml);

[0083] 4. After loading, use 5 to 10 column volumes of a mixture of A and B (10% B solution) to wash away impurities.

[0084] 5. Elute the target protein with 2 column volumes of Solution B and collect;

[0085] 6. The collected target protein was concentrated by centrifugation using a 10 kDa ultrafiltration tube. When the concentration reached 0.5 mL, 5 mL of solution C was added and concentrated again by ultrafiltration. Repeat this process 3-4 times to reduce the imidazole concentration to achieve displacement. Finally, the pure enzyme was obtained by concentrating the protein to 0.5 mL.

[0086] 7. The obtained pure enzyme is quickly frozen with liquid nitrogen and stored at -80°C.

[0087] Example 3 Activity determination of lipase mutants

[0088] The total reaction volume was 0.7 mL, consisting of 0.5 mL of organic phase and 0.2 mL of aqueous phase. The organic phase contained 0.1 M α-pinene, 0.1 M n-heptanoic acid, and 1% (v / v) Tween 80 in toluene. The aqueous phase contained 0.75 M hydrogen peroxide and an appropriate amount of enzyme solution in 100 mM KPB buffer, pH 7.0. The reaction was shaken in a 2 mL Eppendorf tube at 30°C and 200 rpm for 1 hour. The reaction mixture was analyzed by gas chromatography (GC) for the olefin substrate α-pinene and the α-epoxypinene produced by the peroxyacid reaction. The decrease in substrate concentration and the increase in epoxidation product before and after the reaction were measured to determine the catalytic activity of the lipase.

[0089] After the reaction, the organic phase was separated from the aqueous phase and dried over anhydrous sodium sulfate overnight. The sample was centrifuged and analyzed using a Shimadzu GC-2014 equipped with an Rxi-5Sil MS column (30.0 m × 0.25 mm, 0.25 μm). The injector and detector temperatures were both 280°C. A 1 μL sample was injected with a split ratio of 20:1. For the determination of α-pinene and α-epoxypinene, the initial oven temperature was 100°C for 3 minutes, then increased to 250°C at a rate of 10°C / min and held for 1 minute. The retention times of α-pinene and α-epoxypinene were 4.2 minutes and 7.4 minutes, respectively.

[0090] One unit of enzyme activity is defined as the amount of enzyme required to generate 1 μmol of epoxide per minute under the above reaction conditions. Specific activity is defined as the number of activity units per milligram of protein.

[0091] The specific activities of the pure enzymes of the mutants were determined using α-pinene as a substrate. The results are shown in Table 1. In the table below, the sequence numbers refer to the series of sequences following Table 1.

[0092] Table 1. Specific activities of lipase mutants in epoxidation reactions

[0093]

[0094]

[0095] The amino acid sequence of the lipase mutant is one of the following sequences:

[0096] (1) replacing asparagine at position 72 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0097] (2) replacing asparagine at position 72 of the amino acid sequence shown in SEQ ID No. 2 with aspartic acid;

[0098] (3) replacing asparagine at position 72 of the amino acid sequence shown in SEQ ID No. 2 with serine;

[0099] (4) replacing the leucine at position 169 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0100] (5) replacing the leucine at position 169 of the amino acid sequence shown in SEQ ID No. 2 with aspartic acid;

[0101] (6) replacing the leucine at position 169 of the amino acid sequence shown in SEQ ID No. 2 with glutamic acid;

[0102] (7) replacing the leucine at position 169 of the amino acid sequence shown in SEQ ID No. 2 with glycine;

[0103] (8) replacing glutamic acid at position 218 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0104] (9) replacing glutamic acid at position 218 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0105] (10) replacing glutamic acid at position 218 of the amino acid sequence shown in SEQ ID No. 2 with lysine;

[0106] (11) replacing glutamic acid at position 218 of the amino acid sequence shown in SEQ ID No. 2 with leucine;

[0107] (12) replacing the isoleucine at position 219 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0108] (13) replacing the isoleucine at position 219 of the amino acid sequence shown in SEQ ID No. 2 with phenylalanine;

[0109] (14) replacing the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0110] (15) replacing the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 with phenylalanine;

[0111] (16) replacing the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 with leucine;

[0112] (17) The isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the glutamic acid at position 218 is replaced by isoleucine;

[0113] (18) The isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the isoleucine at position 219 is replaced by leucine;

[0114] (19) The isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by methionine, and the glutamic acid at position 218 is replaced by isoleucine;

[0115] (20) The isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by methionine, and the isoleucine at position 219 is replaced by leucine;

[0116] (21) The asparagine at position 72 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the glutamic acid at position 218 is replaced by isoleucine;

[0117] The measurement results in Table 1 show that the activity of the lipase mutant after site-directed mutagenesis is significantly improved compared with the parent enzyme, and the catalytic activity of the optimal mutant is sharply increased from 0.12 U / mg to 1.25 U / mg.

[0118] Preferably, the lipase mutants are selected from (2), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), and (21). In this case, the catalytic activity of the mutants is significantly improved.

[0119] Example 4 Effect of Carboxylic Acid Type on Enzyme-Catalyzed Peroxidation

[0120] Reaction conditions: The total reaction volume was 0.7 mL, consisting of 0.5 mL of organic phase and 0.2 mL of aqueous phase. The organic phase contained 0.1 M α-pinene, 0.1 M carboxylic acid, and 1% (v / v) Tween 80 in toluene. The aqueous phase contained 0.75 M hydrogen peroxide and 10 mg / mL of the lipase mutant TiL-M18 in 100 mM KPB buffer, pH 7.0. The reaction was shaken at 30°C and 200 rpm in a 2 mL vial for 12 h. The reaction mixture was analyzed by gas chromatography (GC) for the olefin substrate α-pinene and the α-epoxypinene produced by the peroxyacid reaction, and the reaction conversion was calculated.

[0121]

[0122] Example 5 Lipase TiL-M18 Mediated Epoxidation of 100 mM α-pinene

[0123] 500 μL of the toluene organic phase (containing 100 mmol / L n-heptanoic acid and 100 mmol / L α-pinene) and 200 μL of aqueous buffer (containing 100 mM KPB, pH 7.0, 1.5 M H₂O₂, and 4 mg of lyophilized enzyme powder from the TiL-M18 lipase mutant) were placed in a 2 mL Eppendorf tube and reacted at 50°C and 200 rpm for 6 h. After the reaction, the organic and aqueous phases were separated and dried over anhydrous sodium sulfate overnight. Gas chromatography-mass spectrometry (GC-MS) analysis revealed a substrate conversion of 99.87% and a 2,3-epoxypinene yield of 82.30%.

[0124] In this embodiment, the lipase TiL-M18 is prepared by replacing the isoleucine at position 316 with alanine and the isoleucine at position 219 with leucine in the amino acid sequence shown in SEQ ID No. 2.

[0125] Example 6 Lipase TiL-M18 mediates the conversion of α-pinene to α-epoxypinene

[0126] A 50 mL toluene organic phase (containing 100 mmol / L n-heptanoic acid and 100 mmol / L α-pinene) and 20 mL aqueous buffer (containing 100 mM KPB, pH 7.0, 1.5 M H₂O₂, and 400 mg of freeze-dried enzyme powder from the TiL-M18 lipase mutant) were placed in a small double-walled glass reaction vial and incubated at 50°C with magnetic stirring. Samples were taken intermittently to monitor the reaction conversion. After 8 hours of reaction, when the conversion exceeded 99%, the reaction was terminated and the product, α-epoxypinene, was isolated and recovered. The reaction solution was allowed to stand in a separatory funnel until the organic and aqueous phases separated. The organic phase was separated, and the remaining aqueous phase was extracted twice with an equal volume of toluene. The combined organic phases were washed with 50 mM sodium carbonate solution, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and separated on a flash silica gel column using a mobile phase of petroleum ether / ethyl acetate in a 6:1 ratio. 0.43 g of a transparent liquid with a purity of 97.2% was obtained.

[0127] In this embodiment, the lipase TiL-M18 is prepared by replacing the isoleucine at position 316 with alanine and the isoleucine at position 219 with leucine in the amino acid sequence shown in SEQ ID No. 2.

[0128] Comparative Example 1: Epoxidation of 100 mM α-pinene mediated by lipase TiL

[0129] 500 μL of the toluene organic phase (containing 100 mmol / L n-heptanoic acid and 100 mmol / L α-pinene) and 200 μL of aqueous buffer (containing 100 mM KPB, pH 7.0, 1.5 M H₂O₂, and 6 mg of lipase TiL freeze-dried enzyme powder) were placed in a 2 mL Eppendorf tube and shaken at 50°C and 200 rpm for 12 hours. After the reaction, the organic and aqueous phases were separated and dried over anhydrous sodium sulfate overnight. Gas chromatography analysis revealed a substrate conversion of 98.94% and a yield of 68.04% for the product 2,3-epoxypinene.

[0130] The sequences involved in the present invention are as follows:

[0131] SEQ ID No.1

[0132] GAATTCATGAGGTTACACTTCCTATCATTTGCTACAGTGATCAGCGCGATTTCG

[0133] ATCTCCGTCTCCCACGCCGCTGTGGTACCGGTTTCAGCCCCACCGACAAGCG

[0134] ATCCGGCATTCGTTACCCCACAAGCGACCCTGGATGCTAATATTAAATGCCCG

[0135] GGGACCAAAGGTGGTTATGCGGCGGTGTCCAATCCGATTTTGTTGGTTCCAG

[0136] GTACGGGCAACACCGGTAGCGAATCGTTCGATTCCACGTACGTGATTCTGACT

[0137] CGCAACCTGGGCTACCAGCCGTGTTATATCTCTCCGCCGCCGTTCATGCTGAA

[0138] CGATAGCCAAATCAACCGGGAGTATGTTGTAAACGCAGTTTCTCGTTTAAATC

[0139] AGGCAGCGGGCAAGAAAATCCCGGTTCTGGGCTGGTCGCAAGGTAACCTGA

[0140] TCATTCAGTGGGCACTCACCTTTTTCCCGAGCACCGTTCAAAAAACCGATCG

[0141] TTTTGTTTCTTTCGCTGGCGACTTCCGTGGTACGTTTCTGGCATACCTGCTGG

[0142] ACGCCCAGCCGCTGGGTATTGCACCGTCCGTGTGGCAACAAAGCACCCTGAG

[0143] CGCGTATCTGACTGCGCTGCGCAACGCTGGCGGTTTAACTGCCAAGGTGCCG

[0144] ACCACCTCTATTTATAGCGTGACGGACGAAATCGTGCAGCCGCAGATCGGTG

[0145] GCCCAGCGTTGGAGAGCTCCTACTTGTTCGGTGACATGGCAATGAACGTGAA

[0146] GGTGCAGGATTACTGCCCGTTATTGATCGTTGAGCATAGCCAACAGCTTTTCA

[0147] ATATCTTTACCTACAGCGTGGCAAAAGCGGCGTTGCAATCCCCGACCGGTAA

[0148] GGCGGAGGCGGGCAGCTTCAGCTCGGCTAAGTGCAGCCTGGCGTTTCCGCC

[0149] GGGTTTGGGCCTGGGTGACCAGCTGGTCGCCCCTACCATTATTACCCAGGCG

[0150] GCGGTGCACATTGTGGCCGGCCCGCGTGTTGCGTGTGAACCGCCTTTGCTGC

[0151] CGTATGCGGTTAAATACTACCCGTTTGCGAAGCAGGCGTGCAATCCGCTGACG

[0152] CAGGTTGTGGACGGCTTTGTCCCGCAAACCCCGGAACAGCTGAACAGCTTC

[0153] GAGGCTATTACCGTTTATATCCTGACCAGAAGCCTGCGTAACATCCTTGGTAC

[0154] CGCTGGTAAGTAAAAGCTT

[0155] SEQ ID No. 2

[0156] MRLHFLSFATVISAISISVSHAAVVPVSAPPTSDPAFVTPQATLDANIKCPGTKGGY

[0157] AAVSNPILLVPGTGNTGSESFDSTYVILTRNLGYQPCYISPPPFMLNDSQINAEYV

[0158] VNAVSRLNQAAGKKIPVLGWSQGNLIIQWALTFFPSTVQKTDRFVSFAGDFRGTF

[0159] LAYLLDAQPLGIAPSVWQQSTLSAYLTALRNAGGLTAKVPTTSIYSVTDEIVQPQI

[0160] GGPALESSYLFGDMAMNVKVQDYCPLLIVEHSQQLFNIFTYSVAKAALQSPTGK

[0161] AEAGSFSSAKCSLAFPPGLGLGDQLVAPTIITQAAVHIVAGPRVACEPPLLPYAVK

[0162] YYPFAKQACNPLTQVVDGFVPQTPEQLNSFEAITVYILTRSLRNILGTAGK

[0163] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A lipase mutant, characterized in that: It is a protein with the following amino acid sequence: (1) replacing the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (2) replacing the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 with phenylalanine; (3) replacing the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 with leucine; (4) the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the glutamic acid at position 218 is replaced by isoleucine; (5) the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the isoleucine at position 219 is replaced by leucine; (6) the isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by methionine, and the glutamic acid at position 218 is replaced by isoleucine; (7) The isoleucine at position 316 of the amino acid sequence shown in SEQ ID No. 2 is replaced by methionine, and the isoleucine at position 219 is replaced by leucine.

2. An isolated nucleic acid, characterized in that The nucleic acid is a nucleic acid molecule encoding the lipase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: Comprising the nucleic acid according to claim 2.

4. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector according to claim 3.

5. A lipase catalyst, characterized in that It is any of the following forms: (1) culturing the recombinant expression transformant according to claim 4 and isolating resting cells containing the lipase mutant according to claim 1; (2) freeze-dried cells obtained by freeze-drying the resting cells containing the lipase mutant described in form (1); (3) disrupting the resting cells of form (1) to obtain a cell disrupted liquid containing the lipase mutant of claim 1; (4) Freeze-dried enzyme powder obtained by freeze-drying the cell disrupted liquid described in form (3).

6. Use of the lipase mutant according to claim 1 or the lipase catalyst according to claim 5 in catalyzing n-heptanoic acid to produce peroxy-n-heptanoic acid, and then synthesizing α-epoxypinene from peroxy-n-heptanoic acid and α-pinene; in the presence of hydrogen peroxide, under the action of the lipase mutant according to claim 1 or the lipase catalyst according to claim 5, catalyzing the peroxidation of n-heptanoic acid to produce peroxy-n-heptanoic acid, and simultaneously converting the hydrogen peroxide into water. The peroxy-n-heptanoic acid then reacts with α-pinene to produce α-epoxypinene and n-heptanoic acid, and the n-heptanoic acid again participates in the enzyme-catalyzed peroxidation reaction as an oxygen carrier.

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