A lipase mutant, immobilized enzyme and application thereof

Abstract

This invention discloses a lipase mutant, an immobilized enzyme, and their applications, belonging to the field of bioengineering. The SpL enzyme mutant K193R / I134V described in this invention exhibits a specific activity of 7-ACA that is 1.6 times higher than before the mutation, and its half-life at 45℃, 50℃, and 55℃ is increased by 1.9 times, 2.6 times, and 3.8 times, respectively. Immobilizing the his-tagged SpL enzyme on a hydrophobically modified nickel-doped SBA-15 mesoporous molecular sieve preserves the enzyme's activity and improves its thermal stability and reusability. Using this immobilized enzyme to catalyze the production of D-7-ACA allows for at least 10 reusable batches, maintaining a conversion rate above 99%.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a lipase mutant, an immobilized enzyme, and its applications. Background Technology

[0002] Lipase (EC 3.1.1.3) is one of the earliest discovered and studied enzymes, also known as triacylglycerol acyl hydrolases, belonging to the hydrolase family. SpL is a lipase from Sphingomonas sp. HXN-200, which was found to be an intracellular enzyme that generates amides from the ammonolysis of esters or acids. SpL can be used not only for the ammonolysis of esters or acids to prepare amides with a wide substrate range, high activity and / or high enantioselectivity (Zeng Shichao, Liu Ji, Ankanbil Sampson, Chen Ming, Guo Zheng, Adams Joseph P., Snajdrova Radka, Li Zhi. Amide Synthesis via Aminolysis of Ester or Acid with an Intracellular Lipase[J]. ACS Catalysis, 2018, 8(9).), but also for the hydrolysis of acetyl compounds.

[0003] D-7-ACA is a novel intermediate for cephalosporin antibiotics with high reactivity, which is beneficial for the synthesis of new cephalosporin products. It simplifies the production process in the synthesis of some cephalosporin varieties and has the advantages of simple modification, easy purification, good product quality, and reduced production costs. When synthesizing cephalosporin products by alkaline hydrolysis, the usual preparation route is: hydrolyze 7-ACA as raw material at -30℃ to -40℃ under high concentration strong alkaline conditions to obtain D-7-ACA solution, and then synthesize cephalosporin products (Nomura H, Fugono T, Hitaka T, et al. Semisynthetic beta-lactam antibiotics. 6.1 Sulfocephalosporins and their antipseudomonal activities. [J]. Journal of Medicinal Chemistry, 1974, 17(12): 1312-5. DOI: 10.1021 / jm00258a017). However, the alkaline hydrolysis method for preparing cephalosporin products requires harsh reaction conditions and results in low conversion rates. In contrast, enzymatic deacetylation offers simpler reaction conditions, higher conversion rates, and higher purity. However, free enzymes are expensive, have poor stability, and are sensitive to environmental factors such as temperature and pH, limiting their industrial application.

[0004] Rational design in protein engineering is based on a certain understanding of the enzymatic information of the target protein, such as its sequence, structure, and catalytic mechanism. Various bioinformatics software are used to predict the impact of mutations at different sites on the stability and catalytic performance of the target protein. Whole plasmid PCR is one of the most widely used site-directed mutagenesis methods, offering advantages such as ease of operation, speed, and efficiency.

[0005] Enzyme immobilization refers to the process of immobilizing enzymes within a defined spatial range, enabling repeated and continuous use. While enzyme-catalyzed reactions typically occur in aqueous solutions, immobilized enzymes are created by treating water-soluble enzymes physically or chemically to render them insoluble in water while retaining their enzymatic activity. This technology has not only solved the problem of product separation and purification but also significantly improved the reusability of enzymes, thus enabling their industrial application. Similarly, due to the rigid structure of immobilized enzymes, they retain enzyme activity comparable to their initial activity during repeated use and can still catalyze reactions under harsh conditions. With increasing demands for enzyme applications, enzyme immobilization technologies require timely updates, with research on immobilization methods and carriers remaining a hot topic. Currently, many methods have been proposed to address the problems associated with free enzymes. For example, patent CN103045580A provides a method for immobilizing wheat malt lipase, using epichlorohydrin-modified silica gel as a carrier, resulting in immobilized enzymes with improved stability and reusability. Patent CN105154428A discloses a method for preparing and applying ordered three-dimensional mesoporous carbon-immobilized lysozyme, resulting in immobilized enzymes with good stability and reusability. Although the carriers used in the above methods can solve the problem of free enzymes to some extent, the preparation process is cumbersome and easily leads to enzyme activity loss. Summary of the Invention

[0006] The purpose of this invention is to solve the problems of low lipase activity and poor adsorption capacity of immobilized carriers for free lipase, which cannot significantly improve the thermal stability and operational stability of the enzyme. Based on this, this invention provides a lipase, a mutant, an immobilized enzyme, and its applications.

[0007] Specifically, this invention first provides a SpL lipase mutant. Guided by the principles of surface amino acid (Gly-Ala, Lys-Arg) substitution and consensus theory, and with the assistance of bioinformatics software GetArea and Consensus Finder, a nitrile hydrolase mutant with high thermal stability and high activity was obtained.

[0008] This invention further provides a functionalized nickel-doped mesoporous silica support, which is used to prepare immobilized enzymes. This solves the problems of poor adsorption capacity of immobilized supports for free lipases and inability to significantly improve the thermal and operational stability of enzymes. The functionalized nickel-doped mesoporous silica support provided in this application has a simple synthesis method and a strong adsorption effect on proteins with histidine tags.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] The first aspect of this invention provides a lipase mutant selected from one of the following:

[0011] (1) Mutate the lysine K at position 193 in the amino acid sequence shown in SEQ ID NO.1 to arginine R;

[0012] (2) Mutate isoleucine I at position 134 of the amino acid sequence shown in SEQ ID NO.1 to valine V;

[0013] (3) The lysine K at position 193 in the amino acid sequence shown in SEQ ID NO.1 is mutated to arginine R, and the isoleucine I at position 134 is mutated to valine V.

[0014] In one embodiment of the present invention, the N-terminus of the lipase mutant expresses a His tag.

[0015] In one embodiment of the present invention, the N and C ends of the lipase mutant express a His tag.

[0016] The amino acid sequence of the parent lipase SpL is shown in SEQ ID NO.1, which is derived from Sphingomonas sp. HXN-200; the encoding gene for lipase SpL is shown in SEQ ID NO.7.

[0017] After expressing a His tag at the N-terminus of the parental lipase SpL, its amino acid sequence is shown in SEQ ID NO.2, and the corresponding nucleotide sequence is shown in SEQ ID NO.8.

[0018] After expressing the His tag at the N and C ends of the parental lipase SpL, the amino acid sequence is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO.9;

[0019] When the lipase mutant is selected from (1), it is represented as SpL. K193R The amino acid sequence is shown in SEQ ID NO.4, and the corresponding nucleotide sequence is shown in SEQ ID NO.10;

[0020] When the lipase mutant is selected from (2), it is represented as SpL. I134V The amino acid sequence is shown in SEQ ID NO.5, and the corresponding nucleotide sequence is shown in SEQ ID NO.11;

[0021] When the lipase mutant is selected from (3), it is represented as SpL. K193R-I134V The amino acid sequence is shown in SEQ ID NO.6, and the corresponding nucleotide sequence is shown in SEQ ID NO.12.

[0022] This invention first uses the principle that Gly is replaced with Ala and Lys is replaced with Arg on the surface of thermophilic proteins. Using GetArea software, a total of 6 Gly and 11 Lys amino acids were found on the surface of SpL, and site-directed mutagenesis was performed on them to obtain a mutant enzyme with improved thermostability. Then, based on the principle that conserved amino acids in homologous sequences are more stable than non-conserved amino acids, multiple sequence alignment of SpL was performed using Consensus Finder software to screen out a total of 6 amino acids with a threshold greater than 70%. After site-directed mutagenesis of these amino acids to the corresponding conserved amino acids, a mutant enzyme with improved activity against 7-ACA hydrolase was obtained.

[0023] After mutating the above two amino acid combinations, a two-site mutant with significantly improved thermal stability and specific activity was obtained, thus improving the thermal stability of the existing SpL.

[0024] The lipase mutant of this invention exhibits significantly improved specific activity and thermostability compared to the parental lipase SpL. It is derived from a site-directed mutagenesis of the parental lipase SpL, *Sphingomonas* sp. HXN-200, by introducing mutations at positions 193 (lysine K) and 134 (isoleucine I) using whole-plasmid PCR. More specifically, the mutation replaces lysine K at position 193 with arginine R and isoleucine I at position 134 with valine V. The nitrile hydrolase mutant K193R / I134V is named using the format "original amino acid abbreviation + mutation position + replacement amino acid abbreviation".

[0025] Compared with the lipase SpL of the parent Sphingomonas HXN-200 (the specific activity of the pure enzyme is 516.32 U / g), the specific activities of the lipase SpL mutants K193R, I134V and K193R / I134V are 527.17 U / g, 739.88 U / g and 857.69 U / g, respectively. Among them, K193R / I134V showed the best specific activity, which was 1.66 times higher than that of the parent.

[0026] Compared with the lipase SpL of the parent sphingomonas HXN-200, the half-life t at 45℃, 50℃, and 55℃ was longer. 1 / 2The half-lives of the lipase SpL mutants K193R, I134V, and K193R / I134V at 45°C were 1.58, 1.03, and 1.87 times that of the parents, respectively (t1 / 2 of 2.36 h, 1.54 h, and 2.79 h, respectively). At 50°C, their half-lives were 1.68, 1.19, and 2.64 times that of the parents, respectively (t1 / 2 of 1.36 h, 0.96 h, and 2.14 h, respectively). At 55°C, their half-lives were 2.39, 1.31, and 3.80 times that of the parents, respectively (t1 / 2 of 1.49 h, 0.81 h, and 0.49 h, respectively). 1 / 2 The durations were 1.17h, 0.64h, and 1.86h, respectively.

[0027] In a second aspect, the present invention also provides an isolated nucleic acid, said nucleic acid being a nucleic acid molecule encoding the lipase mutant.

[0028] In a third aspect, the present invention also provides a recombinant expression vector comprising the nucleic acid sequence of the lipase mutant.

[0029] The recombinant expression vector is obtained by cloning the lipase mutant nucleic acid into various expression vectors using conventional methods in the art. The expression vectors include various vectors conventional in the art, such as commercially available plasmids, bacteriophages, or viral vectors; for example, the vector chosen is plasmid pRSFDuet-1.

[0030] A preferred recombinant expression vector can be obtained by the following example: plasmid pRSFDuet-1 and the DNA fragment of the lipase mutant gene obtained by PCR amplification are digested with restriction endonucleases, respectively. The digested lipase fragment and empty plasmid are recovered and ligated using ligase to construct a recombinant expression vector containing the lipase mutant gene for expression in Escherichia coli.

[0031] In a fourth aspect, the present invention also provides a recombinant expression transformant comprising the lipase mutant gene or its recombinant expression vector.

[0032] The recombinant expression transformant can be prepared by transforming the above-mentioned recombinant expression vector into host cells.

[0033] The host cell is a conventional host cell in the art, as long as it satisfies the requirement that the recombinant expression vector can stably replicate on its own and that the lipase mutant gene it carries can be effectively expressed. The host cell is preferably *Escherichia coli*, and more preferably *E. coli* BL21(DE3). Transforming the recombinant expression vector into *E. coli* BL21(DE3) yields the preferred recombinant expression transformant of this invention.

[0034] In a fifth aspect, the present invention also provides a method for preparing the recombinant lipase mutant.

[0035] The preferred method for preparing the recombinant lipase mutant of the present invention is as follows: culturing the recombinant expression transformant as described above, and isolating the lipase mutant. The culture medium used for culturing the recombinant expression transformant is any culture medium in the art capable of enabling the transformant to grow and produce the recombinant lipase of the present invention. There are no special limitations on the culture method and conditions; appropriate selections can be made according to conventional knowledge in the art, based on factors such as host cell type and culture method, as long as the transformant can grow and produce the lipase mutant.

[0036] The specific procedures for culturing the recombinant expression transformant can be performed according to conventional procedures in the art. Preferably, the present invention provides a method for obtaining the aforementioned highly binding, thermostable, and highly active lipase mutant, comprising the following steps:

[0037] Using plasmid pRSFDuet-1 as a template, primers were designed, and a recombinant plasmid containing the lipase mutant was obtained by PCR. The gene and plasmid encoding the lipase mutant were then obtained, and the lipase mutant was expressed using *E. coli* as the host. Preferably, the *E. coli* seed culture containing the recombinant plasmid was inoculated into a fermentation medium and fermented at 37°C and 200 rpm for 12 hours. The fermentation medium consisted of: 9.4 g / L K₂HPO₄, 2.2 g / L KH₂PO₄, 24 g / L yeast extract, 12 g / L tryptone, and a natural pH (approximately 7.1).

[0038] In a sixth aspect, the present invention also provides a lipase catalyst, which is any one of the following forms:

[0039] (1) Culture the recombinant expression transformant and isolate resting cells containing the lipase mutant;

[0040] (2) Frozen stem cells obtained by freeze-drying the resting cells described in form (1);

[0041] (3) The resting cells described in form (1) are broken up to obtain a cell lysate containing the lipase mutant.

[0042] (4) The freeze-dried enzyme powder obtained by freeze-drying the cell lysate described in form (3).

[0043] After cell culture, the precipitated bacterial cells are collected by centrifugation; these are the resting cells of the recombinant expression transformant. The obtained cells are suspended in potassium phosphate buffer, sonicated, and the lysate is centrifuged. The supernatant is collected to obtain the cell lysate of the recombinant lipase. The resting cells and cell lysate are freeze-dried separately to obtain frozen stem cells and freeze-dried enzyme powder.

[0044] In a seventh aspect, the present invention also provides an immobilized enzyme, comprising an immobilized enzyme carrier and a lipase or a lipase mutant immobilized on the immobilized enzyme carrier, wherein the immobilized enzyme carrier is a functionalized nickel-doped mesoporous silica carrier, wherein the functionalized nickel-doped mesoporous silica carrier refers to a nickel-doped mesoporous silica surface that has been functionalized to give it a certain degree of surface hydrophobicity.

[0045] This invention provides a functionalized nickel-doped mesoporous silica support, which is used to prepare immobilized enzymes. This solves the problems of poor adsorption capacity of immobilized enzyme supports for free lipases and the inability to significantly improve the thermal and operational stability of enzymes. The functionalized nickel-doped mesoporous silica support provided in this application has a simple synthesis method and, in particular, has a strong adsorption effect on proteins with histidine tags.

[0046] In one embodiment of the present invention, the method for preparing the functionalized nickel-doped mesoporous silica support is as follows:

[0047] Step 1: Add template agent P123 to HCl solvent, then add mesitylene, ammonium fluoride and nickel source, stir evenly, then add tetraethyl orthosilicate and the precursor of surface functional groups, and continue stirring the above mixture in a hot water bath to react;

[0048] Step 2: The reaction product and mother liquor are transferred to a hydrothermal crystallization reactor for crystallization. After cooling, the mixture is filtered, dried, template agent removed, filtered again, dried, and calcined to obtain the functionalized nickel-doped mesoporous silica support. In one embodiment of the present invention, the functionalized group precursor in the reaction system is triethoxymethylsilane or triethoxypropylsilane.

[0049] In one embodiment of the present invention, the functionalized group precursor in the reaction system is triethoxymethylsilane.

[0050] In one embodiment of the present invention, the mass ratio of triethoxymethylsilane to silicon source (tetraethyl orthosilicate) in the reaction system is 1% to 9%.

[0051] In one embodiment of the present invention, the mass ratio of triethoxymethylsilane to silicon source in the reaction system is 7%.

[0052] In one embodiment of the present invention, in step two, the calcination temperature is 250℃~550℃ and the calcination time is 6h~15h.

[0053] In one embodiment of the present invention, in step two, the calcination temperature is 300°C and the calcination time is 12 hours.

[0054] In one embodiment of the present invention, in step one, the HCl solvent is 1.6M, and the reaction time in step one is 20h to 24h.

[0055] In an eighth aspect, the present invention also provides a method for preparing the immobilized enzyme described in the seventh aspect, wherein the method comprises mixing a solution containing lipase or a lipase mutant with the immobilized enzyme carrier and then immobilizing the mixture by adsorption, and obtaining the immobilized enzyme by centrifugation.

[0056] In one embodiment of the present invention, in the immobilization system containing lipase or lipase mutant and the above-mentioned immobilized enzyme carrier, the concentration of lipase or lipase mutant is 2-4 mg / ml and the concentration of immobilized carrier is 5-30 mg / ml.

[0057] In one embodiment of the present invention, in the immobilization system containing lipase or a lipase mutant and the above-mentioned immobilized enzyme carrier, the concentration of the enzyme is 3 mg / ml and the concentration of the immobilized carrier is 15 mg / ml.

[0058] In one embodiment of the present invention, the pH of the immobilization system is 6 to 9.

[0059] In one embodiment of the present invention, the pH of the immobilization system is 8.

[0060] In one embodiment of the present invention, the reaction temperature in the immobilization system is 20–40°C and the reaction time is 5–30 min.

[0061] In one embodiment of the present invention, the reaction temperature in the immobilization system is 30°C and the reaction time is 10 min.

[0062] In a ninth aspect, the invention also provides the application of the lipase mutant described in the first aspect or the immobilized enzyme described in the seventh aspect to catalyze the hydrolysis of 7-ACA as a substrate to generate D-7-ACA.

[0063] In one embodiment of the present invention, the reaction was carried out using 7-ACA as a substrate at a substrate concentration of 70 mM, a reaction temperature of 25°C, a pH of 8.0, and an immobilized enzyme amount of 8 g / L. After repeated use for 12 batches, the product was collected and analyzed, and the conversion rate was found to be 99.21%.

[0064] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0065] Guided by the principles of surface amino acid substitution and consensus theory, site-directed mutagenesis was performed on the SpL lipase gene to obtain the SpL mutant enzyme K193R / I134V, which exhibits improved thermostability and specific activity. For 7-ACA, its half-life at 55°C is 3.8 times that of the wild-type enzyme (SpL lipase), and its specific activity is 1.66 times that of the wild-type enzyme. This invention also provides the synthesis of a functionalized nickel-doped mesoporous silica support and its application in immobilizing enzymes. By modifying the support surface to be hydrophobic, the immobilized enzyme shows better loading capacity and higher activity compared to the unfunctionalized support. Compared to enzymes immobilized with an N-terminal His-tagged protein, the immobilized enzyme with the double His tag has a higher protein loading rate and specific activity. Furthermore, compared to the free enzyme, the immobilized enzyme shows significant improvements in thermostability, pH stability, and operational stability, as detailed below:

[0066] By functionalizing the vector, the protein loading rate of the immobilized enzyme can reach 79%, while the protein loading rate of the immobilized enzyme in the unfunctionalized vector is only 63%.

[0067] The protein loading rate of the dual His-tagged immobilized enzyme was 7.5% higher than that of the conventional His-tagged immobilized enzyme.

[0068] The free enzyme retained only 12% of its activity after incubation at 50°C for 24 hours, while the immobilized enzyme retained 57% of its activity after incubation at 50°C for 24 hours.

[0069] The free enzyme retained only 43% of its activity after being incubated at 50°C for 1 hour in 50mM Tris-HCl (pH 9) buffer, while the immobilized enzyme retained 83% of its activity after being incubated at 50°C for 1 hour in 50mM Tris-HCl (pH 9) buffer.

[0070] The immobilized enzyme retained 95% of its conversion rate after being reused 15 times. Attached Figure Description

[0071] Figure 1 : Comparison of specific activity between the mutant constructed in this invention and wild-type SpL.

[0072] Figure 2 includes Figure 2a , 2b 2c: Comparison of the inactivation half-life of the mutant constructed in this invention and wild-type SpL at different temperatures.

[0073] Figure 3 Immobilization effects of different functional groups and different group contents.

[0074] Figure 4The efficiency of fixing single His tags and double His tags.

[0075] Figure 5 Temperature stability of free enzymes and immobilized enzymes.

[0076] Figure 6 pH stability of free and immobilized enzymes.

[0077] Figure 7 The reusability of immobilized enzymes.

[0078] Figure 8 Liquid phase spectrum of product D-7-ACA. Detailed Implementation

[0079] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0080] The culture medium formulations involved in the examples are as follows:

[0081] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0.

[0082] LB solid medium: Add 15 g / L agar to the basic formulation of LB liquid medium.

[0083] Fermentation medium: K2HPO4 9.4 g / L, KH2PO4 2.2 g / L, yeast extract 24 g / L, tryptone 12 g / L, natural pH (pH approximately 7.1).

[0084] Cell lysis and protein extraction:

[0085] The cultured fermentation broth was centrifuged at 12,000 rpm for 10 min, and then washed twice with physiological saline. Based on the relationship between OD value and cell concentration, the cells were resuspended in phosphate buffer (50 mM pH 8) to achieve a cell concentration of 10 g / L. Ultrasonic disruption was performed at 450 W for 3 s, with a 4 s interval, for a total disruption time of 10 min. After one disruption in an ice bath, the cells were centrifuged at 4°C, 12,000 rpm for 30 min to obtain the lysed supernatant.

[0086] Protein content determination:

[0087] Add 0.5 mL of the cell lysis supernatant to a 5 mL EP tube, then add 3.0 mL of Coomassie Brilliant Blue G-250 reagent. Mix immediately on a vortex mixer and let stand for 5 min. Transfer the mixture to a 1 mL cuvette and immediately measure the absorbance at 595 nm. 595 Calculate the protein concentration.

[0088] Protein binding rate determination:

[0089] The supernatant and washing solution after immobilization were combined, and the protein content of the combined solution was measured. The protein binding rate was calculated as D = (A0 - (A1 × B)) / A0 × 100%, where A0 is the protein content before immobilization, A1 is the protein content of the combined solution, and B is the dilution factor of the supernatant after immobilization.

[0090] Assay of lipase SpL activity:

[0091] The activity assay reaction system (1 mL) consisted of: 100 μg of crude enzyme mixed with 0.49 mL of 50 mM PB (pH 8.0) buffer, followed by preheating with 50 mM PB (pH 8.0) buffer containing 0.02 M substrate 7-ACA at 35 °C for 5 min. Then, 0.50 mL of substrate solution was added to the reaction system to bring the final concentration to 10 mM. The mixture was reacted at 35 °C and 1000 rpm for 30 min using a constant temperature mixer. After quenching and mixing with 1 mL of methanol, the mixture was vortexed and centrifuged at 12000 rpm for 2 min. The supernatant was then aspirated using a sterile syringe for HPLC analysis.

[0092] Enzyme activity assay of immobilized lipase SpL:

[0093] The activity assay reaction system (1 mL) consisted of: 0.6 mg of immobilized enzyme mixed with 0.49 mL of 50 mM PB (pH 8.0) buffer, followed by preheating with 50 mM PB (pH 8.0) buffer containing 0.02 M substrate 7-ACA at 35 °C for 5 min. Then, 0.50 mL of substrate solution was added to the reaction system to bring the final concentration to 10 mM. The mixture was reacted at 35 °C and 1000 rpm for 30 min using a constant temperature mixer. After quenching and mixing with 1 mL of methanol, the mixture was vortexed and centrifuged at 12000 rpm for 2 min. The supernatant was then aspirated using a sterile syringe for HPLC analysis.

[0094] Analytical method (HPLC): The chromatographic column was a reversed-phase C18 column (Diamonsil plus, 4.6 mm * 250 mm * 5 μm); the mobile phase was methanol:phosphate (50 mM sodium dihydrogen phosphate, pH 6.5) = 20:80 for 0-10 min; the flow rate was 1.0 mL / min; the detection wavelength was 254 nm; the column temperature was set at 25 °C; and the injection volume was 10 μL. The retention times of D-7-ACA and 7-ACA were 3.5 min and 5.6 min, respectively.

[0095] Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of product (hydroxymethyl-7-aminocephalosporanic acid) per minute at 35°C and pH 8.0.

[0096] Determination of thermal stability: Wild-type and mutant enzyme solutions were incubated in metal water baths at 40℃, 50℃, and 55℃, respectively. Residual enzyme activity was measured at regular intervals. A linear relationship graph was fitted with incubation time as the x-axis and the logarithm of residual enzyme activity as the y-axis. Then, based on t... 1 / 2 =ln2 / k d The half-life is then determined.

[0097] Methods for determining the temperature stability of lipase SpL / immobilized enzyme:

[0098] Take 0.1 mg of free enzyme and 0.1 mg of immobilized enzyme containing protein, and incubate them in 50 mM PB (pH 8) buffer at 45 °C, 50 °C, and 55 °C for 3 h, 6 h, 12 h, and 24 h, respectively. The activity before incubation is taken as 100%, and the residual enzyme activity after incubation is calculated by comparing it with the activity after incubation to examine the temperature stability of free enzyme and immobilized enzyme.

[0099] Methods for determining the pH stability of lipase SpL / immobilized enzyme:

[0100] Take 0.1 mg of free enzyme and 0.1 mg of immobilized enzyme containing protein, and place them in a buffer system of (50 mM PB buffer (pH 6, 6.5, 7, 7.5, 8), 50 mM Tris-HCl buffer (pH 8, 9), 50 mM Gly-NaOH buffer (pH 9, 10, 11)). After incubation at 50 °C for 1 h, the residual enzyme activity is calculated by comparing the activity before incubation with the activity after incubation, with the activity before incubation as 100%, to examine the pH stability of the free enzyme and the immobilized enzyme.

[0101] Methods for determining the reusability of immobilized enzymes:

[0102] Take 8 mg of (CH3)-NiCl2-SBA-15 immobilized enzyme and add it to 0.5 mL of 100 mM PB (pH 8) buffer. Incubate at 25 °C for 3 min, then add 0.5 mL of solution containing 5.44 mg of substrate (20 μmol) and react for 90 min. After centrifugation, collect the supernatant and wash the immobilized enzyme twice. Take 0.5 mL of buffer again and add fresh substrate solution to react. Detect the conversion rate.

[0103] The conversion rate of D-7-ACA is calculated as follows:

[0104] Conversion rate α = (0.1947 * 10) ^(-3)(A-A0)-0.0252)×B / C×100%;

[0105] Where A represents the peak area integrated by external standard method, A0 represents the peak area of ​​spontaneous hydrolysis of the substrate, B represents the dilution factor of the overall reaction system, and C represents the concentration of substrate 7-ACA. The relationship is y = 0.1947 * 10 ^(-3) x-0.0252 is the external standard curve of D-7-ACA.

[0106] The present invention will be further described below with reference to specific embodiments.

[0107] Example 1

[0108] His tag expression at the C-terminus was performed using whole-plasmid PCR.

[0109] Using the recombinant plasmid pRSFDuet-1-SpL as a template, a plasmid containing a His tag at its C-terminus was amplified in vitro by PCR. The primers used to add the His tag were:

[0110] SpL-F1:5'-GCACACCACCACCACCACCACTGAGAATTCGAGCTCGGCGCG CCT-3'

[0111] SpL-F2:5'-TCTCAGTGGTGGTGGTGGTGGTGTGCCGCTCCGGTAGCCTCG GC-3'

[0112] SpL-R1:5'-GCACCTGAAGTCAGCCCCATACGATATA-3'

[0113] SpL-R2:5'-TATATCGTATGGGGCTGACTTCAGGTGC-3'

[0114] The PCR reaction system is as follows: (primer concentration is 10 μmol / L)

[0115]

[0116]

[0117] The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 20 s, 30 cycles; 72℃ extension for 5 min; storage at 4℃.

[0118] After verifying the correctness of the PCR product by nucleic acid electrophoresis, 10 μL of the reaction solution was recovered using a universal DNA purification and recovery kit. The DNA template was then digested with Dpn I restriction enzyme at 37°C for 4 hours. The digested product was then transformed into *E. coli* BL21(DE3) competent cells. The cells were plated on LB resistant solid plates (containing 50 μg / mL Kana) and incubated at 37°C for 12-14 hours. One to three transformants were picked and transferred to LB liquid medium (containing 50 μg / mL Kana) and incubated for 6 hours until the medium became turbid. The bacterial culture was then sent for sequencing.

[0119] Example 2

[0120] SpL enzyme mutant was constructed using whole plasmid PCR.

[0121] Using the recombinant plasmid pRSFDuet-1-SpL as a template, the plasmid containing the mutant gene was amplified in vitro by PCR.

[0122] The primers used for site-directed mutagenesis are (underlined areas indicate mutation sites):

[0123] K193R primer-F:5'-GACGAACCGGCAGCG AGG CCGGTGATC-3'

[0124] K193R primer-R:5'-GCGATCACCGG CGC CGCTGCCGGTTC-3'

[0125] I134V primer-F:5'-GCCGGTCATCGCG GTG GATTATCGCCTCGC-3'

[0126] I134V primer-R:5'-AGTTTCGTTCCGAGCCG CCT GGATGCCTGA-3'

[0127] The PCR reaction system is as follows: (primer concentration is 10 μmol / L)

[0128]

[0129] The PCR amplification program was as follows: 95℃ pre-denaturation for 10 min; 98℃ denaturation for 10 s, 58℃ annealing for 15 s, 72℃ extension for 75 s, 24 cycles; 72℃ extension for 5 min; storage at 4℃.

[0130] After verifying the correctness of the PCR product by nucleic acid electrophoresis, 10 μL of the reaction solution was recovered using a universal DNA purification and recovery kit. The DNA template was then digested with Dpn I restriction enzyme at 37°C for 4 hours. The digested product was then transformed into *E. coli* BL21(DE3) competent cells. The cells were plated on LB resistant solid plates (containing 50 μg / mL Kana) and incubated at 37°C for 12-14 hours. One to three transformants were picked and transferred to LB liquid medium (containing 50 μg / mL Kana) and incubated for 6 hours until the medium became turbid. The bacterial culture was then sent for sequencing.

[0131] A strain of E. coli BL21(DE3) / pRSFDuet-1-SpL with improved thermostability compared to the original strain was obtained through screening for superior strains. K193R (SpL K193R The amino acid sequence is shown in SEQ ID NO.4, and its nucleotide sequence is shown in SEQ ID NO.10), and the strain E. coli BL21(DE3) / pRSFDuet-1-SpL with increased enzyme activity compared to the original strain. I134V (SpL I134V The amino acid sequence is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.11.

[0132] The second round used pRSFDuet-1-SpL K193R Using plasmids as templates and I134V-F and I134V-R as primers, after whole-plasmid PCR, transformation, and plate plating, a superior strain of E. coli, BL21(DE3) / pRSFDuet-1-SpL, with improved enzyme activity and thermostability compared to the original strain, was obtained. K193R-I134V (SpL K193R-I134V The amino acid sequence is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.12.

[0133] Example 3

[0134] Culture and expression of parents and mutants:

[0135] Add 5 mL of LB liquid culture medium to a 15 mL test tube. After autoclaving, first add kanamycin to a final concentration of 50 μL / mL, then add E. coli BL21(DE3)-pRSFDuet-1-SpL and E. coli BL21(DE3) / pRSFDuet-1-SpL. K193R-I134V Transfer 5 μL of glycerol to a tube and add it to the liquid culture medium. Place the tube in a constant temperature shaker at 37°C and 180 rpm and incubate overnight.

[0136] Dilute the cultured bacterial suspension to an appropriate ratio and spread it onto LB solid medium with a final kanamycin concentration of 50 μL / mL. Place the plates in a constant temperature incubator and incubate at 37℃ for 16 hours. Seal the plates with sealing film and store them upside down in a refrigerator at 4℃ until use.

[0137] Pick a single colony from the solid culture medium and transfer it to LB liquid medium containing kanamycin (final concentration 50 μL / mL). Place the medium in a constant temperature shaker at 37°C and 200 rpm for 12 h.

[0138] The seed culture was transferred to the fermentation broth (K₂HPO₄ 9.4 g / L, KH₂PO₄ 2.2 g / L, yeast extract 24 g / L, tryptone 12 g / L) and cultured in a constant temperature shaker (37℃, 200 rpm). After 1.5 h of fermentation, IPTG at a final concentration of 0.25 mM was added to induce recombinant lipase expression, and fermentation was carried out at 22℃, 180 rpm for 15 h to produce enzyme.

[0139] Example 4

[0140] The cell lysis and protein extraction methods are as follows:

[0141] The cultured fermentation broth was centrifuged at 12,000 rpm for 10 min, and then washed twice with physiological saline. Based on the relationship between OD value and cell concentration, the cells were resuspended in phosphate buffer (50 mM pH 8) to achieve a cell concentration of 10 g / L. Ultrasonic disruption was performed at 450 W for 3 s, with a 4 s interval, for a total disruption time of 10 min. After one disruption in an ice bath, the cells were centrifuged at 4°C, 12,000 rpm for 30 min to obtain the lysed supernatant.

[0142] Example 5

[0143] Comparison of the activities of different mutants and parental enzymes

[0144] The enzyme activity of the lysate supernatant was determined according to the method described above, and the protein concentration was determined using the Bradford method. (See attached image.) Figure 1 As shown, the substrate was 7-ACA. The specific activity of the parental SpL was 516.32 U / g, while the specific activities of the mutants K193R, I134V, and K193R / I134V were 527.17 U / g, 739.88 U / g, and 857.69 U / g, respectively, which were 1.02, 1.43, and 1.66 times higher than those of the parental strain.

[0145] Example 6

[0146] Comparison of thermostability of different mutants with parental enzymes

[0147] The thermostability comparison between different mutants and the parent enzyme was mainly determined by measuring the half-life at different temperatures. (See attached image) Figure 2a As shown, the substrate was 7-ACA. At 45°C, the half-life of the parental mutant was 1.5 h, while the half-lives of the mutants K193R, I134V, and K193R / I134V were 0.9 h, 2.9 h, and 3.5 h, respectively, which were 0.6 times, 1.9 times, and 2.3 times that of the parental mutant. (See attached image.) Figure 2b As shown, the half-life of the parental strain at 50℃ is 1.0 h, while the half-lives of the mutants K193R, I134V, and K193R / I134V are 1.5 h, 2.2 h, and 3.3 h, respectively, which are 1.5 times, 2.2 times, and 3.3 times longer than those of the parental strain. (See attached image.) Figure 2c As shown, the half-life of the parental strain at 55℃ was 0.5 h, while the half-lives of the mutants K193R, I134V, and K193R / I134V were 0.8 h, 1.2 h, and 1.9 h, respectively, which were 1.6 times, 2.3 times, and 3.8 times higher than those of the parental strain (see results below). Figure 2a , 2b (2c). As can be seen, the two-site mutant K193R / I134 exhibits the best thermostability at both low and high temperatures, indicating that the combined mutation can further improve the thermostability of the enzyme.

[0148] Example 7

[0149] The synthesis and preparation method of nickel-doped mesoporous silica is as follows:

[0150] Weigh 2g of template agent P123 and add it to 75ml of 1.6mM hydrochloric acid solution. Stir until the template agent is completely dissolved to obtain mixture 1. Add 3g of mesitylene, 23mg of ammonium fluoride, and nickel chloride to mixture 1 and stir at 37℃ for 4h to obtain mixture 2. Add 4.4g of tetraethyl orthosilicate to mixture 2 and stir at 40℃ for 24h to obtain mixture 3. Place mixture 3 in a crystallization kettle and crystallize at 100℃ for 24h to obtain a mixture. Filter and dry the mixture to obtain a white solid. Place the white solid in a muffle furnace and calcine at 550℃ for 5h to obtain 1g of NiCl2-SBA-15.

[0151] The synthesis and preparation method of functionalized nickel-doped mesoporous silica is as follows:

[0152] Weigh 2g of template agent P123 and add it to 75ml of 1.6mM hydrochloric acid solution. Stir until the template agent is completely dissolved to obtain mixture 1. Add 3g of mesitylene, 23mg of ammonium fluoride, and nickel chloride to mixture 1 and stir at 37℃ for 4h to obtain mixture 2. Add 4.4g of tetraethyl orthosilicate and 0.13g of triethoxymethylsilane to mixture 2 and stir at 40℃ for 24h to obtain mixture 3. Place mixture 3 in a crystallization vessel and crystallize at 100℃ for 24h to obtain mixture 4. Filter and dry mixture 4, then add 150mL of anhydrous ethanol and extract by stirring at 70℃ for 6h to obtain mixture 5. Filter, wash, and dry mixture 5 to obtain a white solid. Place the white solid in a muffle furnace and calcine at 300℃ for 12h to obtain 1g of methyl-functionalized NiCl2-SBA-15.

[0153] Example 8

[0154] The immobilization method of lipase SpL is as follows:

[0155] 3 mg of crude enzyme (lipase SpL) and 15 mg of NiCl2-SBA-15 or (CH3)-NiCl2-SBA-15 carrier were adsorbed and immobilized in 1 ml of 50 mM PB (pH 8) at 30 °C and 1000 rpm for 10 min. The immobilized enzyme was obtained by centrifugation and washed twice. The immobilized enzymes were named NiCl2-SBA-15@SpL and (CH3)-NiCl2-SBA-15@SpL.

[0156] Example 9

[0157] Performance comparison of supports modified with different functional groups and original supports

[0158] Following the preparation method of Example 7, the functionalized precursors (methyl, propyl) and their amounts were varied (1%, 3%, 5%, 7%, 9%). 15 mg of different functionalized carriers were immobilized with 3 mg of free enzyme (immobilization conditions: adsorption and immobilization at 30℃ and 1000 rpm for 10 min). Using 7-ACA as a substrate, the performance of the above carriers was examined by liquid chromatography. The results are as follows: Figure 3 As shown, compared with unmodified NiCl2-SBA-15, (CH3)-NiCl2-SBA-15 showed nearly double the specific enzyme activity and 14% higher protein loading rate. It was also found that the content of hydrophobic groups had a significant impact on the reaction of the immobilized enzyme, while different groups had little effect on the reaction.

[0159] Example 10

[0160] 3 mg of crude enzyme with a single His tag and 3 mg of crude enzyme with a double His tag were respectively added to 1 ml of 50 mM PB (pH 8) carrier and immobilized at 30℃ and 1000 rpm for 10 min. The immobilized enzyme was obtained by centrifugation and washed twice. The supernatant and wash buffer were combined, and the protein content in the supernatant was measured to calculate the immobilized protein loading rate. Simultaneously, the activity of the immobilized enzyme containing 0.1 mg of protein was measured. The results are as follows: Figure 4 As shown, enzymes with dual His tags have higher protein loading rates and specific enzyme activities compared to enzymes with single His tags.

[0161] Example 11

[0162] Determination of the thermal stability of lipases / immobilized enzymes

[0163] Take 0.1 mg of free enzyme and 0.1 mg of immobilized enzyme containing protein, and incubate them in 50 mM PB (pH 8) buffer at 45°C, 50°C, and 55°C for 3 h, 6 h, 12 h, and 24 h, respectively. The activity before incubation is taken as 100%, and the residual enzyme activity after incubation is calculated by comparing it to this value, in order to examine the temperature stability of the free and immobilized enzymes. Figure 5 As shown, the residual enzyme activity of free lipase SpL was 12% after incubation at 50℃ for 24 hours, while the residual enzyme activity of immobilized enzyme was 57% under the same conditions. The enzyme activity was significantly improved after immobilization.

[0164] Example 12

[0165] pH stability assay of lipase / immobilized enzyme

[0166] 0.1 mg of free enzyme and 0.1 mg of immobilized enzyme containing protein were placed in a buffer system consisting of 50 mM PB buffer (pH 6, 6.5, 7, 7.5, 8), 50 mM Tris-HCl buffer (pH 8, 9), and 50 mM Gly-NaOH buffer (pH 9, 10, 11). After incubation at 50 °C for 1 h, the residual enzyme activity was calculated by comparing the activity before incubation with the activity after incubation, taking the activity before incubation as 100%, to examine the pH stability of the free and immobilized enzymes. Results are shown below. Figure 6 Under neutral and alkaline conditions, the immobilized enzyme exhibits significantly higher stability than the free enzyme. At PB (pH 8), the immobilized enzyme retains 95% of its residual activity, showing almost no inactivation at the optimal reaction pH. At Tris-HCl (pH 9), the immobilized enzyme also retains 83% of its residual activity, while the free enzyme retains only 43%. At Gly-NaOH (pH 10), the free enzyme retains only 19% of its residual activity, while the immobilized enzyme still retains 42%.

[0167] Example 13

[0168] Reusability assay of immobilized enzymes

[0169] (CH3)-NiCl2-SBA-15-SpL immobilized enzyme containing 8 mg of protein was added to 0.5 mL of 100 mM PB (pH 8) buffer and incubated at 25 °C for 3 min. Then, 0.5 mL of a solution containing 5.44 mg of substrate (20 μmol) was added, and the reaction was carried out for 90 min. The supernatant was collected by centrifugation, and the immobilized enzyme was washed twice. The immobilized enzyme was then resuspended in 0.5 mL of buffer and incubated at 25 °C. A fresh substrate solution was then added, and the reaction was carried out. The conversion rate was then measured. Results are as follows: Figure 7 As shown, the (CH3)-NiCl2-SBA-15 immobilized enzyme SpL can still achieve a substrate conversion rate of over 99% after 13 cycles, and the substrate conversion rate can still reach over 95% after 15 cycles, which shows the strong operational stability of the immobilized enzyme.

[0170] Example 14

[0171] 95.3 mg of 7-ACA was dissolved in 2 mL of phosphate buffer (pH = 8, 200 mM), and the pH was adjusted to 8.0 with ammonia. The prepared immobilized enzyme containing 40 mg of protein was suspended in 3 mL of buffer solution, and substrate solution was added to bring the final substrate concentration to 70 mM. The protein content of the immobilized enzyme was 8 mg / mL. The reaction temperature was 25℃, and 12 batches were reacted. As the reaction progressed, enzyme activity decreased to some extent, requiring an extension of the reaction time. Batches 1-6 were reacted for 1.5 h each, and batches 6-12 were reacted for 2 h each. After the reaction, the immobilized enzyme was washed twice. The supernatants from each batch were combined, and the pH was adjusted to approximately 4 with 6 M HCl at low temperature for crystal growth. After filtration and vacuum drying, the crystals were collected to obtain 0.775 g of a yellow powder solid, with a yield of 80.15%. High-performance liquid chromatography analysis showed a purity greater than 99% (see Appendix). Figure 8 ).

[0172] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any 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 invention should be within the protection scope of the present invention.

Claims

1. A lipase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

6.

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

3. A recombinant expression vector, characterized in that, It contains the nucleic acid as described in claim 2.

4. A recombinant expression transformant, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. An immobilized enzyme, characterized in that, Includes an immobilized enzyme carrier and a lipase mutant immobilized on the immobilized enzyme carrier; The immobilized enzyme carrier is a functionalized nickel-doped mesoporous silica carrier, which refers to the surface of nickel-doped mesoporous silica being functionalized to give it a certain degree of surface hydrophobicity. The amino acid sequence of the lipase mutant is shown in SEQ ID NO.

6.

6. An immobilized enzyme according to claim 5, characterized in that, The method for preparing the functionalized nickel-doped mesoporous silica support is as follows: Step 1: Add template agent P123 to HCl solvent, then add mesitylene, ammonium fluoride and nickel source, stir evenly, then add tetraethyl orthosilicate and the precursor of surface functional groups, and continue stirring the above mixture in a hot water bath to react; Step 2: The reaction product and mother liquor are transferred to a hydrothermal crystallization kettle for crystallization. After cooling, the mixture is filtered, dried, template agent removed, filtered again, dried, and calcined to obtain the functionalized nickel-doped mesoporous silica support.

7. An immobilized enzyme according to claim 6, characterized in that, In the reaction system, the functionalized group precursor is triethoxymethylsilane or triethoxypropylsilane; The mass ratio of triethoxymethylsilane to tetraethyl orthosilicate is 1% to 9%; In step two, the calcination temperature is 250 ℃~550 ℃, and the calcination time is 6 h~15 h; In step one, the reaction time is 20 h to 24 h.

8. The method for preparing an immobilized enzyme according to claim 6, characterized in that, The system containing the lipase mutant and the immobilized enzyme carrier were mixed and then immobilized by adsorption. The immobilized enzyme was obtained by centrifugation. In the immobilization system, the concentration of the lipase mutant and the immobilized enzyme carrier is 2-4 mg / ml and the concentration of the immobilized carrier is 5-30 mg / ml. In the immobilization system, the pH is 6-9 and the reaction temperature is 20-40°C. o C. The time is 5~30 minutes.

9. The application of the lipase mutant according to claim 1 and the immobilized enzyme according to claim 5, characterized in that, The lipase mutant of claim 1 and the immobilized enzyme of claim 5 are used to catalyze the hydrolysis of 7-ACA to generate D-7-ACA.

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