Lipase mutant, immobilized enzyme and application thereof

Abstract

The invention discloses a lipase mutant, an immobilized enzyme and application thereof, and belongs to the field of bioengineering. The specific activity of the SpL enzyme mutant K193R / I134V to 7-ACA is 1.6 times that before mutation, and the half-life period of the SpL enzyme mutant K193R / I134V at 45 DEG C, 50 DEG C and 55 DEG C is prolonged by 1.9 times, 2.6 times and 3.8 times. SpL enzyme with a his label is immobilized on the hydrophobically modified nickel-doped SBA-15 mesoporous molecular sieve, the activity of the enzyme is retained, the thermal stability and reutilization performance of the enzyme are improved, the immobilized enzyme is used for catalyzing to generate D-7-ACA, the D-7-ACA can be repeatedly used for at least 10 batches, and the conversion rate is kept to be 99% or above.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to a lipase mutant, an immobilized enzyme and applications thereof. Background Art

[0002] Lipase (EC 3.1.1.3) is one of the earliest enzymes discovered and studied, also known as triacylglycerol acyl hydrolase, 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 by aminolysis of esters or acids. SpL can not only be used for aminolysis of esters or acids to prepare amides with a wide substrate range, high activity and / or high enantiomeric selectivity (Zeng Shichao, Liu Ji, Anankanbil 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 can also be used for the hydrolysis of acetyl-containing compounds.

[0003] D-7-ACA is a new intermediate of cephalosporin antibiotics, with high reaction activity, which is conducive to the synthesis of new cephalosporin products. When synthesizing some cephalosporin varieties, it simplifies the production process route 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: using 7-ACA as a raw material, hydrolyzing it at -30℃ to -40℃ under high concentration strong alkaline conditions to obtain D-7-ACA solution, and then synthesizing 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 has harsh reaction conditions and low conversion rate for preparing cephalosporin products. The deacetylation reaction using enzymes has simple reaction conditions, high conversion rate and high purity. However, free enzymes have high cost, poor stability and are sensitive to factors such as temperature and pH of the reaction environment, which limits their application in industry.

[0004] Rational design in protein engineering is based on a certain understanding of the sequence, structure, catalytic mechanism and other enzymatic information of the target protein, and with the help of various bioinformatics software, predicts the effects of mutations at different sites on the stability and catalytic performance of the target protein. The whole plasmid PCR method is one of the most widely used site-directed mutagenesis methods, with the advantages of simple operation, rapidity and high efficiency.

[0005] The immobilization of enzymes refers to enzymes that catalyze within a certain spatial range and can be used repeatedly and continuously. Usually, enzyme catalytic reactions are carried out in aqueous solutions, and immobilized enzymes are water-soluble enzymes treated by physical or chemical methods to make them insoluble in water, but still have enzyme activity. The development of this technology not only solves the problem of product separation and purification, but also greatly improves the reuse rate of enzymes, and the industrial application of enzymes has also been realized. Similarly, due to the structural rigidity of the immobilized enzyme, during the process of repeated use, it still has an enzyme activity equivalent to the initial enzyme activity, and can still catalyze reactions in a relatively harsh environment. With the increase in the application demand of enzymes, the immobilization technology for enzymes also needs to be updated in time. Among them, the research on enzyme immobilization methods and carriers has always been a hot topic of research. At present, in order to solve the problems of free enzymes, many methods have been proposed: Patent CN103045580A provides a method for immobilizing wheat malt lipase, which uses epichlorohydrin-modified silica gel as a carrier, and the resulting immobilized enzyme improves stability and the number of reuses. Patent CN105154428A discloses a method for preparing ordered three-dimensional mesoporous carbon immobilized lysozyme and its application, and the obtained immobilized enzyme has good stability and can be reused. Although the carrier used in the above method can solve the problem of free enzyme to a certain extent, the preparation is cumbersome and easily leads to enzyme activity loss. Summary of the invention

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

[0007] Specifically, the present invention first provides a SpL lipase mutant, and with the guidance of surface amino acid (Gly-Ala, Lys-Arg) replacement and consensus theory, with the assistance of bioinformatics software GetArea and Consensus Finder, a nitrilase mutant with high thermal stability and high activity is obtained.

[0008] The present invention further provides a functionalized nickel-doped mesoporous silica carrier, and uses the functionalized nickel-doped mesoporous silica carrier to prepare immobilized enzymes, thereby solving the problems of poor adsorption capacity of the immobilized carrier for free lipase and inability to significantly improve the thermal stability and operational stability of the enzyme. The functionalized nickel-doped mesoporous silica carrier provided in the present application has a simple synthesis method and has a strong adsorption effect on proteins with histidine tags.

[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 selected from one of the following:

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

[0012] (2) mutating the isoleucine I at position 134 in 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, a His tag is expressed at the N-terminus of the lipase mutant.

[0015] In one embodiment of the present invention, the N- and C-termini of the lipase mutant express His tags.

[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 coding gene of the lipase SpL is shown in SEQ ID NO.7;

[0017] After the His tag is expressed at the N-terminus of the parent 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 His tags were expressed at the N and C termini of the parent lipase SpL, the amino acid sequence was shown in SEQ ID NO.3, and the corresponding nucleotide sequence was shown in SEQ ID NO.9;

[0019] When the lipase mutant is selected from (1), it is represented by 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 by 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 by 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] The present invention firstly uses GetArea software to find that there are 6 Gly and 11 Lys on the surface of SpL based on the principle that Gly is replaced by Ala and Lys is replaced by Arg on the surface of thermophilic proteins, and performs site-directed mutagenesis on them respectively to obtain a mutant enzyme with improved thermal stability; then, based on the principle that conservative amino acids in homologous sequences are more stable than non-conservative amino acids, multiple sequence alignment of SpL is performed using Consensus Finder software, and a total of 6 amino acids with a threshold value greater than 70% are screened out, and after site-directed mutagenesis of them into corresponding conservative amino acids, a mutant enzyme with improved 7-ACA hydrolase activity is obtained.

[0023] After mutating the above two amino acids in combination, a two-site mutant with significantly improved thermal stability and specific activity was obtained, which improved the existing thermal stability of SpL.

[0024] The specific activity and thermal stability of the lipase mutant of the present invention are significantly improved compared with the parent lipase SpL. The lipase SpL derived from the parent Sphingomonas sp. HXN-200 is subjected to site-directed mutagenesis, and mutations are introduced into the lysine K at position 193 and the isoleucine I at position 134 of SpL by whole plasmid PCR technology. More specifically, the mutation is to mutate the lysine K at position 193 to arginine R, and the isoleucine I at position 134 to valine V. The nitrilase mutant K193R / I134V is represented by the naming method of "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 which K193R / I134V shows the best specific activity, which is 1.66 times higher than that of the parent.

[0026] Compared with the lipase SpL of parent Sphingomonas HXN-200 (half-life t 1 / 2The half-lives of the lipase SpL mutants K193R, I134V and K193R / I134V at 45°C were 1.58 times, 1.03 times and 1.87 times that of the parent (t1 / 2 were 2.36 h, 1.54 h and 2.79 h, respectively), and the half-lives at 50°C were 1.68 times, 1.19 times and 2.64 times higher than those of the parent (t1 / 2 were 1.36 h, 0.96 h and 2.14 h, respectively), and the half-lives at 55°C were 2.39 times, 1.31 times and 3.80 times higher than those of the parent (t 1 / 2 1.17h, 0.64h and 1.86h respectively).

[0027] The second aspect of the present invention also provides an isolated nucleic acid, wherein the nucleic acid is a nucleic acid molecule encoding the lipase mutant.

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

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

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

[0031] The fourth aspect of 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 recombinant expression vector into a host cell.

[0033] The host cell is a conventional host cell in the art, as long as the recombinant expression vector can stably replicate itself and the gene of the lipase mutant carried by it can be effectively expressed. The host cell is preferably Escherichia coli, and more preferably: Escherichia coli E.coli BL21 (DE3). The recombinant expression vector is transformed into Escherichia coli E.coli BL21 (DE3) to obtain the preferred recombinant expression transformant of the present invention.

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

[0035] The method for preparing the recombinant lipase mutant of the present invention is preferably: culturing the recombinant expression transformant as described above, and isolating and obtaining the lipase mutant. The culture medium used for culturing the recombinant expression transformant is any culture medium in the art that can grow the transformant and produce the recombinant lipase of the present invention. There are no special restrictions on the culture method and culture conditions, and they can be appropriately selected according to the conventional knowledge in the art according to factors such as the host cell type and the culture method, as long as the transformant can grow and produce the lipase mutant.

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

[0037] Using plasmid pRSFDuet-1 as a template, primers are designed, and a recombinant plasmid containing a lipase mutant is obtained by PCR, and a gene and plasmid encoding the lipase mutant are obtained, and the lipase mutant is expressed using Escherichia coli as a host. Preferably, the Escherichia coli seed liquid containing the recombinant plasmid is inoculated into a fermentation medium, and the fermentation time is 12 hours at 37°C and 200rpm; the fermentation medium is composed of: K2HPO4 9.4g / L, KH2PO4 2.2g / L, yeast extract powder 24g / L, tryptone 12g / L, and natural pH (pH about 7.1).

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

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

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

[0041] (3) disrupting the resting cells described in form (1) to obtain a cell disrupted liquid containing the lipase mutant;

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

[0043] After the cell culture is completed, the precipitated bacterial cells are collected by centrifugation, which are the resting cells of the recombinant expression transformant; the obtained cells are suspended in potassium phosphate buffer, ultrasonically disrupted, the disrupted liquid is centrifuged, and the supernatant is collected to obtain the cell disrupted liquid of the recombinant lipase. The resting cells and the cell disrupted liquid are freeze-dried to obtain freeze-dried cells and freeze-dried enzyme powder.

[0044] In the seventh aspect of the present invention, an immobilized enzyme is provided, 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, and the functionalized nickel-doped mesoporous silica carrier refers to a nickel-doped mesoporous silica surface that is functionalized to have a certain surface hydrophobicity.

[0045] The present invention provides a functionalized nickel-doped mesoporous silica carrier, and uses the functionalized nickel-doped mesoporous silica carrier to prepare immobilized enzymes, thereby solving the problems of poor adsorption capacity of the immobilized enzyme carrier for free lipase and failure to significantly improve the thermal stability and operational stability of the enzyme. The functionalized nickel-doped mesoporous silica carrier provided by the present application has a simple synthesis method, and has a strong adsorption effect on proteins with histidine tags, in particular.

[0046] In one embodiment of the present invention, the preparation method of the functionalized nickel-doped mesoporous silica carrier is:

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

[0048] Step 2: Transfer the reaction product and the mother liquor to a hydrothermal crystallization reactor for crystallization, filter after cooling, dry, remove the template, filter, dry, and calcine to obtain the functionalized nickel-doped mesoporous silica carrier. In one embodiment of the present invention, in the reaction system, the functionalized group precursor is triethoxymethylsilane or triethoxypropylsilane.

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

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

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

[0052] In one embodiment of the present invention, in step 2, the calcination temperature is 250° C. to 550° C., and the calcination time is 6 h to 15 h.

[0053] In one embodiment of the present invention, in step 2, the calcination temperature is 300° C. and the calcination time is 12 hours.

[0054] In one embodiment of the present invention, in step 1, the HCl solvent is 1.6 M, and in step 1, the reaction time is 20 h to 24 h.

[0055] In an eighth aspect, the present invention further 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 and a system of the immobilized enzyme carrier, immobilizing the mixture by adsorption, and obtaining the immobilized enzyme by centrifugal separation.

[0056] In one embodiment of the present invention, in the immobilization system, in the 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, in the system containing lipase or 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, in the immobilization system, the pH of the system is 6-9.

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

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

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

[0062] The ninth aspect of the present invention further provides the use of the lipase mutant described in the first aspect or the immobilized enzyme described in the seventh aspect, which is used to catalyze the hydrolysis of 7-ACA as a substrate to produce D-7-ACA.

[0063] In one embodiment of the present invention, 7-ACA is used as a substrate, the substrate concentration is 70 mM, the reaction temperature is 25°C, the pH is 8.0, the amount of immobilized enzyme is 8 g / L, and after repeated use for 12 batches, the product is collected and analyzed, and the conversion rate is 99.21%.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] Guided by the substitution of surface amino acids and consensus theory, the SpL lipase gene was subjected to site-directed mutagenesis to obtain the SpL mutant enzyme K193R / I134V with improved thermal stability and specific activity. For 7-ACA, the half-life at 55°C can reach 3.8 times that of the wild-type enzyme (SpL lipase), and the specific activity is 1.66 times that of the wild-type enzyme. The present invention also provides a synthesis of a functionalized nickel-doped mesoporous silica carrier and its application in immobilized enzymes. By hydrophobic modification of the carrier surface, the immobilized enzyme has a better loading effect than the unfunctionalized carrier, and at the same time exhibits a higher activity during the reaction. Compared with the enzyme with a fixed double His tag and the enzyme with a fixed N-terminal His tag, the immobilized enzyme with a double His tag has a higher protein loading rate and specific enzyme activity; and compared with the free enzyme, the immobilized enzyme has significantly improved thermal stability, pH stability, and operational stability, as follows:

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

[0067] The protein loading rate of the immobilized enzyme with double His tags was 7.5% higher than that of the immobilized enzyme with ordinary His tags.

[0068] The free enzyme had only 12% residual enzyme activity after being incubated at 50°C for 24 hours, while the immobilized enzyme still retained 57% after being incubated at 50°C for 24 hours.

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

[0070] The immobilized enzyme retained 95% conversion after being reused 15 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 : Comparison of specific activities of the mutants constructed by the present invention and wild-type SpL.

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

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

[0074] Figure 4: Immobilization efficiency of single His-tag and double His-tag.

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

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

[0077] Figure 7 : Reusability of immobilized enzymes.

[0078] Figure 8 : Liquid phase spectrum of product D-7-ACA. DETAILED DESCRIPTION

[0079] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0080] The culture medium formula involved in the embodiment is as follows:

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

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

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

[0084] Cell lysis and protein extraction:

[0085] The cultured fermentation broth was centrifuged at 12000rpm for 10min, and then washed twice with physiological saline. According to the relationship between OD value and cell concentration, the bacteria were resuspended with phosphate buffer (50mM pH 8) and the cell concentration was 10g / L. Ultrasonic crushing conditions were used with a power of 450W, a working time of 3s, an interval time of 4s, and a crushing time of 10min. After crushing once in a crushed ice bath, the supernatant of cell lysis was obtained after centrifugation in a high-speed centrifuge (4°C, 12000rpm, 30min).

[0086] Protein content determination:

[0087] Take 0.5 mL of cell lysis supernatant and add it to a 5 mL EP tube, then add 3.0 mL of Coomassie Brilliant Blue G-250 reagent, mix immediately on a vortex mixer, let stand for 5 min, transfer the mixture to a 1 mL cuvette, and immediately measure the light absorption value A at 595 nm. 595 , calculate the protein concentration.

[0088] Protein binding rate determination:

[0089] The supernatant and washing liquid after immobilization were combined, the protein content of the combined liquid was determined, and the protein binding rate was calculated. The obtained binding rate was D = (A0-(A1×B)) / A0×100%, wherein A0 was the protein content before immobilization, A1 was the protein content of the combined liquid, and B was the dilution multiple of the supernatant after immobilization.

[0090] Lipase SpL enzyme activity determination:

[0091] The activity test reaction system is (1mL): 100μg crude enzyme is mixed with 0.49ml 50mM PB (pH 8.0) buffer, and then preheated with 50mM PB (pH 8.0) buffer containing 0.02M substrate 7-ACA at 35℃ for 5min, then 0.50mL substrate solution is added to the reaction system to make its final concentration 10mM, and reacted at 35℃, 1000rpm for 30min on a constant temperature mixer, and then 1mL liquid methanol is added for quenching and mixing. After mixing in a vortex instrument, centrifuge at 12000rpm for 2min, and the supernatant is drawn with a sterile syringe for HPLC analysis.

[0092] Determination of the activity of immobilized lipase SpL:

[0093] The activity test reaction system is (1 mL): 0.6 mg of immobilized enzyme is mixed with 0.49 ml of 50 mM PB (pH 8.0) buffer, and then preheated with 50 mM PB (pH 8.0) buffer containing 0.02 M substrate 7-ACA at 35°C for 5 min, and then 0.50 mL of substrate solution is added to the reaction system to make its final concentration 10 mM. After reacting at 35°C, 1000 rpm for 30 min on a constant temperature mixer, 1 mL of liquid methanol is added for quenching and mixing. After mixing in a vortex instrument, centrifuge at 12000 rpm for 2 min, and the supernatant is drawn with a sterile syringe for HPLC analysis.

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

[0095] Enzyme activity definition: Under the conditions of 35°C and pH 8.0, the amount of enzyme required to generate 1 μmol of product (hydroxymethyl-7-aminocephalosporanic acid) per minute is defined as one enzyme activity unit (U).

[0096] Determination of thermal stability: The enzyme solutions of the wild type and mutants were placed in metal water baths at 40°C, 50°C, and 55°C for insulation, and samples were taken at regular intervals to determine the residual enzyme activity. A linear relationship was fitted with the insulation time as the x-axis and the logarithm of the residual enzyme activity as the y-axis. 1 / 2 =ln2 / k d Determine the half-life.

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

[0098] 0.1 mg of free enzyme and immobilized enzyme containing 0.1 mg of protein were incubated 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. The activity before incubation was taken as 100%, and the residual enzyme activity was calculated by comparing it with the enzyme activity after incubation to examine the temperature stability of free enzyme and immobilized enzyme.

[0099] pH stability determination method of lipase SpL / immobilized enzyme:

[0100] 0.1 mg of free enzyme and immobilized enzyme containing 0.1 mg of protein were placed 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 was calculated by taking the activity before incubation as 100% and comparing it with the enzyme activity after incubation to investigate the pH stability of free enzyme and immobilized enzyme.

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

[0102] Take (CH3)-NiCl2-SBA-15 immobilized enzyme containing 8 mg protein, add it to 0.5 ml 100 mM PB (pH 8) buffer, incubate at 25 ° C for 3 min, add 0.5 mL of a solution containing 5.44 mg substrate (20 μmol) and react for 90 min. Then, collect the supernatant by centrifugation, wash the immobilized enzyme twice, continue to take 0.5 mL of buffer, add new substrate solution to react, and 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 integrated peak area of ​​the external standard method, A0 represents the peak area of ​​the spontaneous hydrolysis of the substrate, B represents the dilution multiple of the total reaction system, and C represents the concentration of the substrate 7-ACA. Relational formula y = 0.1947*10 ^(-3) x-0.0252 is the external standard curve of D-7-ACA.

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

[0107] Example 1

[0108] Expression of His tag at the C-terminus based on whole plasmid PCR

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

[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: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 20 s, 30 cycles; extension at 72°C for 5 min; and storage at 4°C.

[0118] Take 10 μL of the product obtained by PCR and verify it by nucleic acid electrophoresis, then use a universal DNA purification and recovery kit to recover the reaction solution. Use Dpn I endonuclease to digest the DNA template, the enzyme reaction temperature is 37 ° C, the reaction time is 4 hours, and then the enzyme product is transformed into Escherichia coli BL21 (DE3) competent cells. Spread LB resistant solid plate (containing 50 μg / mL Kana), culture at 37 ° C for 12-14 hours, pick 1-3 transformants and transfer them to LB liquid culture medium (containing 50 μg / mL Kana) and culture for 6 hours. After the liquid becomes turbid, take the bacterial solution for sequencing.

[0119] Example 2

[0120] Construction of SpL enzyme mutants based on whole plasmid PCR

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

[0122] The primers used for site-directed mutagenesis are (underlined 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: pre-denaturation at 95°C for 10 min; denaturation at 98°C for 10 s, annealing at 58°C for 15 s, extension at 72°C for 75 s, 24 cycles; extension at 72°C for 5 min; and storage at 4°C.

[0130] Take 10 μL of the product obtained by PCR and verify it by nucleic acid electrophoresis, then use a universal DNA purification and recovery kit to recover the reaction solution. Use Dpn I endonuclease to digest the DNA template, the enzyme reaction temperature is 37 ° C, the reaction time is 4 hours, and then the enzyme product is transformed into Escherichia coli BL21 (DE3) competent cells. Spread LB resistant solid plate (containing 50 μg / mL Kana), culture at 37 ° C for 12-14 hours, pick 1-3 transformants and transfer them to LB liquid culture medium (containing 50 μg / mL Kana) and culture for 6 hours. After the liquid becomes turbid, take the bacterial solution for sequencing.

[0131] The strain E. coli BL21(DE3) / pRSFDuet-1-SpL with improved thermal stability compared with the original strain was obtained by screening the dominant strains K193R (SpL K193R The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.10), and the enzyme activity of the strain E. coli BL21 (DE3) / pRSFDuet-1-SpL is improved compared with the original bacteria I134V (SpL I134V The amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.11).

[0132] The second round of pRSFDuet-1-SpL K193R The plasmid was used as a template, and I134V-F and I134V-R were used as primers. After whole plasmid PCR, transformation, and plating, the dominant strain E. coli BL21(DE3) / pRSFDuet-1-SpL was obtained, which had improved enzyme activity and thermal stability compared with the original strain. K193R-I134V (SpL K193R-I134V The amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.12).

[0133] Example 3

[0134] Culture expression of parental and mutants:

[0135] Add 5 mL of LB liquid culture medium to a 15 mL test tube, sterilize with high pressure steam, add kanamycin at a final concentration of 50 μL / mL, and then add the E. coli BL21(DE3)-pRSFDuet-1-SpL and E. coli BL21(DE3) / pRSFDuet-1-SpL K193R-I134V 5 μL of the culture medium was transferred from the glycerol tube and added to the liquid culture medium. The culture medium was placed in a constant temperature shaker with the temperature set to 37°C and the rotation speed set to 180 rpm for overnight culture.

[0136] Dilute the cultured bacterial solution to an appropriate multiple and apply it on LB solid medium with a final concentration of 50 μL / mL of kanamycin. Place the plate in a constant temperature incubator at 37°C for 16 hours, seal it with sealing film, and place the plate upside down in a 4°C refrigerator for storage.

[0137] A single colony was picked from the solid culture medium, transferred to LB liquid culture medium containing kanamycin (final concentration of 50 μL / mL), placed in a constant temperature shaker, the temperature was set to 37°C, the speed was set to 200 rpm, and cultured for 12 h.

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

[0139] Example 4

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

[0141] The cultured fermentation broth was centrifuged at 12000rpm for 10min, and then washed twice with physiological saline. According to the relationship between OD value and cell concentration, the bacteria were resuspended with phosphate buffer (50mM pH 8) and the cell concentration was 10g / L. Ultrasonic crushing conditions were used with a power of 450W, a working time of 3s, an interval time of 4s, and a crushing time of 10min. After crushing once in a crushed ice bath, the supernatant of cell lysis was obtained after centrifugation in a high-speed centrifuge (4°C, 12000rpm, 30min).

[0142] Example 5

[0143] Comparison of the activities of different mutants and the parent enzyme

[0144] The supernatant was used to determine the enzyme activity according to the above method and the protein concentration was determined by Bradford method. Figure 1 As shown, the substrate was 7-ACA, the specific activity of the parent SpL was 516.32 U / g, and 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 parent.

[0145] Example 6

[0146] Comparison of thermal stability between different mutants and the parent enzyme

[0147] The thermal stability of different mutants compared with the parent enzyme is mainly determined by measuring the half-life at different temperatures. Figure 2a As shown in the figure, the substrate is 7-ACA, the half-life of the parent at 45°C is 1.5h, while the half-lives of the mutants K193R, I134V, and K193R / I134V are 0.9h, 2.9h, and 3.5h, which are 0.6 times, 1.9 times, and 2.3 times that of the parent, respectively. Figure 2b As shown in Figure 2, the half-life of the parent at 50°C was 1.0 h, while the half-lives of the mutants K193R, I134V, and K193R / I134V were 1.5 h, 2.2 h, and 3.3 h, respectively, which were 1.5 times, 2.2 times, and 3.3 times higher than those of the parent. Figure 2c As shown, the half-life of the parent at 55°C 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 parent (see the results of the study). Figure 2a , 2b , 2c). It can be seen that the two-site mutant K193R / I134 showed the best thermal stability at both low and high temperatures, indicating that the thermal stability of the enzyme can be further improved after combined mutation.

[0148] Example 7

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

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

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

[0152] Weigh 2g of template P123, add it to 75ml of 1.6mM hydrochloric acid solution and stir until the template is completely dissolved to obtain mixed solution 1; take 3g of mesitylene, 23mg of ammonium fluoride and nickel chloride, add them to mixed solution 1 and stir at 37℃ for 4h to obtain mixed solution 2; take 4.4g of tetraethyl orthosilicate and 0.13g of triethoxymethylsilane and add them to mixed solution 2 and stir at 40℃ for 24h to obtain mixed solution 3; put mixed solution 3 into a crystallization kettle and crystallize at 100℃ for 24h to obtain mixture 4. After filtering and drying mixture 4, add 150mL of anhydrous ethanol, stir and extract at 70℃ for 6h to obtain mixture 5, and filter, wash and dry mixture 5 to obtain a white solid. The white solid is placed in a muffle furnace and calcined at 300℃ for 12h to obtain 1 gram 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 50 mM PB (pH 8) system at 30°C 1000 rpm for 10 min. The immobilized enzyme was obtained by centrifugation and washed twice. The obtained immobilized enzymes were recorded as NiCl2-SBA-15@SpL and (CH3)-NiCl2-SBA-15@SpL.

[0156] Example 9

[0157] Comparison of performance between carriers modified with different functional groups and original carriers

[0158] Referring to the preparation method of Example 7, the functional group precursor (methyl, propyl) and its amount (1%, 3%, 5%, 7%, 9%) were changed, 15 mg of different functionalized carriers and 3 mg of free enzyme were fixed (fixation conditions: adsorption and immobilization at 30°C and 1000 rpm for 10 min), 7-ACA was used as the substrate, and the performance of the above carriers was investigated by liquid phase analysis. The results are as follows: Figure 3 As shown, compared with the unmodified NiCl2-SBA-15, the specific enzyme activity of (CH3)-NiCl2-SBA-15 increased by nearly one-fold, the protein loading rate increased by 14%, and it was found that the content of hydrophobic groups had a greater influence on the reaction of the immobilized enzyme, and different groups had little effect on the reaction.

[0159] Example 10

[0160] 3 mg of crude enzyme with single His tag and 3 mg of crude enzyme with double His tag were taken, and 15 mg of (CH3)-NiCl2-SBA-15 carrier were adsorbed and immobilized in 1 ml of 50 mM PB (pH 8) system at 30 ° C 1000 rpm for 10 min. The immobilized enzyme was obtained by centrifugation and washed twice. The supernatant and washing liquid were combined, the protein content in the supernatant was determined, and the immobilized protein loading rate was calculated. At the same time, the immobilized enzyme containing 0.1 mg of protein was taken and the activity was determined. The results are shown in Figure 4 As shown in the figure, the double-His-tagged enzyme has a higher protein loading rate and specific enzyme activity than the single-His-tagged enzyme.

[0161] Embodiment 11

[0162] Determination of thermal stability of lipase / immobilized enzyme

[0163] Take 0.1 mg of free enzyme and immobilized enzyme containing 0.1 mg of 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. The activity before incubation is 100%, and the residual enzyme activity is calculated by comparing the enzyme activity after incubation with the activity before incubation to examine the temperature stability of free enzyme and immobilized enzyme. Figure 5 As shown in the figure, the residual enzyme activity of free lipase SpL after incubation at 50°C for 24 hours was 12%, while the residual enzyme activity of immobilized enzyme was 57% under the same conditions. The enzyme activity after immobilization was significantly improved.

[0164] Example 12

[0165] pH stability determination of lipase / immobilized enzyme

[0166] 0.1 mg of free enzyme and immobilized enzyme containing 0.1 mg of protein were placed 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 hour, the activity before incubation was 100%, and the residual enzyme activity after incubation was calculated compared with the activity before incubation to investigate the pH stability of free enzyme and immobilized enzyme. The results are shown in Figure 6 Under neutral and alkaline conditions, the immobilized enzyme showed much higher stability than the free enzyme. Under PB (pH 8), the immobilized enzyme retained 95% of the residual enzyme activity and was almost not inactivated under the optimal reaction pH. Under Tris-HCl (pH 9), the immobilized enzyme also retained 83% of the residual enzyme activity, while the free enzyme only had 43% of the residual enzyme activity. Under Gly-NaOH (pH 10), the free enzyme had only 19% of the residual enzyme activity, while the immobilized enzyme still had 42% of the residual enzyme activity.

[0167] Example 13

[0168] Determination of the reusability of immobilized enzymes

[0169] Take (CH3)-NiCl2-SBA-15-SpL immobilized enzyme containing 8mg protein, add it to 0.5ml 100mM PB (pH8) buffer, incubate at 25℃ for 3min, add 0.5mL solution containing 5.44mg substrate (20μmol) and react for 90min, then collect the supernatant by centrifugation, wash the immobilized enzyme twice, take 0.5mL buffer to resuspend the immobilized enzyme, incubate at 25℃, then add new substrate solution to react and detect the conversion rate. The results are as follows Figure 7 As shown, the (CH3)-NiCl2-SBA-15 immobilized enzyme SpL can still achieve a substrate conversion rate of more than 99% after being recycled 13 times, and the substrate conversion rate can still reach more than 95% after being reused 15 times, which shows that the immobilized enzyme has strong operational stability.

[0170] Embodiment 14

[0171] Weigh 95.3 mg of 7-ACA and dissolve it in 2 mL of phosphate buffer (pH=8, 200 mM), and adjust the pH to 8.0 with ammonia water; suspend the prepared immobilized enzyme containing 40 mg of protein in 3 mL of buffer and add it to the substrate solution to make the final concentration of the substrate 70 mM, the protein amount of the immobilized enzyme 8 mg / mL, the reaction temperature is 25 ° C, and 12 batches are reacted. As the reaction proceeds, the enzyme activity is lost to a certain extent, and the reaction time needs to be extended. Among them, the 1-6 batches are reacted for 1.5 h each, and the 6-12 batches are reacted for 2 h each. After the reaction, the immobilized enzyme is washed twice. Combine the supernatant of each batch, adjust the pH to about 4 with 6M HCl at low temperature, and grow crystals. After filtering and vacuum drying, collect the crystals to obtain 0.775 g of yellow powder solid, with a yield of 80.15%. The purity is greater than 99% after high performance liquid chromatography analysis (see attached Figure 8 ).

[0172] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 selected from one of the following: (1) mutating the lysine K at position 193 in the amino acid sequence shown in SEQ ID NO.1 to arginine R; (2) mutating the isoleucine I at position 134 in the amino acid sequence shown in SEQ ID NO.1 to valine V; (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.

2. A lipase mutant according to claim 1, characterized in that: The N-terminus of the lipase mutant expresses a His tag, or, The N- and C-termini of the lipase mutant express His tags.

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

4. A recombinant expression vector, characterized in that: Comprising the nucleic acid as described in claim 3.

5. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector as described in claim 4.

6. An immobilized enzyme, characterized in that It comprises an immobilized enzyme carrier and a lipase or a lipase mutant immobilized on the immobilized enzyme carrier; The immobilized enzyme carrier is a functionalized nickel-doped mesoporous silica carrier, and the functionalized nickel-doped mesoporous silica carrier refers to the surface of nickel-doped mesoporous silica being functionalized to have a certain surface hydrophobicity; The amino acid sequence of the lipase is shown in SEQ ID NO.1, or the N-terminus of the lipase expresses a His tag, or the N- and C-termini of the lipase express a His tag; The lipase mutant is as shown in claim 1 or 2.

7. An immobilized enzyme according to claim 6, characterized in that: The preparation method of the functionalized nickel-doped mesoporous silica carrier is as follows: Step 1: Add template P123 to HCl solvent, then add mesitylene, ammonium fluoride and nickel source, stir evenly, then add tetraethyl orthosilicate and precursor of surface functional group, and continue to stir the mixture in a hot water bath to react; Step 2: Transfer the reaction product and the mother liquor to a hydrothermal crystallization kettle for crystallization, filter after cooling, dry, remove the template, filter, dry, and calcine to obtain the functionalized nickel-doped mesoporous silica carrier.

8. An immobilized enzyme according to claim 7, characterized in that: In the reaction system, the functional group precursor is triethoxymethylsilane or triethoxypropylsilane; The mass ratio of triethoxymethylsilane to silicon source tetraethyl orthosilicate is 1% to 9%; In step 2, the calcination temperature is 250°C to 550°C, and the calcination time is 6h to 15h; In step 1, the reaction time is 20 h to 24 h.

9. The method for preparing an immobilized enzyme according to claim 6, characterized in that: The solution containing lipase or lipase mutant and the immobilized enzyme carrier system are mixed and then adsorbed and immobilized, and the immobilized enzyme is obtained by centrifugal separation; In the immobilization system, the concentration of the lipase or 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 of the system is 6-9, the reaction temperature is 20-40° C., and the reaction time is 5-30 min.

10. The use of the lipase mutant according to claim 1 or 2 and the immobilized enzyme according to claim 6, characterized in that: The lipase mutant according to claim 1 or 2 and the immobilized enzyme according to claim 6 are used to catalyze the hydrolysis of 7-ACA as a substrate to produce D-7-ACA.

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