Method for improving thermal stability and catalytic activity of trypsin through multi-site mutation

Abstract

The invention discloses a method for improving thermal stability and catalytic activity of trypsin through multi-site mutation, and relates to the technical field of genetic engineering. A mutant takes trypsin shown as SEQ ID NO.3 as a parent; the 14th amino acid of the parent is mutated into glutamine, the 15th amino acid of the parent is mutated into proline, the 16th amino acid of the parent is mutated into leucine, the 17th amino acid of the parent is mutated into serine, the 18th amino acid of the parent is mutated into serine, the 19th amino acid of the parent is mutated into leucine, and the 20th amino acid of the parent is mutated into pyrrole lysine; the amino acid at the 21st site is mutated into pyrrole lysine; the thermal stability of the mutant is remarkably improved, and the catalytic activity of the enzyme is also improved, so that the requirements of industrial production are met, and the mutant has a good industrial application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a method for improving the thermal stability and catalytic activity of trypsin by multi-site mutation. Background Art

[0002] Trypsin is a proteolytic enzyme that can cleave peptide bonds formed by the carboxyl groups of lysine or arginine, and is widely used due to its safety and high efficiency. Trypsin is a pancreatic serine protease, and due to its narrow specificity and selectivity, it has been widely used in food technology, proteomic analysis, regulation of soybean protein allergy, production of antihypertensive peptides, and biomarkers for diseases such as pancreatitis and cystic fibrosis. It is widely used in the food industry in meat tenderization, polypeptide processing, beer clarification, and improvement of bread quality. In industrial production, trypsin can be used for the degradation of protein feed; it is also a safe organic pesticide degrader that can effectively reduce the toxicity of pesticides and insecticides such as diazinon, tetramethrin, deltamethrin, and malathion; it is one of the important enzyme preparations in the leather industry. In the medical field, trypsin can dilute blood, sputum, and pus without causing harm to normal tissues, which is very important in medicine. Applying trypsin at the wound can help the wound recover quickly because trypsin can decompose denatured proteins but has no effect on normal tissues that have not been denatured. Commonly used trypsin is used for debriding and removing stasis from purulent and ulcerated wounds to promote wound healing.

[0003] Enzymes are natural catalysts with catalytic activity and substrate specificity, and have broad application prospects. However, since many enzymes are isolated from mesophilic bacteria, through long-term natural selection, organisms tend to adjust their specific proteins to function effectively at their normal environmental temperature. They have well met the biological requirements for their catalytic performance, which usually means that the protein has a limited temperature range within which it maintains structural integrity. Outside this range, the protein denatures, resulting in the loss of corresponding functions such as enzyme activity. The thermal stability and activity of industrial microbial enzymes are key factors for industrial utilization. Cellulase, lipase, amylase, and protease are collectively called the four major industrial enzymes. Among them, trypsin is widely used in industrial production due to its special polypeptide hydrolysis performance, and its consumption accounts for about 3% of the industrial enzyme preparation market. However, natural trypsin usually cannot withstand the harsh industrial conditions in production. During industrial application, due to the low thermal stability of natural trypsin, it is prone to denaturation and inactivation.

[0004] Trypsin has become a commonly used tool enzyme in the industrial field due to its high efficiency, specificity, and high hydrolysis rate. It has a wide market application and large demand. Under high-temperature conditions, trypsin cannot maintain high enzyme activity and thermal stability. Therefore, through genetic engineering means, heterologous expression and molecular modification of trypsin in microorganisms have strong application prospects to better meet industrial needs.

[0005] The existing methods for increasing enzyme mutations generally use single-point mutation methods. In the present invention, through fragment mutations at consecutive sites, the catalytic activity and thermal stability of trypsin are increased to obtain a new enzyme mutation sequence. Summary of the Invention

[0006] In order to solve the deficiencies of the existing technology, the purpose of the present invention is to provide a method for improving the thermal stability and catalytic activity of trypsin by multi-site mutation, which can provide a trypsin with high enzyme activity and thermal stability.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] A trypsin mutant, the mutant uses the trypsin shown in SEQ ID NO.3 as the parent, and mutates the 14th amino acid of the parent into glutamine, the 15th amino acid into proline, the 16th amino acid into leucine, the 17th amino acid into serine, the 18th amino acid into serine, the 19th amino acid into leucine, the 20th amino acid into pyrrolysine, and the 21st amino acid into pyrrolysine.

[0009] Preferably, the amino acid sequence of the trypsin mutant is as shown in SEQ ID NO.1.

[0010] The present invention also claims a gene encoding the above-mentioned trypsin mutant.

[0011] The present invention also claims a vector, the vector contains the above-mentioned gene.

[0012] The present invention also claims a host cell, the host cell contains the above-mentioned vector, or the genome contains the above-mentioned gene.

[0013] The present invention also claims a method for improving the catalytic activity of trypsin by multi-site mutation, which mutates the 14th amino acid of trypsin with the amino acid sequence shown in SEQ ID NO.3 into glutamine, the 15th amino acid into proline, the 16th amino acid into leucine, the 17th amino acid into serine, the 18th amino acid into serine, the 19th amino acid into leucine, the 20th amino acid into pyrrolysine, and the 21st amino acid into pyrrolysine.

[0014] The present invention also claims a method for improving the thermal stability of trypsin by multi-site mutation, in which the 14th amino acid of trypsin with the amino acid sequence shown in SEQ ID NO.3 is mutated to glutamine, the 15th amino acid is mutated to proline, the 16th amino acid is mutated to leucine, the 17th amino acid is mutated to serine, the 18th amino acid is mutated to serine, the 19th amino acid is mutated to leucine, the 20th amino acid is mutated to pyrrolysine, and the 21st amino acid is mutated to pyrrolysine.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides a trypsin mutant with improved enzyme activity and thermal stability and its application. By using site-directed mutagenesis biotechnology to modify the molecular structure of trypsin and performing mutation verification, a mutant strain with improved enzyme activity and thermal stability is obtained. While the thermal stability of the mutant is significantly improved, the catalytic activity of the enzyme is also enhanced, meeting the requirements of industrial production and having good industrial application prospects. Specific embodiments

[0017] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following further elaborates on the present invention in combination with embodiments. Of course, the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0018] Unless otherwise specified, the chemical reagents and materials in the present invention are purchased through market channels or synthesized from raw materials purchased through market channels.

[0019] The following further elaborates on a method for improving the thermal stability of trypsin by combinatorial mutation provided by the present invention. For the experimental methods without specific conditions indicated in the following embodiments, they are carried out according to conventional methods and conditions, or selected according to the product instructions. The room temperature mentioned herein is the conventional room temperature in the art, and the room temperature range is 20-40°C.

[0020] 1. Materials and instruments

[0021] (1) Test materials

[0022] E.coli BL21 and E.coli DH5α are cultured in Luria Bertani medium (10.0 g NaCl, 10.0 g tryptone, 5.0 g yeast extract). pET-28a(+) is used to construct a plasmid expressing the target gene. Escherichia coli DH5α is used as the host for plasmid cloning, and Escherichia coli BL21 is used as the expression host strain. Enterokinase and Mut The II Fast Mutation Kit V2 was provided by Yuanye Bio-Technology Co., Ltd. and Novoprotein Scientific Inc. respectively. The substrate benzoyl-L-arginine ethyl ester hydrochloride (BAEE) and other reagents and chemicals were all purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).

[0023] (2) Test Instruments

[0024] Table 1 Instrument and Equipment

[0025] Instruments and Equipment Model Manufacturer Laminar Flow Hood ZHJH-1214B Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. High-Speed Refrigerated Centrifuge Sigma3-16PK Sigma Corporation Gel Imaging System Tamon-1600 Haitianneng Technology Co., Ltd. Snowflake Ice Maker XB70 GRANT Thermostatic Orbital Shaker ZHWY-2112B Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Electronic Balance PL6001-S Mettler-Toledo Instruments Co., Ltd. Mini Horizontal Electrophoresis Cell Sub-Cel GT BIO-RAD, USA Micro High-Speed Centrifuge TG16-W Mettler-Toledo Instruments Co., Ltd. UV Spectrophotometer UV-1800 Shimadzu Instruments PCR Instrument sC005675 BIO-RAD, USA Electrothermal Thermostatic Incubator ZSD-2270 Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Electrothermal Thermostatic Water Bath DK-S22 Shanghai Jinghong Experimental Equipment Co., Ltd. Vortex Mixer DL-Sc05 Beijing Daoguo Changsheng Biotechnology Co., Ltd. Ultrasonic Disruptor SCIENTZ-IID Ningbo Xinzhi Biotechnology Co., Ltd.

[0026] Example 1 Construction of Trypsin Vector

[0027] (1) Construction of Mutant Recombinant Plasmid

[0028] Using the trypsin with the amino acid sequence shown in SEQ ID NO.3 as the parent, the 14th amino acid of the parent was mutated to glutamine, the 15th amino acid was mutated to proline, the 16th amino acid was mutated to leucine, the 17th amino acid was mutated to serine, the 18th amino acid was mutated to serine, the 19th amino acid was mutated to leucine, the 20th amino acid was mutated to pyrrolysine, and the 21st amino acid was mutated to pyrrolysine, to obtain the amino acid sequence SEQ ID NO.1.

[0029] SEQ ID NO.1:

[0030] MFPTDDDDKIVGGQPLSSLOOPYQVSLNSGSHFCGGSLINSQWVVSAAHCYKSRIQVRLGEHNIDVLEGNEQFINAAKIITHPNFNGNTLDNDIMLIKLSSPATLNSRVATVSLPRSCAAAGTECLISGWGNTKSSGSSYPSLLQCLKAPVLSDSSCKSSYPGQITGNMICVGFLEGGKDSCQGDSGGPVVCNGQLQGIVSWGYGCAQKNKPGVYTKVCNYVNWIQQTIAANLEHHHHHH

[0031] SEQ ID NO.3:

[0032] MFPTDDDDKIVGGYTCAANSIPYQVSLNSGSHFCGGSLINSQWVVSAAHCYKSRIQVRLGEHNIDVLEGNEQFINAAKIITHPNFNGNTLDNDIMLIKLSSPATLNSRVATVSLPRSCAAAGTECLISGWGNTKSSGSSYPSLLQCLKAPVLSDSSCKSSYPGQITGNMICVGFLEGGKDSCQGDSGGPVVCNGQLQGIVSWGYGCAQKNKPGVYTKVCNYVNWIQQTIAANLEHHHHHH

[0033] The gene sequence of the parent is shown in SEQ ID NO.4.

[0034] SEQ ID NO.4 (with Histag label, restriction enzyme site):

[0035] GAATTCATGTTCCCAACCGATGACGACGATAAGATCGTGGGCGGCTATACCTGTGCCGCTAATAGCATTCCGTACCAGGTTTCCCTGAACTCCGGTAGCCATTTTTGTGGTGGTTCCCTGATCAACTCTCAGTGGGTTGTGTCTGCGGCACATTGTTACAAAAGCCGTATCCAGGTCCGTCTGGGTGAACACAATATCGACGTGCTGGAAGGCAACGAGCAGTTCATCAATGCCGCTAAAATTATCACCCACCCGAACTTCAACGGCAACACCCTGGACAATGATATCATGCTGATCAAGCTGTCCTCTCCAGCGACTCTGAACTCCCGTGTTGCAACTGTCTCTCTGCCGCGTAGCTGCGCGGCCGCAGGTACTGAGTGCCTGATCTCTGGTTGGGGCAATACCAAGTCTTCTGGTAGCAGCTATCCGTCCCTGCTGCAGTGTCTGAAAGCACCAGTTCTGTCTGATTCCTCTTGTAAGAGCTCTTACCCTGGCCAGATTACCGGTAACATGATCTGCGTGGGCTTCCTGGAAGGCGGTAAAGATTCCTGTCAGGGCGACAGCGGTGGTCCAGTTGTTTGCAACGGCCAGCTGCAGGGTATCGTTAGCTGGGGCTATGGCTGTGCGCAAAAAAACAAACCGGGCGTGTACACCAAAGTTTGCAACTACGTTAACTGGATCCAACAGACCATTGCGGCGAACCTGGAACACCACCACCACCATCAC TAAC CTCGAG

[0036] (2) Construction of recombinant engineering bacteria

[0037] The amino acid sequence of the designed trypsin, SEQ ID NO.1, was optimized according to the codon preference of E. coli to synthesize the coding sequence of porcine trypsin, SEQ ID NO.2, with restriction enzyme sites EcoRI and XhoI. Plasmid pET-28a(+) and the synthesized porcine trypsin sequence were digested with EcoRI and XhoI respectively. After recovering the digested pET-28a(+) and porcine trypsin sequence, they were ligated with a ligase to obtain the ligation product. The constructed ligation product was transformed into E. coli DH5α, and after verification, it was transformed into E. coli BL21 to obtain the E. coli BL21(DE3) strain, and then trypsin was expressed.

[0038] SEQ ID NO.2 (with Histag tag, the underlined part is the restriction enzyme site):

[0039] GAATTCATGTTCCCAACCGATGACGACGATAAAATCGTAGGTGGCCAACCGCTGTCTAGCCTGCCTTACCAGGTCTCCCTGAACTCTGGTTCCCACTTCTGTGGCGGCTCTCTGATTAACTCTCAGTGGGTAGTGTCTGCTGCCCACTGCTACAAATCTCGTATCCAGGTTCGCCTGGGTGAGCACAACATCGACGTGCTGGAAGGCAACGAACAGTTCATCAACGCTGCTAAGATTATCACTCACCCGAACTTCAACGGTAACACTCTGGATAACGACATCATGCTGATCAAGCTGTCCTCCCCGGCCACCCTGAACTCTCGCGTTGCAACTGTTAGCCTGCCGCGCTCTTGTGCTGCGGCAGGTACTGAATGTCTGATTTCTGGCTGGGGTAACACCAAAAGCTCTGGCAGCTCCTATCCGTCCCTGCTGCAATGCCTGAAAGCCCCGGTTCTGTCTGACAGCTCTTGCAAATCTTCTTACCCGGGTCAAATCACCGGCAACATGATCTGTGTAGGCTTCCTGGAAGGCGGTAAAGACAGCTGTCAGGGTGACTCTGGTGGTCCAGTAGTATGCAACGGTCAGCTGCAGGGTATTGTATCTTGGGGCTATGGTTGCGCACAAAAAAACAAACCGGGTGTTTACACTAAAGTGTGCAACTACGTTAACTGGATTCAGCAGACTATCGCCGCGAATCTGGAACACCACCACCACCATCACTAAC CTCGAG

[0040] The ligation product was transformed into E. coli DH5α competent cells. The specific transformation steps are as follows:

[0041] (1) First, adjust the temperature of the constant temperature water bath to 42 °C.

[0042] (2) Take out a tube (100 μL) of competent bacteria from the ultra-low temperature freezer at -80 °C, then insert it into ice and perform an ice bath for 10 minutes.

[0043] (3) Add 10 μL of the ligated plasmid pET28α, gently shake it, and then place it on ice for 20 min.

[0044] (4) Gently shake it well and then insert it into a 42°C water bath for 90 s of heat shock, and then quickly put it back on ice and let it stand for 5 min.

[0045] (5) Add 700 μL of LB medium without antibiotics to each of the above tubes and mix gently, and then shake it on a shaker at 37°C for 50 min.

[0046] (6) Drop 300 μL of the above transformation mixture taken out from the ultra-clean workbench onto solid LB agar plates containing appropriate antibiotics respectively, and use a glass spreader that has been burned by an alcohol lamp and cooled to spread it evenly.

[0047] Label the coated petri dishes, first place them in a 37°C constant temperature incubator for 30 - 60 min. After the liquid on the surface has penetrated into the medium, turn them over and place them in a 37°C constant temperature incubator overnight. Identify the recombinant plasmids from the grown single colonies: Pick the single colonies grown on the above LB solid medium, inoculate them into 5.0 mL of LB medium containing kanamycin, culture them overnight at 37°C and 200 r / min. Take part of the bacterial liquid to preserve the bacterial strain with a glycerol tube, and take appropriate amounts of the bacterial liquid respectively to extract the recombinant plasmid according to the instructions of the plasmid extraction kit. Digest the extracted recombinant plasmid with EcoRI and XhoI. The digestion system is the same as that of the double digestion of pET-28a. Analyze the target gene fragment and vector fragment of the product after double digestion by 1% agarose gel electrophoresis. Then sequence the recombinant plasmid identified as positive by double digestion.

[0048] Example 2 Expression of Trypsin

[0049] (1) Prepare the seed solution: Use an inoculation loop to pick up the E. coli BL21(DE3) strain stored at -20°C in the ultra-clean workbench, streak it in three zones on an LB plate containing kanamycin, and culture it inverted in a 37°C constant temperature incubator for 12 h. Pick a single colony on the plate and inoculate it into 50.0 mL of LB containing kanamycin.

[0050] (2) Induced culture: Add 1 mL of the seed solution to an LB shake flask medium containing kanamycin, place it on a shaker at 37°C and 220 rpm for shaking culture. When the OD 600 is between 0.6 and 0.8, add IPTG with a final concentration of 0.5 mM to induce the recombinant bacteria to express the protein, lower the temperature to 18°C, and induce protein expression at low temperature for 10 h. Centrifuge at 6500 rpm for 5.0 min to collect the supernatant and precipitate of the induced bacteria, and take a small amount of the sample for SDS-PAGE analysis of trypsin expression.

[0051] (3) At the same time, collect the recombinant bacterial cell precipitate without adding the inducer IPTG in the same way. Take 1.0 mL of the seed liquid in a sterile laminar flow hood and inoculate it into a shake flask containing LB medium for induction culture for the same period of time. Centrifuge at 12000 rpm for 5.0 min, and collect the supernatant and the cell precipitate. Add bacterial lysis buffer and ultrasonically lyse the cells. Centrifuge at 12000 rpm for 5 min to collect the supernatant and the precipitate respectively.

[0052] Example 3 Obtaining Trypsin

[0053] The expressed trypsin was verified to form inclusion bodies, and the following treatments need to be carried out on the inclusion bodies:

[0054] (1) Ultrasonic lysis: Mix the wet bacterial cells and the lysis buffer in a ratio of 1:10. Add the lysis buffer pre-cooled at 4°C to the wet bacterial cells, stir well to make the bacterial cells fully suspended; ultrasonically lyse the bacterial cells under ice bath conditions. The ultrasonic conditions are: power 70 W, working for 15 min, running for 5 s, and stopping for 7 s; perform ultrasonic treatment twice, and centrifuge at 10000 rpm and 4°C for 15 min to collect the precipitate.

[0055] (2) Wash the inclusion bodies: Wash the precipitate with washing buffer I and buffer II respectively. Suspend the inclusion bodies by adding 10.0 mL of the washing solution per 1 g of the precipitate, stir with a glass rod until completely dissolved, and centrifuge at 10000 rpm and 4°C for 15 min to collect the precipitate. The collected precipitate is the wet inclusion body.

[0056] (3) Lyse the inclusion bodies: Mix the wet inclusion bodies and the lysis solution in a ratio of 1:20, stir well to make the inclusion bodies fully dissolved, and it can also be placed in a water bath at 37°C until the inclusion bodies are completely dissolved in the lysis solution. Centrifuge at 10000 rpm and 4°C for 15 min to collect the supernatant. The supernatant is the lysis solution of the inclusion bodies. Take a sample to measure the protein concentration.

[0057] (4) Dilute and refold the lysis solution: Dilute the protein content to about 0.2 mg / mL with the refolding buffer. Obtain the refolding solution by refolding at 4°C for 24 h.

[0058] Example 4 Purification of Trypsin

[0059] Use Ni 2+ affinity chromatography to purify the target protein. Utilize that metal ions can bind to the imidazole ring of histidine. Ni 2+ The specific steps for purifying the protein by affinity chromatography are as follows:

[0060] (1) Column packing: First, pack the Ni-NTA purification resin into the chromatography column. Load 1.0 mL of the resin into a total volume of 15.0 mL, let the resin settle freely, and equilibrate the resin with 10 column volumes of Wash Buffer.

[0061] (2) Sample loading: Filter the trypsin solution obtained after dilution and refolding through a 0.22 μm filter membrane to prevent clogging of the pore size between the resins, resulting in a slow flow rate. Control the flow rate at 0.5 mL / min and collect it with a centrifuge tube at the bottom of the chromatography column. Then pour the obtained solution back into the chromatography column to allow the target protein to fully bind to the resin.

[0062] (3) Washing: Slowly add Wash Buffer to the chromatography column, control the flow rate at 1 mL / min, and wash away the miscellaneous proteins that have not bound to the resin with approximately ten times the volume of Wash Buffer. Use Wash Buffer as the blank until the value at A280 in the eluate from the chromatography column no longer changes and approaches zero.

[0063] (4) Elution: Use Elution Buffer to elute the target protein. The eluate is detected by a spectrophotometer. Stop collecting when the value at A280 in the eluate no longer changes. Perform SDS PAGE electrophoresis on the collected solution, and the electrophoresis result of the collected solution shows a single protein band.

[0064] (5) Cleaning: Use Elution Buffer with a high concentration of imidazole to clean the column, about ten column volumes, and then use 10 column volumes of ddH2O to clean the column for the next use.

[0065] (6) Preservation: Seal the column with 20% ethanol for subsequent use.

[0066] Example 5 Enzyme Activity Assay

[0067] (1) Preparation of substrate solution: Use N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) as the substrate to determine the activity of trypsin. The prepared substrate solution needs to be used within two hours. Keep it at a constant temperature of 25.0 ± 0.5 °C, use water as the blank, and measure the absorbance at a wavelength of 253 nm. If necessary, adjust it with the above substrate stock solution or phosphate buffer to make the absorbance between 0.575 and 0.585.

[0068] (2) Sample treatment: The trypsin sample needs to be dissolved in 0.001 mol / L hydrochloric acid solution and quantitatively diluted to prepare a solution containing 50 - 60 trypsin units per 1 mL.

[0069] (3) Measurement method: Use ultraviolet spectrophotometry for measurement. When N-benzoyl-L-arginine ethyl ester hydrochloride is used as the reaction substrate of trypsin, it can react with trypsin to generate the product H-benzoyl-L-arginine (BA).

[0070] The total reaction system is 3.2 mL. Take 3.0 mL of the substrate solution, add 200 μL of 0.001 M hydrochloric acid solution, mix well, and use it as the blank. Additionally, take 200 μL of the sample solution, add 3.0 mL of the substrate solution (kept at a constant temperature of 25.0 °C), mix well and start timing immediately; read the absorbance at 253 nm every 30 s for a total of 5 min. Use the absorbance and time as the vertical and horizontal coordinates respectively to plot a graph; the change in each absorbance reading should be maintained between 0.015 and 0.018, and the linear relationship time should be no less than 3 min. If the above requirements are not met, the concentration of the test substance solution should be adjusted and measured again. In the above-mentioned absorbance-time relationship graph, take the absorbance of the linear part and calculate according to the following formula. Under the above conditions, an absorbance change of 0.003 per minute is equivalent to 1 trypsin unit.

[0071] Trypsin activity unit df is the dilution factor.

[0072] Example 6 Determination of the thermal stability of trypsin

[0073] After trypsin is activated by enterokinase, it is incubated at 50 °C for 0 min, 30 min, 60 min, 120 min, and 180 min respectively, and then the enzyme activity is measured after ice bath for 60 s. The activity before incubation is 100%. Calculate the percentage of residual activity at different incubation times. Set the natural logarithm of the residual activity as the vertical coordinate and the incubation time as the horizontal coordinate. Perform linear fitting, and the slope is the inactivation rate constant k d . Calculate the half-life t according to the following formula 1 / 2 :

[0074]

[0075] The specific enzyme activity of the continuously mutated trypsin was measured to be 5950.0 U / mg, the half-life was measured to be 682.5 min, and the optimal temperature was measured to be 88.6 °C.

[0076] Comparative Example 1

[0077] The parental sequence (amino acid sequence is SEQ ID NO.3, encoding sequence is SEQ ID NO.4) was expressed and purified in the same manner as in Example 1. The specific enzyme activity was measured to be 3122.5 U / mg, the half-life was measured to be 420.1 min, and the optimal temperature was measured to be 80 °C. This sequence was obtained by gene synthesis.

[0078] Comparative Example 2

[0079] The amino acid sequence of the enzyme was changed to SEQ ID NO.5, and the corresponding coding sequence was SEQ ID NO.6. The other operation methods were the same as those in Example 1. The specific activity of the mutant trypsin was measured to be 5500.4 U / mg, the half-life was measured to be 532.6 min, and the optimal temperature was measured to be 84 °C.

[0080] SEQ ID NO.5:

[0081] MFPTDDDDKIVGGYTCAALOOPYQVSLNSGSHFCGGSLINSQWVVSAAHCYKSRIQVRLGEHNIDVLEGNEQFINAAKIITHPNFNGNTLDNDIMLIKLSSPATLNSRVATVSLPRSCAAAGTECLISGWGNTKSSGSSYPSLLQCLKAPVLSDSSCKSSYPGQITGNMICVGFLEGGKDSCQGDSGGPVVCNGQLQGIVSWGYGCAQKNKPGVYTKVCNYVNWIQQTIAANLEHHHHHH

[0082] SEQ ID NO.6 (with Histag label, restriction enzyme cleavage site):

[0083] GAATTCATGTTCCCGACGGATGATGATGATAAGATCGTGGGCGGCTATACCTGCGCCGCTCTGCCATACCAGGTTTCCCTGAACAGCGGCAGCCACTTCTGCGGCGGTAGCCTGATTAACAGCCAGTGGGTTGTGTCTGCCGCACACTGTTACAAAAGCCGCATCCAGGTTCGTCTGGGTGAGCACAACATTGATGTCCTGGAAGGCAACGAGCAGTTTATCAACGCAGCGAAGATCATCACCCATCCGAACTTCAACGGCAACACCCTGGATAACGACATTATGCTGATCAAGCTGAGCAGCCCGGCTACCCTGAACAGCCGCGTTGCGACTGTTTCTCTGCCTCGTTCCTGCGCAGCTGCGGGCACTGAATGTCTGATCTCTGGCTGGGGCAACACTAAATCCAGCGGTTCCAGCTATCCGTCTCTGCTGCAGTGCCTGAAAGCTCCGGTCCTGAGCGACTCCTCCTGCAAAAGCTCTTACCCAGGCCAGATTACCGGTAACATGATCTGCGTTGGCTTCCTGGAAGGTGGTAAAGACTCTTGTCAAGGTGACTCCGGTGGCCCAGTCGTATGCAATGGTCAGCTGCAGGGCATTGTTTCTTGGGGTTACGGCTGTGCGCAGAAAAACAAACCGGGCGTGTACACCAAAGTATGTAACTACGTGAACTGGATTCAGCAGACCATCGCCGCAAACCTGGAACACCACCACCATCACCATTAA CTCGAG

[0084] Comparative Example 3

[0085] The amino acid sequence of the enzyme was changed to SEQ ID NO.7, and the corresponding coding sequence was SEQ ID NO.8. Other operation methods were the same as in Example 1. The specific activity of the mutant trypsin was measured to be 5231.6 U / mg, the half-life was measured to be 680.5 min, and the optimum temperature was measured to be 87.9 °C.

[0086] SEQ ID NO.7:

[0087] MFPTDDDDKIVGGQPLAANSIPYQVSLNSGSHFCGGSLINSQWVVSAAHCYKSRIQVRLGEHNIDVLEGNEQFINAAKIITHPNFNGNTLDNDIMLIKLSSPATLNSRVATVSLPRSCAAAGTECLISGWGNTKSSGSSYPSLLQCLKAPVLSDSSCKSSYPGQITGNMICVGFLEGGKDSCQGDSGGPVVCNGQLQGIVSWGYGCAQKNKPGVYTKVCNYVNWIQQTIAANLEHHHHHH

[0088] SEQ ID NO.8 (with Histag label, restriction enzyme cleavage site):

[0089] GAATTCATGTTCCCAACCGACGATGACGACAAAATCGTTGGCGGTCAGCCGCTGGCCGCCAATAGCATTCCGTACCAGGTTTCCCTGAACAGCGGTTCCCACTTTTGTGGCGGTAGCCTGATCAATTCCCAATGGGTCGTGTCTGCTGCGCACTGTTACAAAAGCCGTATCCAGGTTCGTCTGGGTGAACACAACATCGACGTTCTGGAGGGTAACGAACAGTTCATCAACGCAGCTAAGATCATTACGCACCCGAACTTCAACGGCAACACCCTGGACAACGACATCATGCTGATCAAACTGTCTTCTCCGGCTACCCTGAACAGCCGTGTGGCTACCGTTTCCCTGCCGCGTTCTTGCGCAGCTGCGGGTACCGAATGCCTGATCTCCGGTTGGGGTAATACGAAATCCTCCGGTTCTTCCTATCCGTCCCTGCTGCAGTGTCTGAAAGCACCGGTACTGTCTGATAGCAGCTGCAAATCCTCCTACCCAGGCCAGATCACTGGTAACATGATCTGCGTTGGCTTCCTGGAAGGTGGTAAAGACTCCTGCCAGGGCGATTCTGGTGGCCCGGTCGTGTGCAACGGTCAACTGCAAGGTATCGTTTCCTGGGGCTACGGTTGCGCTCAGAAGAACAAACCGGGTGTCTATACCAAAGTCTGTAACTACGTTAACTGGATTCAACAAACGATCGCGGCGAATCTGGAACACCACCACCACCACCATTAA CTCGAG

[0090] As described above, it is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

Claims

1. A trypsin mutant, characterized in that, The mutant is obtained by using the trypsin shown in SEQ ID NO.3 as the parent, and mutating the 14th amino acid of the parent into glutamine, the 15th amino acid into proline, the 16th amino acid into leucine, the 17th amino acid into serine, the 18th amino acid into serine, the 19th amino acid into leucine, the 20th amino acid into pyrrolysine, and the 21st amino acid into pyrrolysine respectively.

2. The trypsin mutant according to claim 1, characterized in that, The amino acid sequence of the trypsin mutant is as shown in SEQ ID NO.

1.

3. A gene encoding the trypsin mutant according to claim 1.

4. A carrier, characterized in that, The vector contains the gene according to claim 3.

5. A host cell, characterized in that, The host cell contains the vector according to claim 4, or contains the gene according to claim 3 in its genome.

6. A method for improving the catalytic activity of trypsin by multi-site mutation, characterized in that, Mutate the 14th amino acid of the trypsin with the amino acid sequence shown in SEQID NO.3 into glutamine, the 15th amino acid into proline, the 16th amino acid into leucine, the 17th amino acid into serine, the 18th amino acid into serine, the 19th amino acid into leucine, the 20th amino acid into pyrrolysine, and the 21st amino acid into pyrrolysine.

7. A method for improving the thermal stability of trypsin by multi-site mutation, characterized in that, Mutate the 14th amino acid of the trypsin with the amino acid sequence shown in SEQID NO.3 into glutamine, the 15th amino acid into proline, the 16th amino acid into leucine, the 17th amino acid into serine, the 18th amino acid into serine, the 19th amino acid into leucine, the 20th amino acid into pyrrolysine, and the 21st amino acid into pyrrolysine.

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