Construction method of trypsin with improved thermal stability

Abstract

The invention discloses a construction method of trypsin with improved thermal stability. According to a mutant, trypsin with an amino acid sequence as shown in SEQ ID NO.3 is taken as a parent; the amino acid at the 41st site of the parent mutates into proline, the amino acid at the 87th site of the parent mutates into proline, the amino acid at the 193rd site of the parent mutates into tyrosine, the amino acid at the 195th site of the parent mutates into leucine, and the amino acid at the 200th site of the parent mutates into serine. According to the invention, a trypsin molecular structure is modified through a site-directed mutagenesis biotechnology, and a mutant strain with improved thermal stability is obtained after mutation verification. The thermal stability of the mutant is obviously improved, and the catalytic activity of the enzyme is not influenced or even improved. The mutant can be industrially produced at a relatively high temperature, meets the requirements of industrial production, and 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 constructing a trypsin with improved thermal stability. Background Art

[0002] 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 its structure remains intact. However, the industrial environment often differs greatly from the natural environment and is more demanding.

[0003] 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.

[0004] Trypsin is a pancreatic serine protease. Due to the advantages of high catalytic efficiency and selectivity, it has been widely used in food technology, regulating the allergenicity of soy proteins, generating antihypertensive peptides, and as 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 bread quality improvement. In industrial production, trypsin can be used for the degradation of protein feed; it is also a safe organic pesticide degrading agent that can effectively reduce the toxicity of pesticides and insecticides such as diazinon, deltamethrin, and malathion; it is one of the important enzyme preparations in the leather industry.

[0005] Trypsin has become a commonly used tool enzyme in the industrial field due to its high efficiency, specificity, and high hydrolysis rate, with wide market applications and large demand. However, in a high-temperature environment, trypsin cannot maintain high enzyme activity and thermal stability, and natural trypsin usually cannot withstand the harsh industrial conditions in production. Due to the low thermal stability of natural trypsin, it is prone to denaturation and inactivation, and thus cannot meet the requirements of industrial applications. Summary of the Invention

[0006] The main object of the present invention is to propose a method for constructing a trypsin with improved thermal stability, aiming to provide a trypsin with high thermal stability that can meet the requirements of industrial applications.

[0007] To achieve the above object, the present invention provides a trypsin mutant, which is obtained by mutating the 41st amino acid of trypsin with the amino acid sequence shown in SEQ ID NO.3 to proline, the 87th amino acid to proline, the 193rd amino acid to tyrosine, the 195th amino acid to leucine, and the 200th amino acid to serine, respectively, using the trypsin as a parent.

[0008] Optionally, the amino acid sequence of the trypsin mutant is as shown in SEQ ID NO.1.

[0009] The present invention also provides a gene encoding the above-mentioned trypsin mutant.

[0010] The present invention also provides a vector containing the above-mentioned gene.

[0011] The present invention also provides a host cell containing the above-mentioned vector or having the above-mentioned gene in its genome.

[0012] The present invention also provides a method for constructing a trypsin with improved thermal stability, which comprises mutating the 41st amino acid of trypsin with the amino acid sequence shown in SEQ ID NO.3 to proline, the 87th amino acid to proline, the 193rd amino acid to tyrosine, the 195th amino acid to leucine, and the 200th amino acid to serine.

[0013] The present invention also provides a method for constructing a trypsin with improved enzyme activity, which comprises mutating the 41st amino acid of trypsin with the amino acid sequence shown in SEQ ID NO.3 to proline, the 87th amino acid to proline, the 193rd amino acid to tyrosine, the 195th amino acid to leucine, and the 200th amino acid to serine.

[0014] The present invention also provides the application of the above-mentioned mutant, or the above-mentioned gene, or the above-mentioned vector, or the above-mentioned host cell in the fields of industry, medicine, biochemistry and food.

[0015] The beneficial effects of the present invention are as follows:

[0016] In the technical solution provided by the present invention, the molecular structure of trypsin is modified by site-directed mutagenesis biotechnology, and a mutant strain with improved thermal stability is obtained after mutation verification. While the thermal stability of the mutant is significantly improved, the catalytic activity of the enzyme is not affected and even increased. The mutant can be used for industrial production at a higher temperature, meeting the requirements of industrial production, and having good industrial application prospects. Detailed Embodiments To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Reagents or instruments without indicating the manufacturer can be obtained as conventional products through commercial purchase. In addition, the meaning of "and / or" appearing throughout the text includes three parallel options. Taking "A and / or B" as an example, it includes option A, option B, or the option where both A and B are satisfied simultaneously. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of those of ordinary skill in the art to implement. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention. The experimental methods without specific conditions in the following embodiments 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.

[0017] 1. Description of Materials and Instruments

[0018] (1) Test materials E.coli BL21 and E.coli DH5α were cultured in Luria Bertani medium (10.0 g NaCl, 10.0 g tryptone, 5.0 g yeast extract). pET-28a(+) was used to construct a plasmid expressing the target gene. Escherichia coli DH5α was used as the host for plasmid cloning, and Escherichia coli BL21 was used as the expression host strain. Enterokinase and Mut II Quick Mutagenesis Kit V2 were provided by Yuanye Biotechnology 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 Sangon Biotech Co., Ltd. (Shanghai, China).

[0019] (2) Test Instruments

[0020]

[0021] Example 1 Construction of Trypsin Vector

[0022] 1. Construction of Mutant Recombinant Plasmid

[0023] Using trypsin with the amino acid sequence shown in SEQ ID NO.3 as the parent, the S at position 41 of the parent was mutated to P, the G at position 87 was mutated to P, the N at position 193 was mutated to Y, the Q at position 195 was mutated to L, and the V at position 200 was mutated to S, resulting in the amino acid sequence SEQ ID NO.1.

[0024] SEQ ID NO.1:

[0025] MFPTDDDDKIVGGYTCAANSIPYQVSLNSGSHFCGGSLINPQWVVSAAHC

[0026] YKSRIQVRLGEHNIDVLEGNEQFINAAKIITHPNFNPNTLDNDIMLIKLSSPA

[0027] TLNSRVATVSLPRSCAAAGTECLISGWGNTKSSGSSYPSLLQCLKAPVLSDS

[0028] SCKSSYPGQITGNMICVGFLEGGKDSCQGDSGGPVVCYGLLQGISSWGYG

[0029] CAQKNKPGVYTKVCNYVNWIQQTIAANLEHHHHHH

[0030] SEQ ID NO.3:

[0031] MFPTDDDDKIVGGYTCAANSIPYQVSLNSGSHFCGGSLINSQWVVSAAHC

[0032] YKSRIQVRLGEHNIDVLEGNEQFINAAKIITHPNFNGNTLDNDIMLIKLSSPA

[0033] TLNSRVATVSLPRSCAAAGTECLISGWGNTKSSGSSYPSLLQCLKAPVLSDS

[0034] SCKSSYPGQITGNMICVGFLEGGKDSCQGDSGGPVVCNGQLQGIVSWGYG

[0035] CAQKNKPGVYTKVCNYVNWIQQTIAANLEHHHHHH

[0036] The gene sequence encoding the parent is shown in SEQ ID NO.4.

[0037] SEQ ID NO.4 (with Histag tag, the underlined part is the restriction enzyme cleavage site): GAATTC ATGTTCCCAACCGATGACGACGATAAGATCGTGGGCGGCTATACCTGTGCCGCTAATAGCATTCCGTACCAGGTTTCCCTGAACTCCGGTAGCCATTTTTGTGGTGGTTCCCTGATCAACTCTCAGTGGGTTGTGTCTGCGGCACATTGTTACAAAAGCCGTATCCAGGTCCGTCTGGGTGAACACAATATCGACGTGCTGGAAGGCAACGAGCAGTTCATCAATGCCGCTAAAATTATCACCCACCCGAACTTCAACGGCAACACCCTGGACAATGATATCATGCTGATCAAGCTGTCCTCTCCAGCGACTCTGAACTCCCGTGTTGCAACTGTCTCTCTGCCGCGTAGCTGCGCGGCCGCAGGTACTGAGTGCCTGATCTCTGGTTGGGGCAATACCAAGTCTTCTGGTAGCAGCTATCCGTCCCTGCTGCAGTGTCTGAAAGCACCAGTTCTGTCTGATTCCTCTTGTAAGAGCTCTTACCCTGGCCAGATTACCGGTAACATGATCTGCGTGGGCTTCCTGGAAGGCGGTAAAGATTCCTGTCAGGGCGACAGCGGTGGTCCAGTTGTTTGCAACGGCCAGCTGCAGGGTATCGTTAGCTGGGGCTATGGCTGTGCGCAAAAAAACAAACCGGGCGTGTACACCAAAGTTTGCAACTACGTTAACTGGATCCAACAGACCATTGCGGCGAACCTGGAACACCACCACCACCATCAC TAAC CTCGAG

[0038] 2. Construction of recombinant engineering bacteria

[0039] 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.

[0040] SEQ ID NO.2 (with Histag tag, the underlined part is the restriction enzyme site):

[0041] GAATTCATGTTCCCAACGGACGACGACGACAAGATCGTAGGCGGCTATACCTGCGCTGCCAACTCTATTCCGTACCAGGTTTCCCTGAACAGCGGTAGCCACTTTTGTGGCGGCAGCCTGATCAACCCACAGTGGGTAGTTTCCGCTGCGCACTGCTATAAATCTCGTATCCAGGTACGTCTGGGCGAACACAACATCGACGTGCTGGAAGGTAACGAACAGTTCATCAACGCGGCTAAGATCATCACCCATCCGAACTTCAACCCGAATACCCTGGACAACGACATCATGCTGATTAAACTGTCTAGCCCGGCGACTCTGAACTCCCGTGTTGCTACTGTATCTCTGCCACGTTCCTGCGCAGCGGCAGGTACTGAATGTCTGATTTCTGGCTGGGGTAACACGAAATCTAGCGGTTCTAGCTACCCGAGCCTGCTGCAGTGTCTGAAAGCGCCTGTCCTGTCCGATAGCTCCTGTAAATCCAGCTACCCAGGCCAGATTACTGGCAACATGATTTGTGTTGGCTTCCTGGAAGGTGGTAAGGATTCTTGTCAAGGTGACTCTGGTGGTCCAGTGGTTTGCTACGGTCTGCTGCAGGGTATTTCTAGCTGGGGCTATGGCTGTGCGCAGAAAAATAAACCGGGCGTTTACACTAAAGTGTGCAACTACGTTAACTGGATCCAGCAGACTATCGCAGCTAACCTGGAGCACCACCACCACCACCATTAAC CTCGAG The ligation product was transformed into E. coli DH5α competent cells, and the specific transformation steps are as follows:

[0042] (1) First, adjust the temperature of the constant temperature water bath to 42 °C.

[0043] (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.

[0044] (3) Add 10 μL of the ligated plasmid pET28α, gently shake it, and then place it on ice for 20 min.

[0045] (4) Shake it gently and then insert it into a 42°C water bath for 90 s of heat shock, then quickly put it back on ice and let it stand for 5 min.

[0046] (5) Add 700 μL of LB medium without antibiotics to each of the above tubes and mix gently. Then shake it on a shaker at 37°C for 50 min.

[0047] (6) Drop 300 μL of the above transformation mixture taken out from the laminar flow hood 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 evenly.

[0048] Label the spread plates and 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 of 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 plasmid extraction kit instructions. Digest the extracted recombinant plasmid with EcoRI and XhoI, which is the same system as the double digestion of pET-28a. Analyze the target gene fragment and vector fragment of the double-digested product by 1% agarose gel electrophoresis. Then sequence the recombinant plasmid identified as positive by double digestion.

[0049] Example 2 Expression of Trypsin

[0050] (1) Prepare the seed liquid: Use an inoculation loop to pick up the E. coli BL21(DE3) strain stored at -20°C in the laminar flow hood, streak it in three zones on an LB plate containing kanamycin, and culture it upside down 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.

[0051] (2) Induce culture: Add 1 mL of the seed liquid 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, 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.

[0052] (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 shake flask of LB medium in a sterile laminar flow hood and induce it for the same time. Centrifuge at 12,000 rpm for 5.0 min, and collect the supernatant and cell precipitate. Add bacterial lysis buffer and ultrasonically lyse the cells. Centrifuge at 12,000 rpm for 5 min to collect the supernatant and precipitate respectively.

[0053] Example 3 Obtaining Trypsin

[0054] The expressed trypsin was verified to form inclusion bodies, and the following treatments need to be carried out on the inclusion bodies:

[0055] (1) Ultrasonic lysis: Mix the wet bacterial cells and 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; ultrasonicate twice, and centrifuge at 10,000 rpm and 4°C for 15 min to collect the precipitate.

[0056] (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 washing solution per 1 g of precipitate, stir with a glass rod until completely dissolved, and centrifuge at 10,000 rpm and 4°C for 15 min to collect the precipitate. The collected precipitate is the wet inclusion body.

[0057] (3) Lyse the inclusion bodies: Mix the wet inclusion bodies and 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 10,000 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.

[0058] (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.

[0059] Example 4 Purification of Trypsin

[0060] 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:

[0061] (1) Column packing: First, pack the Ni-NTA purification resin into the chromatography column. Load 1.0 mL of 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.

[0062] (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 under the chromatography column. Then pour the obtained solution into the chromatography column again to allow the target protein to fully bind to the resin.

[0063] (3) Washing: Slowly add Wash Buffer to the chromatography column, control the flow rate at 1 mL / min, and wash away the unbound impurity proteins with approximately ten times the volume of Wash Buffer. Use Wash Buffer as the blank until the value at A280 in the eluent from the chromatography column no longer changes and approaches zero.

[0064] (4) Elution: Use Elution Buffer to elute the target protein, and detect the eluate with a spectrophotometer. Stop collecting when the value at A280 in the eluate no longer changes. Perform SDS-PAGE electrophoresis on the collected solution, showing a single protein band.

[0065] (5) Cleaning: Wash the column with Elution Buffer containing high-concentration imidazole, approximately ten column volumes, and then wash the column with 10 column volumes of ddH2O for future use.

[0066] (6) Preservation: Seal the column with 20% ethanol for subsequent use.

[0067] Example 5 Enzyme Activity Assay

[0068] (1) Preparation of substrate solution: Use N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) as the substrate to determine the trypsin activity. The prepared substrate solution needs to be used within two hours, kept 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 with the above substrate stock solution or phosphate buffer to make the absorbance between 0.575 and 0.585.

[0069] (2) Sample treatment: The trypsin sample needs to be dissolved with 0.001 mol / L hydrochloric acid solution and quantitatively diluted to prepare a solution containing 50 - 60 trypsin units per 1 ml.

[0070] (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).

[0071] 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 every 30 s at 253 nm for a total of 5 min. Use the absorbance and time as the vertical and horizontal coordinates respectively to plot the absorbance-time curve; the change in each absorbance reading should be maintained between 0.015 and 0.018, and the time showing a linear relationship should be no less than 3 min. Under the above conditions, an absorbance change of 0.003 per minute is equivalent to 1 trypsin unit.

[0072]

[0073] Example 6 Determination of the Thermal Stability of Trypsin

[0074] 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 the 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 :

[0075]

[0076] The specific activity of the mutant trypsin (S41P, G87P, N193Y, Q196L, V200S) was measured to be 5951.68 U / mg, the half-life was measured to be 683.1 min, and the optimal temperature was measured to be 88.5 °C.

[0077] The parent sequence (amino acid sequence is SEQ ID NO.3, encoding sequence is SEQ ID NO.4) was expressed and purified as above. The specific activity was measured to be 3122.8 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] In summary, the present invention modifies the molecular structure of trypsin through site-directed mutagenesis biotechnology. While the thermal stability of the mutant is significantly improved, the catalytic activity of the enzyme is also enhanced.

[0079] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the patent protection scope of the present invention.

Claims

1. A trypsin mutant, characterized in that, The mutant is obtained by using trypsin with an amino acid sequence as shown in SEQ ID NO.3 as the parent, and mutating the 41st amino acid of the parent into proline, the 87th amino acid into proline, the 193rd amino acid into tyrosine, the 195th amino acid into leucine, and the 200th amino acid into serine, respectively.

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

1.

3. A gene encoding the trypsin mutant as claimed in claim 1.

4. A carrier, characterized in that, The vector contains the gene as claimed in claim 3.

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

6. A method for constructing trypsin with improved thermal stability, characterized in that, Mutate the 41st amino acid of trypsin with an amino acid sequence as shown in SEQ IDNO.3 into proline, the 87th amino acid into proline, the 193rd amino acid into tyrosine, the 195th amino acid into leucine, and the 200th amino acid into serine.

7. A method for constructing trypsin with improved enzyme activity, characterized in that, Mutate the 41st amino acid of trypsin with an amino acid sequence as shown in SEQ ID NO.3 into proline, the 87th amino acid into proline, the 193rd amino acid into tyrosine, the 195th amino acid into leucine, and the 200th amino acid into serine.

Images