A method for improving the thermostability of alkaline protease, the mutant ThAPT3-M5, and its application.

Abstract

This invention relates to the fields of genetic engineering and protein engineering. Specifically, it relates to a method for improving the thermostability of alkaline protease, the mutant ThAPT3-M5, and its applications. This invention uses alkaline protease PA3 as the parent protease and obtains the protease mutant ThAPT3-M5 with significantly improved thermostability. The thermostability of the parent protease PA3 is... m The value was 68.2±0.3℃, while the mutant's T... m The value increased to 69.5 ± 0.2℃. The mutant's t-value at 55℃ and 60℃... 1 / 2 The activity was increased to 2.3 times and 2.8 times that of the parent, respectively. After treatment at 60°C for 10 min, the residual enzyme activity of the parent was 1.9%, while that of the mutant M5 was 34.9%. Furthermore, the specific activity of the mutant increased from the initial 3078.3 ± 82.5 U / mg to 3701.9 ± 42.5 U / mg. Therefore, the alkaline protease mutant provided by this invention exhibits better thermal stability and catalytic activity compared to the wild type, and has better prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein engineering. Specifically, it relates to a method for improving the thermal stability of alkaline protease, the mutant ThAPT3-M5, and its applications. Background Technology

[0002] Serine proteases are important peptide bond hydrolases, accounting for one-third of all known proteases. The subtilisin family, also known as the S8 family of serine proteases, is the second largest family of serine proteases. Therefore, they possess broad cleavage specificity and excellent organic solvent stability, making them widely used in various industrial sectors such as food, leather, medicine, and animal feed. Proteases hold a significant market share in enzyme preparations. However, proteases exhibit poor thermal stability; even the most widely used commercial protease in industry, Proteinase K, shows unsatisfactory stability at 60°C. Therefore, the stability of alkaline proteases at high temperatures is a major bottleneck limiting their further industrial applications. Summary of the Invention

[0003] The purpose of this invention is to provide a mutant ThAPT3-M5 with improved thermal stability and specific activity obtained by using the alkaline protease PA3 derived from Torrubiella hemipterigena as a parent.

[0004] Another object of the present invention is to provide the gene for the above-mentioned alkaline protease mutant.

[0005] Another object of the present invention is to provide a recombinant vector comprising the above-described alkaline protease mutant.

[0006] Another object of the present invention is to provide a recombinant strain comprising the above-described alkaline protease mutant.

[0007] Another object of the present invention is to provide a method for preparing an alkaline protease with improved thermal stability.

[0008] Another object of the present invention is to provide the application of the above-mentioned protease mutant.

[0009] This invention uses the alkaline protease PA3 derived from Torrubiella hemipterigena as a parent to mutate and obtain a thermostable alkaline protease mutant. The amino acid sequence of PA3 is shown in SEQ ID NO:1. (Underlined parts indicate the amino acid sequence of the leader peptide.)

[0010] SEQ ID NO:1:

[0011] APSLARREEPAPLLEARGAQAIPGKFIVKLREGSPLAALQQAMSLLGGKADHVFQNV

[0012] FSGFAASMNPAVIELMRNHPDVEYIEQDGKVNIN AYTTQTGAPWGLGRISHRAKGST

[0013] SYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGS

[0014] KTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDYKTRGCPKGAVASMSLGG

[0015] GKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRST

[0016] FSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYLMGKDGAVAAGLCAKIAQTATRNVLRNIPAGTINALAFNGNPSG*.

[0017] According to the thermostability-enhanced alkaline protease mutant ThAPT3-M5 of this application, the amino acid sequence of the mutant is shown in SEQ ID NO:2 or SEQ ID NO:3, and the mutation site of the shown amino acid sequence is: loop. 19 The region exhibits a three-point mutation: Arg at position 350 is mutated to Leu, Ala at position 358 is mutated to Glu, and Ser at position 371 is mutated to Asn. (Underlined amino acid sequences indicate the precursor peptide, while bold italics indicate the mutated amino acids.)

[0018] SEQ ID NO: 2 (containing leader peptide sequence):

[0019]

[0020] SEQ ID NO: 3 (excluding leader peptide sequence):

[0021]

[0022] According to a specific embodiment of the present invention, the gene sequence of alkaline protease PA3 is shown in SEQ ID NO:4.

[0023] SEQ ID NO:4:

[0024] GCTCCATCCTTGGCTAGAAGAGAAGAACCAGCTCCTTTGTTGGAAGCTAGAGGTG

[0025] CTCAAGCTATCCCAGGTAAGTTCATCGTCAAGTTGAGAGAGGGTTCTCCATTGGCT

[0026] GCATTGCAACAAGCTATGTCCTTGCTTGGTGGTAAGGCTGACCACGTTTTCCAGAA

[0027] CGTTTTCTCTGGTTTCGCCGCCTCTATGAACCCAGCTGTTATTGAGTTGATGAGAA

[0028] ACCATCCAGACGTCGAGTACATTGAGCAGGAC GGTAAGGTTAACATCAACGCCTA

[0029] CACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGG

[0030] GTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTA

[0031] TCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGAT

[0032] CAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGT

[0033] ACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCA

[0034] AGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTGAC

[0035] TTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTTGTTGCCTCTA

[0036] TGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCA

[0037] ACGTGCCGGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTA

[0038] ATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGAC

[0039] GACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGG

[0040] TACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTC

[0041] TATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTGATGGGTAAAGATGGTG

[0042] CAGTTGCTGCAGGTTTGTGTGCTAAGATTGCTCAGACTGCCACCAGAAACGTCCT

[0043] GAGAAATATTCCAGCTGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCATCTGGTTAA.

[0044] This invention provides a gene encoding the above-mentioned protease mutant.

[0045] According to a specific embodiment of the present invention, the gene sequence encoding the alkaline protease mutant ThAPT3-M5 is shown in SEQ ID NO:5 or SEQ ID NO:6.

[0046] SEQ ID NO:5 (containing the leader peptide sequence):

[0047]

[0048]

[0049] SEQ ID NO:6 (excluding the leader peptide sequence):

[0050]

[0051] The present invention provides a recombinant vector pPICZαA-ThAPT3-m5 containing the above-mentioned alkaline protease mutant encoding gene.

[0052] The present invention also provides a recombinant strain GS115-ThAPT3-M5 containing the above-mentioned alkaline protease mutant encoding gene.

[0053] The present invention also provides a method for preparing a protease with improved thermal stability, the method comprising the following steps:

[0054] 1) Pichia pastoris GS115 host cells were transformed with a recombinant vector containing the gene encoding an alkaline protease mutant to obtain a recombinant strain;

[0055] 2) Cultivate recombinant strains and induce alkaline protease expression;

[0056] 3) The recombinant alkaline protease was recovered and purified.

[0057] The beneficial effects of this invention are:

[0058] This invention uses the alkaline protease PA3 as the parent protein and employs rational design methods such as segment substitution and site-directed mutagenesis to conduct molecular modification research on the protease, thereby obtaining the protease mutant ThAPT3-M5 with significantly improved thermostability. The thermostability of the parent protein PA3 is... m The value was 68.2±0.3℃, while the mutant's T... m The value increased to 69.5 ± 0.2℃. The mutant's t-value at 55℃ and 60℃... 1 / 2 The activity was increased to 2.3 times and 2.8 times that of the parent, respectively. After treatment at 60°C for 10 min, the residual enzyme activity of the parent was 1.9%, while that of the mutant M5 was 34.9%. Furthermore, the specific activity of the mutant increased from the initial 3078.3 ± 82.5 U / mg to 3701.9 ± 42.5 U / mg. Therefore, the alkaline protease mutant provided by this invention exhibits better thermal stability and catalytic activity compared to the wild type, and has better prospects for industrial application. Attached Figure Description

[0059] Figure 1 Gel staining images of alkaline protease PA3 and the mutant ThAPT3-M5 Coomassie protein;

[0060] Figure 2 The specific activities of alkaline protease PA3 and single-point mutants were shown at different temperatures;

[0061] Figure 3 The thermal stability of alkaline protease PA3 and single-point mutants was demonstrated after treatment at 60°C.

[0062] Figure 4 The optimal temperature for alkaline protease PA3 and the three-point combination mutant ThAPT3-M5 is shown.

[0063] Figure 5 The thermostability of alkaline protease PA3 and the three-point combination mutant ThAPT3-M5 is shown. Detailed Implementation

[0064] Experimental materials and reagents:

[0065] 1. Strains and vectors: The expression host was Pichiapastoris GS115, and the expression plasmid vector was pPICZαA.

[0066] 2. Enzymes and other biochemical reagents: Restriction endonucleases and other reagents can be purchased from general biochemical reagent companies.

[0067] 3. Culture medium:

[0068] (1) Escherichia coli culture medium low-salt LB (LLB) (1% peptone, 0.5% yeast extract, 0.5% NaCl, natural pH);

[0069] (2) Pichia pastoris culture medium YPD (1% yeast extract, 2% peptone, 2% glucose, pH natural);

[0070] (3) BMGY medium (1% yeast extract, 2% peptone, 1% glycerol, 1.34% YNB, 0.00004% biotin, pH natural);

[0071] (4) BMMY medium (1% yeast extract, 2% peptone, 0.5% methanol, 1.34% YNB, 0.00004% biotin, pH natural);

[0072] Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0073] Example 1: Preparation of the alkaline protease mutant recombinant vector pPICZαA-ThAPT3-m5

[0074] The parental alkaline protease PA3 (pre-mutation) sequence fragment (with signal peptide removed) was cloned into the expression vector pPIC-ZαA, and the recombinant vector was named pPICZαA-ThAPT3-wt. Using the recombinant vector pPICZαA-ThAPT3-wt as a template, it was amplified by primers carrying mutation sites to obtain recombinant vectors carrying individual single-point mutant sequences and combined mutant sequences, respectively.

[0075] Table 1. Specific primers for the alkaline protease mutant ThAPT3-m5

[0076]

[0077] Example 2: Construction of alkaline protease mutant expression strain

[0078] (1) Expression vector electroconversion to expression host

[0079] The expression vector was linearized using the restriction endonuclease Dra I, electroporated into the expression host GS115 competent cells, incubated with sorbitol for 30 min, and then plated on YPD plates containing bleomycin resistance (100 μL / 100 mL) and cultured at 30 °C for 2 days.

[0080] (2) Screening of transformants with high protease activity

[0081] Using a sterilized toothpick, pick a single colony from a YPD plate containing transformants and inoculate it onto a milk double-layer screening solid plate. Incubate overnight at 30°C. Once a visible milk hydrolysis clear zone appears on the milk plate, select the transformant with the largest hydrolysis zone and inoculate it into 30ml of LYPD medium.

[0082] Example 3: Expression and purification of recombinant protease mutants

[0083] (1) Shake-flask level expression of the protease mutant ThAPT3-M5

[0084] Transformants with high enzyme activity were inoculated at a 1% inoculum rate into 300 mL of BMGY medium. After incubation at 30°C and 200 rpm for 48 h with shaking, the culture was centrifuged at 4500 rpm for 5 min, and the supernatant was discarded. The culture was resuspended in 200 mL of BMGY medium and incubated at 30°C and 200 rpm for another 48 h with shaking. During the incubation period, 0.5% methanol was added to the medium every 24 h. After the incubation period, the supernatant fermentation broth was collected by centrifugation at 12000 rpm for 10 min for purification.

[0085] (2) Purification of the protease mutant ThAPT3-M5

[0086] The collected fermentation broth was concentrated to 15 mL using a 5 kDa membrane bag. Salt ions were removed by overnight dialyzing in 20 mM citrate-phosphate buffer (pH 5.5) using a 3 kDa dialysis bag. Purification was performed by cation exchange chromatography using 20 mM citrate-phosphate buffer (pH 5.5) solutions A and B. Enzyme activity was detected and analyzed by SDS-PAGE electrophoresis of the 1 M NaCl linear elution buffer, and staining was performed using Coomassie Brilliant Blue G250. Figure 1 The mutant protein size is consistent with that of the parent.

[0087] Example 4: Enzymatic Properties Analysis of Alkaline Protease PA3 and Single-Spot Mutants

[0088] The enzymatic properties of the protease of this invention were determined using the Folin-Ciocalteu method. Casein was used as the substrate. 500 μL of substrate (1% w / w) and 500 μL of enzyme solution of appropriate dilution were reacted at different temperatures for 20 min. The reaction was terminated by adding 1 mL of 0.4 M trichloroacetic acid solution. The reaction system was transferred to a 2 mL EP tube, centrifuged at 12000 rpm for 3 min, and 500 μL of the supernatant was added to 2.5 mL of 0.4 M sodium carbonate solution, followed by 500 μL of Folin-Ciocalteu reagent. The mixture was incubated at 40 °C for 30 min. After cooling, 250 μL of the incubation system was read, and the absorbance at 680 nm was recorded. The protease activity unit is defined as the amount of enzyme required to break down casein to produce 1 μmol of tyrosine per minute under certain conditions.

[0089] (1) Comparison of specific activity of the parental line and single-point mutants R350L, A358E and S371N at different temperatures

[0090] In a borax-NaOH buffer system, the specific activities of the purified enzyme solution were determined at 55℃, 60℃, and 65℃, respectively, for the parental PA3 and the mutant ThAPT3-M5. Figure 2 As shown, compared with the wild type, the optimal temperature for the single-point mutants remained consistent with the parents at 60℃, but the relative activity of the single-point mutants at 65℃ was significantly higher than that of the parents. Furthermore, the specific activity of the three single-point mutants was increased to some extent compared with the parents.

[0091] (2) Comparison of the thermal stability of the parental lines and single-point mutants R350L, A358E and S371N at 60℃

[0092] The purified parental enzyme and each single-point mutant enzyme solutions were diluted to 100 ng / ml, and 200 μL was placed in 1.5 mL EP tubes. After incubation at 60℃ for 10 min, the remaining activity was measured. The enzyme solution without heat treatment was used as a control, and its remaining relative activity was calculated. Results are as follows: Figure 3 As shown, the parental PA3 exhibits poor thermal stability at 60℃ and is completely inactivated after 10 minutes of heat treatment, while the thermal stability of each single-point mutant is significantly improved.

[0093] Example 5: Enzymatic Properties Analysis of Alkaline Protease PA3 and its Mutant ThAPT3-M5

[0094] (1) Determination of optimal temperature

[0095] In a borax-NaOH buffer system at pH 9.5, the enzyme activities of PA3 and the mutant ThAPT3-M5 were determined at different temperatures (30℃, 40℃, 50℃, 55℃, 60℃, 65℃, 70℃, and 80℃) to determine their optimal temperatures. The activity corresponding to the optimal temperature was defined as 100%, and the relative activities at the other temperatures were calculated. Figure 4 As shown, the optimal temperature for the parent PA3 is 60℃, and the optimal temperature for the mutant M5 is consistent with it.

[0096] (2) Thermal stability

[0097] The purified enzyme solutions of the parent and mutant ThAPT3-M5 were diluted to 100 ng / ml, and 200 μL was placed in 1.5 mL EP tubes. The tubes were incubated at 55℃ and 60℃ for 2 min, 5 min, 10 min, 20 min, 30 min, and 60 min, respectively, and the residual activity was measured. The enzyme solution without heat treatment was used as a control, and its residual relative activity was calculated. The results are as follows: Figure 5 As shown, the mutant ThAPT3-M5 exhibited significantly improved thermal stability compared to its parent. After incubation at 55℃ for 1 hour, it retained 40% of its residual activity, while the parent was essentially inactivated after 1 hour of treatment. After treatment at 60℃ for 10 minutes, it retained 40% of its residual activity, which was also significantly better than the parent.

[0098] Half-life t 1 / 2 Thermostatus is a commonly used parameter for characterizing enzyme thermostability; a higher value indicates better enzyme thermostability. It refers to the time required for the initial activity to decrease by 50% at a given temperature, and is calculated using the following formula:

[0099]

[0100] Where, k d The deactivation rate constant can be obtained using the following formula:

[0101]

[0102] In the formula, A t A0 refers to the residual activity, A0 is the initial activity, and t refers to the treatment time at a given treatment temperature.

[0103] Melting temperature (T) m The temperature at which a protein unfolds to 50% is called the T0. Differential scanning calorimetry (DSC) is commonly used to determine the T0 of proteins. m .

[0104] t1 / 2 and T m The analysis results are shown in Table 2. The thermostability of the mutant ThAPT3-M5 is significantly better than that of the parent. m The value increased by 1.3℃ at 55℃. 1 / 2 Increased to 2.3 times that of the parent, at 60℃ t 1 / 2 It is 2.8 times that of the parent.

[0105] Table 2. T values ​​of parents and the mutant ThAPT3-M5 m value and t 1 / 2 parameter

[0106]

[0107] (3) Determination of kinetic constants

[0108] Casein was prepared at concentrations of 0.5, 0.8, 1.0, 1.3, 1.5, 2.0, 2.5, 5.0, 8.0, and 10.0 mg / mL as substrates. The purified enzyme solutions were appropriately diluted and reacted with different concentrations of substrate at the optimal temperature and pH for 10 min, respectively. Enzyme activity was then measured. The Michaelis-Menten equation constants were fitted using GraphPad Prism version 5.01 software, and K was calculated. m and V max The results are shown in Table 3. The mutant ThAPT3-M5 exhibits higher catalytic activity and efficiency compared to the parent, but K... m The value did not change significantly.

[0109] Table 3 Kinetic parameters of the parent and mutant ThAPT3-M5

[0110]

[0111] The above embodiments are only used to explain the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method for improving the thermal stability and catalytic activity of alkaline protease, characterized in that, The method comprises the following steps: mutating the 350th Arg of the alkaline protease parent PA3 with the amino acid sequence shown in SEQ ID NO: 1 into Leu, mutating the 358th Ala into Glu, and mutating the 371st Ser into Asn.

2. A mutant of alkaline protease characterized in that, The amino acid sequence of the alkaline protease mutant is shown in SEQ ID NO: 2 or SEQ ID NO:

3.

3. A basic protease gene, characterized in that, The alkaline protease gene encodes the alkaline protease mutant of claim 2.

4. The alkaline protease gene according to claim 3, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO: 5 or SEQ ID NO:

6.

5. A recombinant expression vector comprising the alkaline protease gene of claim 3.

6. A recombinant strain comprising the alkaline protease gene of claim 3.

7. A process for the preparation of alkaline protease characterized by, The method comprises the following steps: transforming the host cell with the recombinant expression vector of claim 5; inducing the host cell to express; obtaining the alkaline protease by separation and purification.

8. Use of the alkaline protease mutant of claim 2 in hydrolyzing casein.

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