Method for improving heat stability of alkaline protease, mutant thapt3-m3, thapt3-m4 and application thereof

Abstract

The present application relates to the field of genetic engineering, in particular to a method for improving the heat stability of alkaline protease and mutant ThAPT3-M3, ThAPT3-M4 and application. The present application takes alkaline protease PA3 as parent, and obtains protease mutants with significantly improved heat stability through site-directed mutagenesis. After 10 min and 20 min of treatment at 60 DEG C, the residual enzyme activity of the parent PA3 is 1.9% and 1.6% respectively. After 10 min of treatment at 60 DEG C, the residual enzyme activity of the mutants ThAPT3-M3 and ThAPT3-M4 is 58% and 68% respectively; after 20 min of treatment at 60 DEG C, the residual enzyme activity of the mutants ThAPT3-M3 and ThAPT3-M4 is 31% and 45% respectively. In addition, the specific activity of the mutant ThAPT3-M4 is increased by 25%. Therefore, the alkaline protease mutants provided by the present application have better heat stability and catalytic activity compared with the wild type, and have better industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to a method for improving the thermal stability of alkaline protease, mutant ThAPT3-M3, ThAPT3-M4 and application. BACKGROUND

[0002] Serine proteases are important peptide bond hydrolytic enzymes, and 1 / 3 of the known proteases belong to the serine protease family. The subtilisin family is also known as the S8 family of serine proteases, which is the second largest family of serine proteases. Therefore, it has wide cutting specificity and excellent organic solvent stability, and is widely used in food, leather, medical and feed industries. Protease occupies a high market share in the field of enzyme preparations. However, the thermal stability of protease is poor, and the most widely used commercial protease Proteinase K in industry has a poor stability at 60°C. Therefore, the stability of alkaline protease at high temperature is the main bottleneck limiting its further industrial application.

[0003] Protein engineering is one of the most effective means to optimize the enzymatic properties of protease, and rational design methods such as segment replacement or site-directed mutation can efficiently improve the stability and catalytic activity of protease. SUMMARY

[0004] The purpose of the present application is to provide mutant ThAPT3-M3 and ThAPT3-M4 with improved thermal stability and specific activity, which are obtained from Torrubiella hemipterigena-derived alkaline protease PA3 as parent.

[0005] Another purpose of the present application is to provide a gene of the above-mentioned alkaline protease protease mutant.

[0006] Another purpose of the present application is to provide a recombinant vector comprising the gene of the above-mentioned alkaline protease protease mutant.

[0007] Another purpose of the present application is to provide a recombinant strain comprising the gene of the above-mentioned alkaline protease protease mutant.

[0008] Another purpose of the present application is to provide a method for preparing alkaline protease with improved thermal stability.

[0009] Another purpose of the present application is to provide the application of the above-mentioned protease mutant.

[0010] The present application mutates Torrubiella hemipterigena-derived alkaline protease PA3 as parent to obtain alkaline protease mutant with improved thermal stability, wherein the amino acid sequence of PA3 is shown in SEQ ID NO: 1. (The underlined amino acid sequence corresponds to the leader peptide)

[0011] SEQ ID NO: 1:

[0012] APSLARREEP AP LLEARGAQA IPGKFIVKLREGSPLAALQQAMSLLGGKADHVFQNVFSGFAASMNPA VIELMRNHPDVEYIEQDGKVNIN AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDYKTRGCPKGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYLMGKDGAVAAGLCAKIAQTATRNVLRNIPAGTINALAFNGNPSG*.

[0013] The heat-stable alkaline protease mutant ThAPT3-M3 according to the present application, wherein the amino acid sequence of the mutant is shown as SEQ ID NO: 2 or SEQ ID NO: 3, and the mutation site of the shown amino acid sequence is: single-point mutation of Ala at position 345 in the a8-helix region into Gin. (The amino acid sequence corresponding to the leader peptide is marked with an underline, and the mutated amino acid is marked in bold and italic)

[0014] SEQ ID NO: 2 (containing a leader peptide):

[0015]

[0016] SEQ ID NO: 3 (without a leader peptide):

[0017]

[0018] The heat-stable alkaline protease mutant ThAPT3-M4 according to the present application, wherein the amino acid sequence of the mutant is shown as SEQ ID NO: 4 or SEQ ID NO: 5, and the mutation site of the shown amino acid sequence is: double-point combined mutation of Ala at position 342 in the a8-helix region into Glu and Ala at position 345 into Gin.

[0019] SEQ ID NO: 4 (containing a leader peptide):

[0020]

[0021]

[0022] SEQ ID NO: 5 (without a leader peptide):

[0023]

[0024] According to the specific embodiment of the present application, the gene sequence of alkaline protease PA3 is as shown in SEQ ID NO: 6 or SEQ ID NO: 6.

[0025] SEQ ID NO: 6:

[0026] GCTCCATCCTTGGCTAGAAGAGAAGAACCAGCTCCTTTGTTGGAAGCTAGAGGTG

[0027] CTCAAGCTATCCCAGGTAAGTTCATCGTCAAGTTGAGAGAGGGTTCTCCATTGGCT

[0028] GCATTGCAACAAGCTATGTCCTTGCTTGGTGGTAAGGCTGACCACGTTTTCCAGAA

[0029] CGTTTTCTCTGGTTTCGCCGCCTCTATGAACCCAGCTGTTATTGAGTTGATGAGAA

[0030] ACCATCCAGACGTCGAGTACATTGAGCAGGAC GGTAAGGTTAACATCAACGCCTA

[0031] CACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGG

[0032] GTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTA

[0033] TCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGAT

[0034] CAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGT

[0035] ACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCA

[0036] AGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGAC

[0037] TTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTA

[0038] TGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCA

[0039] ACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTA

[0040] ATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGAC

[0041] GACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGG

[0042] TACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTC

[0043] TATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTGATGGGTAAAGATGGTG

[0044] CAGTTGCTGCAGGTTTGTGTGCTAAGATTGCTCAGACTGCCACCAGAAACGTCCT

[0045] GAGAAATATTCCAGCTGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCATCTGGTTAA。

[0046] The application provides a gene encoding the above-mentioned protease mutant.

[0047] According to the specific embodiment of the application, the gene sequence encoding the alkaline protease mutant ThAPT3-M3 is shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0048] SEQ ID NO: 7 (containing a leader peptide):

[0049]

[0050] SEQ ID NO: 8 (without a leader peptide):

[0051]

[0052]

[0053] According to the specific embodiment of the present application, the coding gene sequence of the alkaline protease mutant ThAPT3-M4 is shown as SEQ ID NO: 9 and SEQ ID NO: 10.

[0054] SEQ ID NO: 9 (containing a leader peptide):

[0055]

[0056]

[0057] SEQ ID NO: 10 (not containing a leader peptide):

[0058]

[0059] The present application provides recombinant vectors pPICZαA-ThAPT3-m3 and pPICZαA-ThAPT3-m4 containing the coding gene of the above alkaline protease mutant.

[0060] The present application also provides recombinant strains GS115-ThAPT3-M3 and GS115-ThAPT3-M4 containing the coding gene of the above alkaline protease mutant.

[0061] The present application also provides a method for preparing a protease mutant with improved thermal stability, which comprises the following steps:

[0062] 1) transforming Pichia pastoris GS115 host cells with a recombinant vector containing the gene of the alkaline protease mutant to obtain a recombinant strain;

[0063] 2) culturing the recombinant strain to induce the expression of the alkaline protease;

[0064] 3) recovering and purifying the recombinant expressed alkaline protease.

[0065] The present application has the following beneficial effects:

[0066] The application takes alkaline protease PA3 as a parent, and carries out molecular improvement research on the protease through segment replacement and site-directed mutation, so that the protease mutants ThAPT3-M3 and ThAPT3-M4 with significantly improved thermal stability are obtained. After the parent PA3 is treated at 60 DEG C for 10 min and 20 min, the residual enzyme activities are 1.9% and 1.6% respectively. After the mutants ThAPT3-M3 and ThAPT3-M4 are treated at 60 DEG C for 10 min, the residual enzyme activities are 58% and 68% respectively; after the mutants ThAPT3-M3 and ThAPT3-M4 are treated at 60 DEG C for 20 min, the residual enzyme activities are 31% and 45% respectively. In addition, the specific activity of the mutant ThAPT3-M4 is increased by 25%. Therefore, the alkaline protease mutants provided by the application have better thermal stability and catalytic activity than the wild type, and have better industrial application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 A Coomassie protein staining gel showing alkaline protease PA3 and mutants ThAPT3-M3 and M4;

[0068] Figure 2 A graph showing the specific activity of alkaline protease PA3 and mutant ThAPT3-M3 at different temperatures;

[0069] Figure 3 A graph showing the thermal stability of alkaline protease PA3 and mutant ThAPT3-M3 treated at 60 DEG C;

[0070] Figure 4 A graph showing the optimum temperature of alkaline protease PA3 and mutant ThAPT3-M4;

[0071] Figure 5 A graph showing the thermal stability of alkaline protease PA3 and mutant ThAPT3-M4. DETAILED DESCRIPTION

[0072] Test materials and reagents:

[0073] 1. Strains and vectors: the expression host is Pichia pastoris GS115, and the expression plasmid vector is pPICZ alpha A.

[0074] 2. Enzymes and other biochemical reagents: restriction endonuclease.

[0075] 3. Culture medium:

[0076] (1) Escherichia coli culture medium low salt LB (LLB) (1% peptone, 0.5% yeast extract, 0.5% NaCL, pH natural);

[0077] (2) Pichia pastoris culture medium YPD (1% yeast extract, 2% peptone, 2% glucose, pH natural);

[0078] (3) BMGY culture medium (1% yeast extract, 2% peptone, 1% glycerol, 1.34% YNB, 0.00004% biotin, pH natural);

[0079] (4) BMMY culture medium (1% yeast extract, 2% peptone, 0.5% methanol, 1.34% YNB, 0.00004% biotin, pH natural);

[0080] The molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in the book "Molecular Cloning Experiment Guide" (third edition) by J. Sambrook, or according to the kit and product instructions.

[0081] Example 1 Preparation of recombinant vectors pPICZαA-ThAPT3-m3 and pPICZαA-ThAPT3-m4 of alkaline protease mutants

[0082] The alkaline protease parent PA3 (before mutation) sequence fragment (signal peptide removed) was cloned into the expression vector pPIC-ZαA, and the recombinant vector was named pPICZαA-ThAPT3-wt. The recombinant vector pPICZαA-ThAPT3-wt was used as a template, and a primer pair carrying a mutation site was used to amplify it, obtaining a recombinant vector carrying a mutant sequence, named pPICZαA-ThAPT3-m3 and pPICZαA-ThAPT3-m4.

[0083] Table 1. Specific primers of alkaline protease mutants ThAPT3-m3 and ThAPT3-m4

[0084]

[0085] Example 2 Construction of alkaline protease mutant expression strain

[0086] (1) Electroporation of expression vector into expression host

[0087] The expression vector was linearized using the restriction enzyme Dra I, and was electroporated into the expression host GS115 competent cells. After 30 min of sorbitol incubation, it was plated on a YPD plate with bleomycin resistance (100 μL / 100 mL), and was cultured at 30°C for 2 days.

[0088] (2) Screening of high protease activity transformants

[0089] The single colony of the transformed YPD plate was picked up with a sterilized toothpick and inoculated on a double-layer screening solid plate with milk, and incubated at 30°C overnight. The milk plate appeared a visible transparent circle of milk hydrolysis, and the transformant with the largest hydrolysis circle was selected and inoculated in 30 mL YPD medium.

[0090] Example 3 Expression and purification of recombinant protease mutants

[0091] (1) Shake flask level expression of protease mutants ThAPT3-M3 and ThAPT3-M4

[0092] The transformant with high enzyme activity screened was inoculated in 300 mL BMGY medium at 1% inoculation amount, and cultured at 30°C, 200 rpm for 48 h. Then, the supernatant was removed by centrifugation at 4500 rpm for 5 min. The culture was resuspended in 200 mL BMMY medium, and cultured at 30°C, 200 rpm for another 48 h. During the culture, 0.5% methanol was added every 24 h. After the culture, the fermentation broth was collected by centrifugation at 12000 rpm for 10 min, and used for purification.

[0093] (2) Purification of protease mutants ThAPT3-M3 and ThAPT3-M4

[0094] The collected fermentation broth was concentrated to 15 mL using a 5 kDa membrane bag. The salt ions were removed by dialysis overnight using a 3 kDa dialysis bag in 20 mM citric acid-phosphate buffer (pH 5.5). The cation exchange chromatography was performed using 20 mM citric acid-phosphate buffer (pH 5.5) A and B solutions. The enzyme activity detection and SDS-PAGE electrophoresis analysis were performed on 1 M NaCl linear eluent, and the staining was performed using Coomassie brilliant blue staining solution G250 (). Figure 1 The mutant and the parent had the same protein size.

[0095] Example 4 Enzymatic property analysis of alkaline protease PA3 and mutant ThAPT3-M3

[0096] The enzymatic property of the protease is determined by using the Folin phenol method, casein is used as the substrate, 500 μL of the substrate (1% W / W) and 500 μL of the enzyme solution diluted by a proper multiple are reacted for 20 min at different temperatures, 1 mL of 0.4M trichloroacetic acid solution is added to terminate the reaction. The reaction system is transferred into a 2 mL EP tube, after centrifugation at 12000 rpm for 3 min, 500 μL of the supernatant is taken and added into 2.5 mL of 0.4M sodium carbonate solution, then 500 μL of Folin phenol reagent is added, coloration is carried out at 40°C for 30 min, after cooling, 250 μL of the coloration system is taken to read the absorbance value at 680 nm. The definition of the protease activity unit is that the amount of the enzyme required for decomposing 1 μmol of casein to generate tyrosine per minute under certain conditions is 1 activity unit (U).

[0097] (1) Comparison of the specific activity of the parent and mutant ThAPT3-M3 at different temperatures

[0098] The specific activity of the purified parent PA3 and mutant ThAPT3-M3 is determined at 55, 60 and 65°C in a borax-NaOH buffer system. As shown in Table 1, compared with the wild type, the optimum temperature of the mutant M3 is increased from 60°C to 65°C, and the enzyme activity at 60°C and 65°C is not much different. In addition, the specific activity of the mutant M3 at 60°C is increased from 3078.3±93.3 U / mg to 3355.426094±55.2 U / mg. Figure 2

[0099] (2) Comparison of the heat stability of the parent and mutant ThAPT3-M3 at 60°C

[0100] The enzyme solution of the purified parent and mutant ThAPT3-M3 is diluted to 100 ng / ml, 200 μL is taken in a 1.5 mL EP tube, and after incubation at 60°C for 10 min and 20 min respectively, the residual activity is detected. The enzyme solution without heat treatment is used as a control, and the residual relative activity is calculated. As shown in Table 2, the heat stability of the parent PA3 at 60°C is poor, and it is completely inactivated after heat treatment for 10 min, while the heat stability of the mutant ThAPT3-M3 is obviously improved, and it retains 59% and 32% of the catalytic activity after heat treatment for 10 min and 20 min respectively. Figure 3

[0101] Example 5 Enzymatic property analysis of alkaline protease PA3 and mutant ThAPT3-M4

[0102] (1) Optimum temperature determination

[0103] ​​The enzyme activity of PA3 and mutant ThAPT3-M4 was determined at different temperatures (30℃, 40℃, 50℃, 55℃, 60℃, 65℃, 70℃ and 80℃) in a borate-NaOH buffer system at pH 9.5 to determine the optimum temperature. The activity corresponding to the optimum temperature was defined as 100%, and the relative activity at the remaining temperatures was calculated. As shown in Figure 4 , the optimum temperature of the parent PA3 was 60℃, and the optimum temperature of the mutant M4 was increased by 5℃.

[0104] (2) Thermal stability

[0105] The purified enzyme solution of the parent and mutant ThAPT3-M4 was diluted to 100 ng / ml, and 200 μL was taken into a 1.5 mL EP tube and incubated at 55℃ and 60℃ for 2 min, 5 min, 10 min, 20 min, 30 min and 60 min, respectively. The residual activity was detected. The enzyme solution without heat treatment was used as a control to calculate the residual relative activity. As shown in Figure 5 , the thermal stability of the mutant ThAPT3-M4 was greatly improved compared with the parent, and more than 60% of the residual activity was retained after 1 h of incubation at 55℃, and more than 40% of the residual activity was retained after 20 min of treatment at 60℃.

[0106] Half-life t 1 / 2 is one of the commonly used parameters for characterizing the thermal stability of enzymes, and the larger the value, the better the thermal stability of the enzyme. It refers to the time required for the initial activity to decrease by 50% at a given temperature, which is calculated by the following formula:

[0107]

[0108] where k d is the inactivation rate constant, which can be obtained by the following formula:

[0109]

[0110] In the formula, A t refers to the residual activity, A0 is the initial activity, and t refers to the treatment time at a given treatment temperature.

[0111] Melting temperature (T m ) refers to the temperature at which 50% of the protein is unfolded. Differential scanning calorimetry (DSC) can be used to determine the T m of a protein.

[0112] t 1 / 2 and T mThe analysis results are shown in Table 2. The thermostability of the mutant ThAPT3-M4 is significantly better than that of the parent. m The value increased by 4℃, at t at 60℃ 1 / 2 It also increased by more than 7 times.

[0113] Table 2. T values ​​of parents and the mutant ThAPT3-M4 m value and t 1 / 2 parameter

[0114]

[0115] (3) Determination of kinetic constants

[0116] 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-M4 exhibits higher catalytic activity and efficiency compared to the parent, but K... m The value did not change significantly.

[0117] Table 3

[0118]

[0119] 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 of alkaline protease, characterized in that, The method comprises the following steps: mutating the Ala at position 345 of the alkaline protease parent PA3 with the amino acid sequence shown in SEQ ID NO: 1 into Gln.

2. A method for improving the thermal stability and catalytic activity of alkaline protease, characterized in that, The method comprises the following steps: mutating the Ala at position 342 and the Ala at position 345 of the alkaline protease parent PA3 with the amino acid sequence shown in SEQ ID NO: 1 into Glu and Gln respectively.

3. A mutant of alkaline protease characterized in that, The amino acid sequence of the alkaline protease mutant is shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

4. A basic protease gene, characterized by, The alkaline protease gene encodes the alkaline protease mutant of claim 3.

5. The alkaline protease gene according to claim 4, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO:

10.

6. A recombinant expression vector comprising the alkaline protease gene of claim 4.

7. A recombinant strain comprising the alkaline protease gene of claim 4.

8. A process for the preparation of alkaline protease characterized by, The method comprises the following steps: transforming a host cell with the recombinant expression vector of claim 6; inducing the host cell to express; isolating and purifying to obtain the alkaline protease.

9. Use of the alkaline protease mutant of claim 3 for hydrolyzing casein.

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