Alkaline protease mutant thapt3-m10 with improved optimum temperature, thermal stability and self-degradation resistance and application thereof

Abstract

The application relates to the field of agricultural biotechnology, in particular to a basic protease mutant ThAPT3-M10 with improved optimal temperature, thermal stability and self-degradation resistance and application. The application takes the basic protease ThAPT3-M9 as a parent, introduces three mutation sites in a surface loop region by a site-directed mutation method, and the optimal temperature of the mutant ThAPT3-M10 is increased from 65 DEG C to 70 DEG C. m The t 1 / 2 From 62.4 min to 111.8 min (about 1.8 times). In addition, the self-degradation phenomenon of ThAPT3-M10 at 70 DEG C is obviously improved compared with the parent. Therefore, the basic protease mutant provided by the application has better thermal stability and self-degradation resistance compared with the parent, and has a better industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to a ThAPT3-M10 alkaline protease mutant with improved optimal temperature, thermal stability and self-degradation resistance and application thereof. 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 accounts for a high market share in the enzyme preparation field. However, the thermal stability of protease is poor, and the most widely used commercial protease Proteinase K in industry has poor stability at 60℃. Therefore, the stability of alkaline protease at high temperature is the main bottleneck limiting its further industrial application.

[0003] Unlike other enzymes, proteases will undergo undesirable self-degradation reaction with themselves as substrates. The self-degradation reaction seriously affects the transportation, storage and application of proteases. Therefore, improving the self-degradation phenomenon of proteases and improving the stability of proteases by means of protein engineering is an effective strategy to reduce production costs. SUMMARY

[0004] The present application aims to provide a ThAPT3-M10 alkaline protease mutant with improved optimal temperature, thermal stability and self-degradation resistance, which is obtained by mutating ThAPT3-M9 as a parent.

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

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

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

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

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

[0010] The present application mutates ThAPT3-M9 as a parent to obtain a ThAPT3-M10 alkaline protease mutant with improved optimal temperature, thermal stability and self-degradation resistance, wherein the amino acid sequence of ThAPT3-M9 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 AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDYKTRGCPKGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYLMTLNRTTAANACEKIQQTATLNVLRNIPEGTINALAFNGNPNG*.

[0013] The alkaline protease mutant ThAPT3-M10 with improved optimum temperature, thermal stability and self-degradation resistance according to the present application, wherein the amino acid sequence of the mutant is shown in SEQ ID NO: 2, and the mutation sites of the amino acid sequence are: the 222nd amino acid is mutated from S to N, the 223rd amino acid is mutated from M to L, and the 280th amino acid is mutated from V to N. (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 the leader peptide sequence):

[0015]

[0016] SEQ ID NO: 3 (not containing the leader peptide sequence):

[0017]

[0018] According to the specific embodiment of the present application, the gene sequence of the parent alkaline protease ThAPT3-M9 is shown in SEQ ID NO: 4.

[0019] SEQ ID NO. 4:

[0020] GCTCCATCCTTGGCTAGAAGAGAAGAACCAGCTCCTTTGTTGGAAGCTAGAGGTGCTCAAGCTATCCCA GGTAAGTTCATCGTCAAGTTGAGAGAGGGTTCTCCATTGGCTGCATTGCAACAAGCTATGTCCTTGCTTGGTGGTAA GGCTGACCACGTTTTCCAGAACGTTTTCTCTGGTTTCGCCGCCTCTATGAACCCAGCTGTTATTGAGTTGATGAGAA ACCATCCAGACGTCGAGTACATTGAGCAGGACGGTAAGGTTAACATCAACGCCTACACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGGGTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTATCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGATCAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGTACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCAAGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGACTTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTATGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCAACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTAATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGACGACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGGTACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTCTATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTGATGACTTTGAACAGAACTACTGCTGCCAACGCATGTGAAAAGATTCAACAGACTGCCACCTTGAACGTCCTGAGAAATATTCCAGAAGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCAAACGGTTAA

[0021] The present application provides a gene encoding the above-mentioned protease mutant.

[0022] According to the specific embodiment of the present application, the gene sequence encoding the alkaline protease mutant ThAPT3-M10 is shown in SEQ ID NO: 5 or SEQ ID NO: 6.

[0023] SEQ ID NO: 5 (containing the sequence encoding the leader peptide):

[0024]

[0025]

[0026] SEQ ID NO: 6 (not containing the sequence encoding the leader peptide):

[0027]

[0028] The present application provides a recombinant vector pPICZ alpha A-ThAPT3-m10 containing the gene encoding the above-mentioned alkaline protease mutant.

[0029] The present application also provides a recombinant strain GS115-ThAPT3-M10 containing the gene encoding the above-mentioned alkaline protease mutant.

[0030] The present application also provides a method for preparing a protease mutant with improved thermal stability, which comprises the following steps:

[0031] 1) transforming a host cell with a recombinant vector containing the gene encoding the above-mentioned alkaline protease mutant to obtain a recombinant strain;

[0032] 2) culturing the recombinant strain to induce the expression of alkaline protease;

[0033] 3) recovering and purifying the recombinant expressed alkaline protease.

[0034] The present application has the following advantages:

[0035] The present application takes alkaline protease ThAPT3-M9 as the parent, introduces three mutation sites in the surface loop region by site-directed mutagenesis, which are the mutation of the 222nd amino acid from S to N, the mutation of the 223rd amino acid from M to L, and the mutation of the 280th amino acid from V to N, to obtain a combination mutant ThAPT3-M10. Through enzymatic property detection, the optimum temperature of the mutant ThAPT3-M10 is increased from 65℃ to 70℃. m The T value is increased by 3℃ compared with the parent. 1 / 2 The t value at 60℃ is increased from 62.4min to 111.8min (≈1.8 times). In addition, the self-degradation phenomenon of ThAPT3-M10 at 70℃ is obviously improved compared with the parent. Therefore, the alkaline protease mutant provided by the present application has better thermal stability and self-degradation resistance compared with the parent, and has better industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1Optimum temperature of parent ThAPT3-M9 and mutant ThAPT3-M10 was shown.

[0037] Figure 2 Thermostability of parent ThAPT3-M9 and mutant ThAPT3-M10 was shown.

[0038] Figure 3 Self-degradation of parent ThAPT3-M9 incubated at 70℃ for different time was shown.

[0039] Figure 4 Self-degradation of mutant ThAPT3-M10 incubated at 70℃ for different time was shown. DETAILED DESCRIPTION

[0040] Test materials and reagents:

[0041] 1. Strains and vectors: expression host was Pichia pastoris GS115, and expression plasmid vector was pPICZαA.

[0042] 2. Enzymes and other biochemical reagents: restriction endonuclease.

[0043] 3. Culture medium:

[0044] (1) Escherichia coli culture medium low salt LB (LLB) (1% peptone, 0.5% yeast extract, 0.5% NaCl, pH natural);

[0045] (2) Pichia pastoris culture medium YPD (1% yeast extract, 2% peptone, 2% glucose, pH natural)

[0046] (3) BMGY culture medium (1% yeast extract, 2% peptone, 1% glycerol, 1.34% YNB, 0.00004% biotin, pH natural);

[0047] (4) BMMY culture medium (1% yeast extract, 2% peptone, 0.5% methanol, 1.34% YNB, 0.00004% biotin, pH natural).

[0048] In the following examples, the molecular biology experimental methods not specifically described were carried out according to the specific methods listed in the book "Molecular Cloning Experiment Guide" (third edition) J. Sambrook, or according to the reagent kit and product instructions.

[0049] Example 1 Preparation of alkaline protease mutant recombinant vector pPICZαA-ThAPT3-m10

[0050] The vector pPICZαA-ThAPT3-m9 carrying the parent alkaline protease was used as a template, and a primer carrying a mutation site was used to amplify the template to obtain a recombinant vector carrying the mutant sequence, which was named pPICZαA-ThAPT3-m10.

[0051] Table 1. Specific primers of alkaline protease mutant ThAPT3-m10

[0052]

[0053] Example 2 Construction of an alkaline protease mutant expression strain

[0054] (1) Electroporation of an expression vector into an expression host

[0055] The expression vector was linearized using the restriction enzyme Dra I and was introduced into the competent cells of the expression host GS115 by electroporation. After 30 min of sorbitol incubation, the cells were plated on a YPD plate containing 100 μL / 100 mL of bleomycin and were cultured at 30°C for 2 days.

[0056] (2) Screening of a high protease activity transformant

[0057] Single colonies were picked from the YPD plate with the transformant using a high-temperature sterilized toothpick and were inoculated on a double-layer solid milk screening plate. The plate was cultured at 30°C overnight. A visible transparent circle of milk hydrolysis appeared on the milk plate, and the transformant with the most obvious hydrolysis circle was inoculated in 30 mL of YPD medium.

[0058] Example 3 Expression and purification of a recombinant protease mutant

[0059] (1) Shake flask level expression of the protease mutant ThAPT3-M10

[0060] The transformant with high enzyme activity screened was inoculated in 300 mL of BMGY medium at a 1% inoculation amount. After 48 h of culture at 30°C and 200 rpm, the supernatant was removed by centrifugation at 4500 rpm for 5 min. The cells were resuspended in 200 mL of BMMY medium and were further cultured at 30°C and 200 rpm for 48 h. During the culture, 0.5% methanol was added to the medium every 24 h. After the culture, the fermentation broth was collected by centrifugation at 12000 rpm for 10 min, and was used for purification.

[0061] (2) Purification of the protease mutant ThAPT3-M10

[0062] 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). Purification was performed using cation exchange chromatography using 20 mM citric acid-phosphate buffer (pH 5.5) A and B. Enzyme activity detection and SDS-PAGE electrophoresis analysis were performed on a 1 M NaCl linear eluent, and staining was performed using Coomassie Brilliant Blue staining solution G250, and the protein size of the mutant was consistent with that of the parent.

[0063] Example 4 Enzymatic property analysis of the parent ThAPT3-M9 and the three-point superimposed mutant ThAPT3-M10

[0064] The enzymatic properties of the protease of the present application were determined using the Folin phenol method, with casein as the substrate, 500 μL of the substrate (1% W / W) and 500 μL of the enzyme solution appropriately diluted, and the reaction was carried out 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 then 500 μL of the supernatant was taken and added to 2.5 mL of 0.4 M sodium carbonate solution. Then 500 μL of Folin phenol reagent was added, and the color was developed at 40°C for 30 min. After cooling, 250 μL of the color developing system was taken to read the absorbance at 680 nm. The definition of protease activity unit: the amount of enzyme required to decompose 1 μg of casein to generate tyrosine per minute under certain conditions is defined as 1 unit (U).

[0065] (1) Determination of the optimum temperature of the parent M9 and the three-point superimposed mutant M10

[0066] In a borax-NaOH buffer system with pH 9.5, the purified enzyme solution was diluted by an appropriate number of times, and the catalytic activity of the parent M9 and the mutant M10 was determined at 30, 40, 50, 60, 65, 70, 75°C and 80°C, respectively. The results are shown in Figure 1 The optimum temperature of the mutant M10 was increased by 5°C compared with the parent M9, and the relative activity at 75°C was also significantly higher than that of the parent.

[0067] (2) Comparison of the thermal stability of the parent M9 and the three-point superimposed mutant M10 at 60, 65 and 70°C

[0068] The purified enzyme solution of the parent and the mutant was diluted to 100 ng / ml, and 200 μL was taken in a 1.5 mL EP tube. After incubation at 60, 65 and 70°C 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, and the residual relative activity was calculated. The results are shown in Figure 2As shown, the mutant has a significant improvement in thermal stability compared to the parent, it can retain more than 75% of the activity after incubation at 60℃ for 1h, while the parent only retains 50% of the activity under the same conditions; the parent almost loses all activity after incubation at 65℃ for 1h, while the mutant retains 30% of the activity under the same conditions; the parent also has a significant advantage over the parent in the remaining enzyme activity after short-term incubation at 70℃.

[0069] (3) Thermodynamic and kinetic stability parameters of the parent M9 and the three-point superimposed mutant M10

[0070] Half-life t 1 / 2 is one of the commonly used characterization parameters of enzyme thermal stability, 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:

[0071]

[0072] where k d is the inactivation rate constant, which can be obtained by the following formula:

[0073]

[0074] In the formula, A t refers to the remaining activity, A0 is the initial activity, and t refers to the treatment time at a given treatment temperature.

[0075] Melting temperature (T m ) is the temperature at which 50% of the protein unfolds. Differential scanning calorimetry (DSC) can be used to determine the T m of a protein. The results are shown in Table 2, the T m value and t 1 / 2 at 60℃ of the parent are significantly improved compared to the parent. Among them, the T m value is increased by 3.1℃, and the t 1 / 2 at 60℃ is increased from 62.4min to 111.8min (≈1.8 times).

[0076] Table 2 T m values and t 1 / 2 parameters of the parent and the mutants ThAPT3-M6, M7 and M8

[0077]

[0078] (4) Differences in the extent of autodegradation of the parent M9 and the three-point superimposed mutant M10 at high temperatures

[0079] The concentration of heat treatment of parent and mutant M10 was 0.5 mg / mL, the treatment system was 50 μL, and the treatment temperature was 70℃. The treatment time was 2, 5, 10, 20, 30, and 60 min, respectively. After heat treatment, protein denaturation electrophoresis loading buffer was immediately added to observe the autodegradation of parent and mutant at high temperature. The results are shown in Figure 3 and Figure 4 As shown, the intact protease band of parent and mutant gradually disappeared at 70℃ as the incubation time was prolonged. After incubation at 70℃ for 60 min, the protein band of parent M9 could not be observed, and it was completely degraded. However, the intact protease band of mutant M10 could still be observed at the protein electrophoresis level after the same incubation time. Therefore, the autodegradation resistance of mutant M10 formed by three-point combined mutation of the surface loop region was greatly improved at high temperature. The improvement of autodegradation resistance of M10 can effectively prolong its action time in the enzymatic reaction process.

[0080] The above examples are only used to explain the technical solutions of the present application, and do not limit the protection scope of the present application.

Claims

1. A method for improving the temperature optimum, thermal stability and resistance to autodegradation of an alkaline protease, characterized in that, The method comprises the following steps: mutating the amino acid at position 222 of the alkaline protease parent Th The amino acid at position 222 of APT3-M9 is mutated from S to N, the amino acid at position 223 is mutated from M to L, and the amino acid at position 280 is mutated from V to N.

2. A mutant of alkaline protease having improved optimum temperature, thermal stability and resistance to self-degradation Th APT3-M10, characterized in that, The alkaline protease mutant Th The amino acid sequence of APT3-M10 is set forth in SEQ ID NO: 2 or SEQ ID NO:

3.

3. A basic protease gene, characterized in that, A mutant alkaline protease having increased temperature optimum, thermal stability and resistance to autodegradation according to claim 2 Th APT3-M10.

4. The alkaline protease gene according to claim 3, characterized in that, The nucleotide sequence of the gene is shown as 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 preparing alkaline protease having improved optimum temperature, thermostability and resistance to autodegradation, characterized by, The method comprises the following steps: transforming a host cell with the recombinant vector of claim 5 to obtain a recombinant strain; culturing the recombinant strain to induce expression of alkaline protease; recovering and purifying the recombinantly expressed alkaline protease.

8. The basic protease mutant of claim 1 Th Use of APT3-M10 to hydrolyze casein.

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