CN116064490B

CN116064490B - A method for improving the thermostability of alkaline protease, and mutants ThAPT3-M3 and ThAPT3-M4 and their applications.

Info

Publication number: CN116064490B
Application number: CN202310199722.3A
Authority: CN
Inventors: 罗会颖; 王宵; 秦星; 姚斌; 王亚茹; 黄火清; 柏映国; 苏小运; 王苑; 涂涛; 张�杰; 王晓璐; 张红莲; 于会民
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2023-03-05
Filing date: 2023-03-05
Publication date: 2025-12-12
Anticipated expiration: 2043-03-05
Also published as: CN116064490A

Abstract

This invention relates to the field of genetic engineering, specifically to a method for improving the thermostability of alkaline proteases, and mutants ThAPT3-M3 and ThAPT3-M4, and their applications. Using alkaline protease PA3 as the parent, this invention obtains protease mutants with significantly improved thermostability through site-directed mutagenesis. After treatment at 60°C for 10 min and 20 min, the residual enzyme activity of parental PA3 was 1.9% and 1.6%, respectively. After treatment at 60°C for 10 min, the residual enzyme activities of mutants ThAPT3-M3 and ThAPT3-M4 were 58% and 68%, respectively; after treatment at 60°C for 20 min, the residual enzyme activities were 31% and 45%, respectively. Furthermore, the specific activity of mutant ThAPT3-M4 was increased by 25%. Therefore, the alkaline protease mutants provided by this invention have better thermostability and catalytic activity than the wild type, and have better prospects for industrial applications.

Description

Method for improving thermal stability of alkaline protease, mutant ThAPT-M3, thAPT3-M4 and application

Technical Field

The invention relates to the field of genetic engineering, in particular to a method for improving thermal stability of alkaline protease, mutants ThAPT-M3 and ThAPT-M4 and application thereof.

Background

Serine proteases are important peptide bond hydrolases, and 1/3 of the currently known proteases belong to the serine protease family. The subtilisin family, also known as the serine protease S8 family, is the second largest family of serine proteases. Therefore, the cutting agent has wide cutting specificity and excellent organic solvent stability, and is widely applied to various industrial fields such as food, leather, medical treatment, feed and the like. Proteases have a high market share in the field of enzyme preparations. However, the protease has poor heat stability, and the most widely used commercial protease K in industry has poor stability performance at 60 ℃. Stability of alkaline proteases in high temperature environments is therefore a major bottleneck limiting their further industrial application.

Protein engineering is one of the most effective means for optimizing the enzymatic properties of protease at present, and the stability and catalytic activity of the protease can be efficiently improved by a rational design method of segment substitution or site-directed mutagenesis.

Disclosure of Invention

The invention aims to provide mutants ThAPT-M3 and ThAPT3-M4 with improved thermostability and specific activity, which are obtained by taking Torrubiella hemipterigena-derived alkaline protease PA3 as a parent.

It is still another object of the present invention to provide a gene of the above alkaline protease mutant.

It is still another object of the present invention to provide a recombinant vector comprising the above alkaline protease mutant gene.

It is still another object of the present invention to provide a recombinant strain comprising the above alkaline protease mutant gene.

It is a further object of the present invention to provide a process for preparing an alkaline protease with improved thermostability.

It is a further object of the present invention to provide the use of the protease mutants described above.

The invention takes Torrubiella hemipterigena alkaline protease PA3 as parent to mutate, and obtains alkaline protease mutant with improved heat stability, wherein the amino acid sequence of PA3 is shown as SEQ ID NO. 1. (underlined indicates the corresponding amino acid sequence of the leader peptide)

SEQ ID NO:1:

APSLARREEPAPLLEARGAQAIPGKFIVKLREGSPLAALQQAMSLLGGKADHVFQNVFSGFAASMNPAVIELMRNHPDVEYIEQDGKVNINAYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDYKTRGCPKGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYLMGKDGAVAAGLCAKIAQTATRNVLRNIPAGTINALAFNGNPSG*.

The alkaline protease mutant ThAPT-M3 with improved thermostability is characterized in that 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 amino acid sequence is single point mutation from 345 th Ala mutation of alpha ₈ -helix region to Gln. (underlined indicates the corresponding amino acid sequence of the leader peptide, bold italics indicates the mutant amino acid

SEQ ID NO. 2 (containing leader peptide):

SEQ ID NO. 3 (NO leader peptide):

The alkaline protease mutant ThAPT-M4 with improved thermostability provided by the application has the amino acid sequence shown as SEQ ID NO. 4 or SEQ ID NO. 5, and the mutation site of the amino acid sequence is a double-point combined mutation of alpha ₈ -helix region, wherein the 342 th Ala is mutated to Glu, and the 345 th Ala is mutated to Gln.

SEQ ID NO. 4 (containing leader peptide):

SEQ ID NO. 5 (NO leader peptide):

According to a specific embodiment of the invention, the gene sequence of alkaline protease PA3 is shown in SEQ ID NO.6 or SEQ ID NO. 6.

SEQ ID NO:6:

GCTCCATCCTTGGCTAGAAGAGAAGAACCAGCTCCTTTGTTGGAAGCTAGAGGTG

CTCAAGCTATCCCAGGTAAGTTCATCGTCAAGTTGAGAGAGGGTTCTCCATTGGCT

GCATTGCAACAAGCTATGTCCTTGCTTGGTGGTAAGGCTGACCACGTTTTCCAGAA

CGTTTTCTCTGGTTTCGCCGCCTCTATGAACCCAGCTGTTATTGAGTTGATGAGAA

ACCATCCAGACGTCGAGTACATTGAGCAGGACGGTAAGGTTAACATCAACGCCTA

CACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGG

GTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTA

TCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGAT

CAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGT

ACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCA

AGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGAC

TTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTA

TGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCA

ACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTA

ATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGAC

GACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGG

TACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTC

TATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTGATGGGTAAAGATGGTG

CAGTTGCTGCAGGTTTGTGTGCTAAGATTGCTCAGACTGCCACCAGAAACGTCCT

GAGAAATATTCCAGCTGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCATCTGGTTAA。

The present invention provides a gene encoding the above protease mutant.

According to a specific embodiment of the invention, the coding gene sequences of alkaline protease mutants ThAPT-M3 are shown in SEQ ID NO. 7 and SEQ ID NO. 8.

SEQ ID NO. 7 (containing leader peptide):

SEQ ID NO. 8 (NO leader peptide):

according to a specific embodiment of the invention, the coding gene sequences of alkaline protease mutants ThAPT-M4 are shown in SEQ ID NO 9 and SEQ ID NO 10.

SEQ ID NO 9 (containing leader peptide):

SEQ ID NO 10 (NO leader peptide):

The invention provides recombinant vectors pPICZ alpha A-ThAPT-m 3 and pPICZ alpha A-ThAPT-m 4 containing the alkaline protease mutant coding genes.

The invention also provides recombinant strains GS115-ThAPT3-M3 and GS115-ThAPT3-M4 containing the alkaline protease mutant coding genes.

The present invention also provides a method for preparing a protease mutant with improved thermostability, the method comprising the steps of:

1) Transforming Pichia pastoris GS115 host cells by using a recombinant vector containing an alkaline protease mutant gene to obtain a recombinant strain;

2) Culturing the recombinant strain and inducing alkaline protease expression;

3) Recovering and purifying the recombinant expressed alkaline protease.

The invention has the beneficial effects that:

The invention takes alkaline protease PA3 as a parent, and carries out molecular improvement research on protease through segment replacement and site-directed mutagenesis, thereby obtaining protease mutants ThAPT-M3 and ThAPT-M4 with obviously improved thermal stability. After treatment of the parent PA3 at 60 ℃ for 10min and 20min, the residual enzyme activities were 1.9% and 1.6%, respectively. The residual enzyme activities were 58% and 68% after treatment of mutants ThAPT-M3 and ThAPT3-M4 at 60℃for 10min, respectively, and 31% and 45% after treatment of mutants ThAPT-M3 and ThAPT-M4 at 60℃for 20min, respectively. In addition, the specific activity of the mutant ThAPT-M4 is improved by 25%. Therefore, compared with the wild type, the alkaline protease mutant provided by the invention has better thermal stability and catalytic activity, and has better industrial application prospect.

Drawings

FIG. 1 shows a chart of alkaline protease PA3 and mutant ThAPT-M3, M4 Coomassie protein staining gums;

FIG. 2 shows the specific activities of alkaline protease PA3 and mutants ThAPT-M3 at different temperatures;

FIG. 3 shows the thermostability of alkaline protease PA3 and mutants ThAPT-M3 treated at 60 ℃;

FIG. 4 shows the optimum temperature for alkaline protease PA3 and mutants ThAPT-M4;

FIG. 5 shows the thermostability of alkaline protease PA3 and mutants ThAPT-M4.

Detailed Description

Test materials and reagents:

1. The strain and the vector are characterized in that the expression host is Pichiapastoris GS-115, and the expression plasmid vector is pPICZ alpha A.

2. Enzymes and other biochemical reagents, restriction enzymes.

3. Culture medium:

(1) Coli medium low salt LB (LLB) (1% peptone, 0.5% yeast extract, 0.5% nacl, ph natural);

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

(3) BMGY medium (1% yeast extract, 2% peptone, 1% glycerol, 1.34% YNB,0.00004% biotin, pH Nature);

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

The molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.

EXAMPLE 1 preparation of alkaline protease mutant recombinant vectors pPICZ alpha A-ThAPT-m 3 and pPICZ alpha A-ThAPT-m 4

Cloning basic protease parent PA3 (pre-mutation) sequence fragment (without signal peptide) onto expression vector pPIC-Zalpha A, naming the recombinant vector pPICZ alpha A-ThAPT-wt, using recombinant vector pPICZ alpha A-ThAPT-wt as template, amplifying it by primer carrying mutation site to obtain recombinant vector carrying mutant sequence, naming pPICZ alpha A-ThAPT-m 3 and pPICZ alpha A-ThAPT-m 4.

TABLE 1 specific primers for alkaline protease mutants ThAPT3-m3 and ThAPT3-m4

EXAMPLE 2 construction of alkaline protease mutant expression Strain

(1) Electrotransformation of expression vectors into expression hosts

The expression vector was linearized with the restriction enzyme Dra I, shocked into expression host GS115 competent cells, incubated with sorbitol for 30min and plated on YPD plates with bleomycin resistance (100. Mu.L/100 mL), cultured at 30℃for 2 days.

(2) Screening of transformants with high protease Activity

Single colonies were picked from YPD plates with transformants using sterilized toothpicks and inoculated onto milk double-layer screening solid plates and incubated overnight at 30 ℃. A macroscopic transparent milk hydrolysis ring appears on the milk plate, and the transformant with the largest hydrolysis ring is selected and inoculated in 30mLYPD culture medium.

EXAMPLE 3 expression and purification of recombinant protease mutants

(1) Shake flask level expression of protease mutants ThAPT-M3 and ThAPT-M4

Transformants with higher enzyme activities obtained by screening are inoculated into 300mL of BMGY culture medium according to the inoculation amount of 1%, shake-cultured for 48 hours at 30 ℃ and 200rpm, centrifuged for 5min at 4500rpm, and the supernatant is removed. The suspension was resuspended in 200mLBMMY medium with shaking, and shaking culture was continued at 30℃and 200rpm for 48h. During the cultivation, 0.5% methanol was added to the medium every 24 hours. After the completion of the culture, the supernatant broth was collected by centrifugation at 12000rpm for 10min for purification.

(2) Purification of protease mutants ThAPT-M3 and ThAPT-M4

The collected fermentation broth was protein concentrated using a 5kDa membrane pack to 15mL. The salt ions were removed by dialysis overnight against 20mM citrate-phosphate buffer (pH 5.5) using a 3kDa dialysis bag. Purification was performed by cation exchange chromatography using 20mM citrate-phosphate buffer (pH 5.5) solution A and B. The 1M NaCl linear eluate was subjected to enzyme activity detection and SDS-PAGE electrophoresis analysis, and stained with Coomassie brilliant blue stain G250 (FIG. 1), the mutant was consistent with the parent protein size.

Example 4 enzymatic Property analysis of alkaline protease PA3 and mutants ThAPT-M3

The protease of the invention is subjected to enzymatic property measurement by using Fu Lin Fenfa, and is reacted for 20min at different temperatures by using casein as a substrate, 500 mu L of the substrate (1% W/W) and 500 mu L of enzyme solution with proper dilution, and 1mL of 0.4M trichloroacetic acid solution is added to terminate the reaction. The reaction system is transferred into a 2mL EP tube, centrifuged for 3min at 12000rpm, and then 500. Mu.L of supernatant is absorbed and added into 2.5mL of 0.4M sodium carbonate solution, then 500. Mu.L of Fu Lin Fen reagent is added, color development is carried out for 30min at 40 ℃, and after cooling, 250. Mu.L of color development system is absorbed to read the absorbance at 680 nm. Protease Activity Unit definition the amount of enzyme required to break down the substrate casein to l. Mu. Mol tyrosine per minute under certain conditions is 1 activity unit (U).

(1) Comparison of specific Activity of parent and mutant ThAPT-M3 at different temperatures

In a borax-NaOH buffer system, the specific activities of the parent PA3 and mutants ThAPT-M3 are measured at 55, 60 and 65 ℃ respectively. As shown in FIG. 2, the optimal temperature of mutant M3 was increased from 60℃to 65℃and the enzyme activities at 60℃and 65℃were not much different from those of the wild type. In addition, the specific activity of the mutant M3 at 60 ℃ is improved from 3078.3 +/-93.3U/mg to 3355.426094 +/-55.2U/mg.

(2) Comparison of heat stability of parent and mutant ThAPT-M3 at 60C

The purified enzyme solution of parent and mutant ThAPT-M3 was diluted to 100ng/mL, 200. Mu.L was placed in a 1.5mL EP tube, incubated at 60℃for 10min and 20min, respectively, and the remaining activity was detected. The remaining relative activity was calculated using the enzyme solution without heat treatment as a control. As shown in FIG. 3, the heat stability of the parent PA3 at 60 ℃ is poor, the parent PA3 is completely inactivated after 10min of heat treatment, and the heat stability of the mutant ThAPT-M3 is obviously improved, and 59% and 32% of catalytic activity are respectively maintained after 10min and 20min of heat treatment.

Example 5 enzymatic Property analysis of alkaline protease PA3 and mutants ThAPT-M4

(1) Determination of optimum temperature

In a borax-NaOH buffer system at pH 9.5, the enzyme activities of PA3 and mutants ThAPT-M4 were measured at different temperatures (30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and 80 ℃) to determine the optimum temperature, the activity corresponding to the optimum temperature was defined as 100%, and the relative activities at the rest of the temperatures were calculated. As shown in fig. 4, the optimum temperature of the parent PA3 was 60 ℃, and the optimum temperature of the mutant M4 was increased by 5 ℃.

(2) Thermal stability

The purified enzyme solution of the parent and mutant ThAPT-M4 was diluted to 100ng/mL, 200. Mu.L was placed in a 1.5mL EP tube, incubated at 55℃and 60℃for 2min,5min,10min,20min,30min and 60min, respectively, and the remaining activity was detected. The remaining relative activity was calculated using the enzyme solution without heat treatment as a control. As shown in FIG. 5, the heat stability of the mutant ThAPT-M4 is greatly improved compared with that of the parent, and the mutant ThAPT-M4 can retain more than 60% of residual activity after being incubated at 55 ℃ for 1h and more than 40% of residual activity after being treated at 60 ℃ for 20 min.

Half-life t _1/2 is one of the common characterization parameters of the thermostability of enzymes, and the larger the value, the better the thermostability of the enzyme. It refers to the time required for 50% reduction of the initial activity at a given temperature, calculated from the following formula:

where k _d is the deactivation rate constant, can be obtained by the following formula:

Where A _t denotes the residual activity, A ₀ denotes the initial activity, and t denotes the treatment time at a given treatment temperature.

The heat denaturation midpoint temperature (melting temperature, T _m) refers to the temperature at which the protein unfolds by 50%. Differential Scanning Calorimetry (DSC) can be used to determine the T _m of a protein in general.

The analysis results of T _1/2 and T _m are shown in table 2, the thermal stability of the mutant ThAPT3-M4 is obviously better than that of the parent, the T _m value is improved by 4 ℃, and the T _1/2 at 60 ℃ is also improved by more than 7 times.

TABLE 2T _m values and T _1/2 parameters of the parent and mutant ThAPT-M4

(3) Kinetic constant determination

Casein with the concentration of 0.5, 0.8, 1.0, 1.3, 1.5, 2.0, 2.5, 5.0, 8.0 and 10.0mg/mL is respectively prepared as a substrate, the purified enzyme solution is properly diluted and reacts with the substrates with different concentrations for 10min at the optimal temperature and pH, and the enzyme activity is measured. K _m and V _max were calculated using GRAPHPAD PRISM version 5.01 software to fit the michaelis equation constants. The results are shown in Table 3, where mutants ThAPT-M4 have higher catalytic activity and catalytic efficiency than the parent, but the K _m value is not significantly altered.

TABLE 3 Table 3

The above embodiments are only for explaining the technical solution of the present application, and do not limit the protection scope of the present application.

Claims

1. A method for improving the thermostability of alkaline protease, characterized in that the method comprises the following steps: mutating Ala at position 345 of the parental alkaline protease PA3, whose amino acid sequence is as shown in SEQ ID NO:1, to 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 amino acid sequence of the parental alkaline protease PA3, as shown in SEQ ID NO:1, from Ala at position 342 to Glu and from Ala at position 345 to Gln.

3. An alkaline protease mutant, characterized in that the amino acid sequence of the alkaline protease mutant is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

4. An alkaline protease gene, characterized in that 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 as 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 method for preparing alkaline protease, characterized by comprising the following steps:

Transform the host cell with the recombinant expression vector of claim 6;

Inducing host cell expression;

Alkaline protease was isolated and purified.

9. The application of the alkaline protease mutant of claim 3 to hydrolyze casein.