CN118685387B - A method for improving the thermal stability of serine protease ScAprE, mutants and applications thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a method for improving the thermal stability of serine protease ScAprE, a mutant and application thereof. The invention carries out molecular improvement on the high-temperature alkaline protease ScAprE so as to improve the thermal stability of the protease, has important significance for improving the comprehensive performance of the protease and reducing the use cost of the protease, and provides an effective technical method for improving the property of the protease.

Description

A method for improving the thermal stability of serine protease ScAprE, mutants and applications

Technical Field

The present invention relates to the field of biotechnology, and in particular to a method for improving the thermal stability of serine protease ScAprE, a mutant and an application thereof.

Background Art

Protease has important application value in the washing, feed, food, brewing and pharmaceutical industries. In the feed industry, scientific supplementation of exogenous protease has a positive effect on controlling feed formula costs, improving protein digestibility, promoting animal intestinal health, improving animal production performance, and reducing environmental pollution.

In order to meet the needs of the feed industry, the ideal protease should have the characteristics of high specific activity, good thermal stability, strong acid resistance, etc. However, the commercial proteases currently on sale have poor thermal stability and cannot meet the production needs of pellet feed. There is an urgent need to develop new heat-resistant feed proteases.

The serine protease ScAprE from Shouchella clausii has the effects of improving animal production performance, protecting animal intestinal health, and reducing feed costs, but its heat resistance cannot meet the production application requirements. Therefore, the discovery and improvement of high temperature and acid-base resistant proteases are of great significance.

Summary of the invention

The object of the present invention is to provide a serine protease ScAprE mutant with improved thermal stability.

Another object of the present invention is to provide a serine protease gene.

Another object of the present invention is to provide the use of the above-mentioned serine protease ScAprE mutant with improved thermal stability.

Another object of the present invention is to provide a method for improving the thermal stability of serine protease ScAprE.

According to the method for improving the thermal stability of the serine protease ScAprE of the present invention, the method comprises subjecting the serine protease ScAprE whose amino acid sequence of the mature protein is shown in SEQ ID NO:1 to the following mutations:

N74D, S126G, S151A, Q176S, V199I, Q200L; or

N18A, R19A, N74D, S85G, V199I, Q200L.

SEQ ID NO: 1:

AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLRIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTWPGSTYASLNGTSMATPHVA GAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

According to the serine protease ScAprE mutant of the present invention, the amino acid sequence of its mature protein is shown in SEQ ID NO:2 or SEQ ID NO:3.

SEQ ID NO:2:

AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLRIRGGASFVPGEPSTQDGNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGGPSPSATLEQAVNSATSRGVLVVAAAGNSGAGSISYPARYANAMAVGATDSNNNRASFSQYGAGLDIVAPGVNILSTWPGSTYASLNGTSMATPHVA GAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

SEQ ID NO:3:

AQSVPWGISRVQAPAAHAAGLTGSGVKVAVLDTGISTHPDLRIRGGASFVPGEPSTQDGNGHGTHVAGTIAALDNSIGVLGVAPGAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNILSTWPGSTYASLNGTSMATPHVA GAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

The serine protease gene of the serine protease according to the present invention encodes the above-mentioned serine protease ScAprE mutant.

The nucleotide sequence of the serine protease gene of the serine protease according to the present invention is shown in SEQ ID NO:4 or SEQ ID NO:5.

SEQ ID NO:4:

ATGAAGAAACCACTTGGAAAAATAGTAGCCTCTACAGCATTGTTAATCTCTGTGGCGTTTTCCTCATCTATTGCTTCTGCTGCAGAGGAAGCCAAAGAGAAATATCTCATTGGGTTCAACGAACAAGAGGCAGTGTCCGAGTTTGTTGAACAAGTCGAAGCAAACGATGAAGTAGCGATACTGTCAGAGGAAGAAGAGGTCGAAATTGAACTTCTGCATGAGTTTGAAACCATACCGGTTTTGTCAGTTGAACTGTCACCT GAAGATGTCGATGCTCTTGAATTA GACCCGGCGATTTCTTACATAGAGGAAGATGCTGAAGTCACAACTATGGCACAGAGCGTACCTTGGGGAATAAGTAGAGTACAAGCGCCGGCCGCACACAACCGTGGACTGACTGGCAGCGGAGTCAAGGTGGCCGTCCTGGACACAGGCATCTCTACACATCCGGATCTGCGTATTAGAGGTGGAGCATCGTTTGTGCCGGGGGAACCGTCTACACAAGATGGGAACGGACATGGCACACACGTCGCAGGAACAATTGCGGCGCT TGATAATAGCATTGGCGTG TTAGGAGTGGCACCGTCCGCGGAGCTGTACGCCGTCAAGGTTTCTTGGCGCTTCAGGCTCAGGCTCCGTGAGCTCAATTGCGCAGGGTTTAGAGTGGGCGGGTAATAACGGCATGCATGTGGCCAACCTTAGCCTTGGCGGACCAAGCCCTAGTGCAACTCTGGAACAGGCGGTAAACAGTGCTACCAGCAGAGGCGTCTTGGTCGTCGCTGCTGCCGGAAACAGTGGTGCGGGAAGTATATCGTATCCGG CGCGCTATGCGAATGCAATGGCAGTAGGAGCCACA GACAGCAATAATAACCGGGCATCTTTCTCACAGTATGGTGCAGGCCTTGACATTGTGGCGCCGGGAGTGAACATATTAAGCACATGGCCAGGAAGCACTTACGCTAGCCTTAATGGCACTTCGATGGCGACACCGCATGTAGCTGGAGCAGCTGCCCTTGTAAAGCAAAAAAACCCGTCATGGTCCAATGTCCAGATCCGCAACCACCTGAAAAATACGGCAACGAGCTTAGGGAGTACTAATCTTTACGGATCTGGCCTT GTCAATGCCGAAGCCGCGACGCGC.

SEQ ID NO:5:

ATGAAGAAACCACTTGGAAAAATAGTAGCCTCTACAGCATTGTTAATCTCTGTGGCGTTTTCCTCATCTATTGCTTCTGCTGCAGAGGAAGCCAAAGAGAAATATCTCATTGGGTTCAACGAACAAGAGGCAGTGTCCGAGTTTGTTGAACAAGTCGAAGCAAACGATGAAGTAGCGATACTGTCAGAGGAAGAAGAGGTCGAAATTGAACTTCTGCATGAGTTTGAAACCATACCGGTTTTGTCAGTTGAACTGTCACCT GAAGATGTCGATGCTCTTGAATTA GACCCGGCGATTTCTTACATAGAGGAAGATGCTGAAGTCACAACTATGGCACAATCAGTTCCGTGGGGCATATCACGCGTTCAGGCACCTGCAGCTCATGCCGCCGGCCTGACAGGATCAGGAGTCAAAGTCGCTGTTTTAGACACGGGGATCAGCACACACCCAGACCTGCGTATCCGTGGAGGAGCAAGTTTCGTTCCGGGAGAACCTAGCACTCAGGACGGGAACGGACATGGAACACATGTTGCTGGCACGATAGCTG CCCTGGATAACTCGATCGGAGTT CTGGGAGTTGCGCCTGGGGCGGAACTGTACGCTGTAAAGGTACTTGGTGCTAGCGGCTCTGGCAGTGTGTCTAGCATAGCGCAGGGACTGGAATGGGCTGGGAATAACGGCATGCATGTGGCTAACCTGTCTTTGGGGTCCCCATCTCCTTCTGCCACTTTAGAACAAGCTGTCAATTCAGCGACAAGTCGCGGGGTCTTGGTCGTCGCTGCAAGCGGCAATTCAGGCGCCGGATCTATTTCGTACCCAGCACGTTA CGCGAACGCAATGGCGGTCGGAGCAACG GACCAAAATAACAATCGCGCGAGCTTTAGCCAGTACGGAGCCGGATTGGACATTGTTGCGCCGGGTGTTAACATTTTGTCTACCTGGCCGGGTAGTACATACGCTTCGCTGAATGGCACAAGCATGGCCACCCCTCATGTCGCCGGTGCTGCTGCGCTTGTTAAACAGAAAAACCCGTCCTGGTCTAATGTGCAGATCCGTAACCACCTTAAGAATACCGCTACGTCACTGGGTAGCACGAATCTTTATGGATCCGGGC TGGTTAATGCCGAAGCTGCGACGAGA.

The present invention performs molecular modification on the high-temperature alkaline protease ScAprE to improve its thermal stability, which is of great significance for improving the comprehensive performance of the protease and reducing the use cost of the protease, and provides an effective technical method for improving the properties of the protease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the thermal stability test of ScAprE and its mutants;

FIG2 shows the enzymatic property detection of recombinant ScAprE and its mutant ScAprEMut1, wherein A: the effect of temperature on enzyme activity; B: the effect of pH on enzyme activity; C: pH stability.

DETAILED DESCRIPTION

The experimental materials and methods involved in the following examples are:

Strains and reagents: Bacillus subtilis SCK6, commercially available; pHT 43 vector; pEASY-Blunt simple vector; high-fidelity DNA polymerase; DNA gel recovery kit; bacterial genome extraction kit and plasmid extraction kit; T4 DNA ligase and restriction endonuclease.

Culture medium:

LB liquid medium: peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L;

LB solid medium: peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, agar powder 15 g/L;

Fermentation medium: soybean meal 10 g, corn flour 5 g, K ₂ HPO ₃ 1 g, gelatin 10 g, total volume 300 mL;

Skim milk powder solid medium: 4% skim milk powder, 2% agar powder;

LBS medium: peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, sorbitol 91.1 g/L;

Electroporation washing medium (SMG): sorbitol 91.1 g/L, mannitol 91.1 g/L, glycerol 100 g/L;

Recovery medium (LBSM): peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, sorbitol 91.1 g/L, mannitol 69.2 g/L.

Example 1 Design, construction and expression of ScAprE multi-point mutants

1.1 Design of ScAprE multi-point mutants

ScAprE (SEQ ID No: 1) is a serine protease of the S8 family from Shouchella clausii . In order to improve the thermal stability of ScAprE, mutation analysis was performed. The specific method is as follows:

The ScAprE homologous sequence was searched by Jackhmmer tool, and the sequence 20 classification model based on MP-Bert was constructed by transfer learning. Then, the ScAprE sequence was randomly masked at 10% of different positions 100,000 times, and the masked sequence was predicted by the sequence model to generate 100,000 mutant sequences. A temperature regression model based on MP-BERT was constructed to test whether the bert-based regression fine-tuning model was better than the model in the literature. These two models were used to predict the above mutant sequences, and the sequences with Ogt value as the main and Tm value as the auxiliary were arranged in descending order. The top five multi-point mutants with higher Tm value and optimal reaction temperature prediction values were selected (I8V, T37P, I43V, T56A, I77V, S139Y, S158A, I159V, I192L, W203Y, A224V, W2 35L; T37P, T56A, H118D, N167S, A168V, S253Y, S259Y; Q107A, H118D, R143K, I159V, Q185N, Q200L, W203Y, K229L, S259A; N74D, S126G, S151A, Q176S, V199I, Q200L; N18A, R19A, N74D, S85G, V199I, Q200L).

1.2 Construction and expression of ScAprE multi-point mutants

According to the codon preference of the Bacillus expression system, the codons of the coding sequences of ScAprE wild-type and mutant proteins were optimized and synthesized, and constructed into the Bam HI site of the expression vector pHT43. The expression plasmids of ScAprE wild-type protein and mutant were transformed into Bacillus SCK6 strain for induced expression. The single clones grown on the transformation plate were spotted on the skim milk powder solid culture medium with the help of sterilized toothpicks. After culturing at 37 ^o C for 30 hours, the formation of transparent circles was observed, and the diameters of the transparent circles of the transformants and the control strains were compared to screen the positive transformants. The positive transformants and the control strain, i.e., the SCK6 strain, were inoculated into LB culture medium respectively. Tetracycline was added to the culture medium of the positive transformants, and tetracycline was not added to the SCK6 culture medium. The culture was shaken at 200 rpm and 37 ^o C. After overnight culture, the inoculation amount was transferred to the fermentation medium at 4% for fermentation culture. After three days of culture, the fermentation broth was collected and the recombinant protease was purified.

The induced expressed protein was purified using a Ni column. The recombinant ScAprE protein and all its mutants were subjected to thermal stability tests. The experimental results showed that the thermal stability of the mutant proteins ScAprEMut1 (SEQ ID NO: 2) and ScAprEMut2 (SEQ ID NO: 3) was significantly improved compared with the wild-type ScAprE protein. After being treated at 70 ^o C for 1 hour, the residual enzyme activity of ScAprE was only 0.1%, while the residual enzyme activity of ScAprEMut2 was still above 65% after being treated at 70 ^o C for 1 hour, showing excellent thermal stability (Figure 1).

Example 2 Thermal stability test of ScAprE multi-point mutants

The alkaline protease activity was determined according to the Folin method described in the National Standard of the People's Republic of China GB/T 23527–2009. 0.5 mL of pH 10.5, 1% casein solution was added to the test tube, preheated at 40 ^o C for 3 min, 0.5 mL of appropriately diluted enzyme solution was added, mixed evenly, reacted at 40 ^o C for 10 min, and 1 mL of 0.4 M trichloroacetic acid solution was added to terminate the reaction. The reaction solution was transferred to a 2 mL EP tube and centrifuged at 12,000 rpm for 10 min. 1 mL of the supernatant was taken, 5 mL of sodium carbonate solution and 1 mL of Folin reagent solution were added in sequence, and after oscillation and mixing, it was placed in a 40 ^o C water bath for color development for 20 min. The absorbance was measured at 680 nm.

In order to detect the comprehensive enzymatic performance of ScAprEMut2, ScAprE and ScAprEMut2 were subjected to enzyme activity assays under different temperature and pH conditions. The results showed that the optimum temperature and pH value of ScAprEMut2 were similar to those of ScAprE, with the optimum temperature being 60 ^o C and the optimum pH being around 11 (Figure 2A, Figure 2B). ScAprEMut2 has stronger acid-base tolerance than the wild-type ScAprE protein and has a wide pH stability range. After being treated in a pH 3.5 buffer for 24 hours, ScAprEMut2 still retained more than 90% of its enzyme activity. After being treated in a pH 12 buffer for 24 hours, more than 85% of its enzyme activity was still retained (Figure 2C).

The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.

Claims (8)

1. A method for improving the thermostability of serine protease ScAprE, comprising mutating serine protease ScAprE having the amino acid sequence of the mature protein as set forth in SEQ ID No. 1 by:

N74D, S126G, S151A, Q176S, V I and Q200L; or (b)

N18A, R19A, N74D, S85G, V199I and Q200L.

2. The serine protease ScAprE mutant is characterized in that the amino acid sequence of the mature protein of the serine protease ScAprE mutant is shown as SEQ ID NO. 2 or SEQ ID NO. 3.

3. A serine protease gene, characterized in that, the serine protease gene encodes the serine protease ScAprE mutant of claim 2.

4. The serine protease gene according to claim 3, wherein the nucleotide sequence of the serine protease gene is shown in SEQ ID NO.4 or SEQ ID NO. 5.

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

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

7. A method of preparing a serine protease, the method comprising the steps of:

transforming a host strain with the recombinant expression vector of claim 5 to obtain a recombinant strain;

Inducing the recombinant strain to express serine protease;

separating and purifying to obtain serine proteinase.

8. Use of the serine protease ScAprE mutant according to claim 2 as feed additive.