Alkaline protease mutant as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and discloses an alkaline protease mutant as well as a coding gene and application thereof. The alkaline protease mutant has alkaline protease activity; the alkaline protease mutant comprises a mutant T37V compared to a parent alkaline protease having an amino acid sequence as shown in SEQ ID NO: 1, and the alkaline protease variant has at least 97% and less than 100% sequence identity to SEQ ID NO: 1. Compared with parent alkaline protease, the alkaline protease provided by the invention has higher enzyme activity; and the heat resistance and / or the laundry detergent tolerance are / is obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to an alkaline protease mutant and its encoding gene and application. Background Art

[0002] Subtilisin (EC number 3.4.21.62), first obtained from Bacillus subtilisin, belongs to the S8 peptidase family and has a catalytic triad in the order of Asp, His and Ser in its amino acid sequence. It is also known as serine protease or alkaline protease.

[0003] Alkaline proteases are widely used in detergents, leather, silk, feed, medicine, food, and other fields, possessing significant industrial and economic value. They represent the largest proportion of industrial enzymes. Currently, alkaline proteases are the best-selling detergent additives on the market, significantly enhancing the cleaning effectiveness of detergents, particularly against protein-based stains such as blood, sweat, milk, and oil. However, maintaining high enzyme concentration and stability, particularly in detergents, remains a key technical challenge in alkaline protease development. Therefore, genetic engineering to modify alkaline proteases with enhanced activity and resistance to high temperatures and / or detergents is of great significance to the laundry industry. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides an alkaline protease mutant, its encoding gene, and its application. The alkaline protease mutant provided by the present invention can further improve enzyme activity, high temperature tolerance, and / or laundry detergent tolerance, thereby better meeting the needs of the daily chemical detergent industry.

[0005] The present invention provides alkaline protease mutants.

[0006] Specifically, the alkaline protease mutant has alkaline protease activity; compared with the parent alkaline protease with the amino acid sequence shown in SEQ ID NO: 1, the alkaline protease mutant includes the mutation T37V, and the alkaline protease variant has a sequence identity of at least 97% and less than 100% with SEQ ID NO: 1.

[0007] SEQ ID NO:1:AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHP DLNIRGGASFVPGEPSYQDGNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGSSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNS GAGSISYPARYANVMAVGAVDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKYPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

[0008] In some embodiments of the present invention, the parent alkaline protease is derived from Bacillus lentus.

[0009] In some embodiments of the present invention, the alkaline protease mutant, compared with the parent alkaline protease with the amino acid sequence shown in SEQ ID NO: 1, further comprises at least one of the following mutations: G113V, G116M, S182P, Q200Y, S210F, G223A, K229W, S253D, N255D, N255W.

[0010] In some embodiments of the present invention, compared to the parent alkaline protease having the amino acid sequence shown in SEQ ID NO: 1, the alkaline protease mutant comprises at least one of mutations (1)-(20):

[0011] (1)T37V(2)T37V+G113V, (3)T37V+G113V+S253D, (4)T37V+G113V+N255W, (5)T37V+G113V+N 255D, (6)T37V+G113V+S253D+N255D, (7)T37V+G113V+S253D+N255W, (8)T37V+G113V+G116M+ S253D+N255W, (9)T37V+G113V+S182P+S253D+N255W, (10)T37V+G113V+Q200Y+S253D+N255W, (11)T37V+G113V+S210F+S253D+N255W, (12)T37V+G113V+G223A+S253D+N255W, (13)T37V+G1 13V+K229W+S253D+N255W, (14)T37V+G113V+G116M+K229W+S253D+N255W, (15)T37V+G113V+ S182P+K229W+S253D+N255W, (16)T37V+G113V+Q200Y+K229W+S253D+N255W, (17)T37V+G113V +S210F+K229W+S253D+N255W, (18)T37V+G113V+G223A+K229W+S253D+N255W, (19)T37V+G113 V+G116M+G223A+K229W+S253D+N255W, (20)T37V+G113V+S182P+G223A+K229W+S253D+N255W.

[0012] In some embodiments of the present invention, the alkaline protease mutant has improved properties, including increased enzymatic activity and / or increased thermal stability.

[0013] In some embodiments of the present invention, the increased thermal stability comprises increased stability compared to a parent alkaline protease after heat treatment at 50°C for 24 hours.

[0014] The present invention also provides a nucleic acid molecule.

[0015] Specifically, a nucleic acid molecule, which encodes the above alkaline protease mutant.

[0016] The present invention also provides a recombinant expression vector.

[0017] Specifically, a recombinant expression vector comprises the above nucleic acid molecule.

[0018] In some embodiments of the present invention, the carrier of the recombinant expression vector is a plasmid; preferably, the plasmid includes a pBE-S plasmid.

[0019] The present invention also provides a recombinant bacterium.

[0020] Specifically, a recombinant bacterium comprises the above nucleic acid molecule or recombinant expression vector.

[0021] In some embodiments of the present invention, the recombinant bacteria are selected from Escherichia coli cells or Bacillus cells.

[0022] The present invention also provides an enzyme-containing composition.

[0023] Specifically, an enzyme-containing composition comprises the above-mentioned alkaline protease mutant.

[0024] The present invention also provides the application of the alkaline protease mutant.

[0025] Specifically, the alkaline protease mutant is used in detergents, including but not limited to laundry detergents, such as laundry detergents for removing protein stains.

[0026] The invention also provides a detergent.

[0027] Specifically, a detergent comprises the above alkaline protease mutant.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The alkaline protease provided by the present invention has higher enzyme activity and thermal stability than the parent alkaline protease.

[0030] (2) Compared with the parent alkaline protease, the alkaline protease mutants provided by the present invention have been significantly improved in heat resistance and / or laundry detergent tolerance. Most alkaline protease mutants have excellent heat resistance and laundry detergent tolerance, and have better decontamination effects on protein-stained cloth, which can better meet the needs of the daily chemical washing industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the enzyme activity retention rate of the parent alkaline protease and the alkaline protease mutant after treatment at 50℃ for 24h. DETAILED DESCRIPTION

[0032] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0033] Unless otherwise specified, the biological materials, reagents, and devices used in the following examples can be obtained from conventional commercial sources or by known methods. Molecular biology experimental procedures not specifically described in the following examples were performed according to the methods outlined in J. Sambrook's Molecular Cloning: A Laboratory Manual (3rd edition), or according to the kits and product instructions.

[0034] definition

[0035] Alkaline protease: The term "alkaline protease" refers to an enzyme that, under alkaline conditions, can cleave peptide bonds within protein molecules, converting them into small peptides and amino acids. Alkaline protease activity can be determined using methods known in the art (e.g., the Folin method as described in "Protease Preparations GB / T 23527-2009").

[0036] According to the present invention, the variant that demonstrates improved characteristic under at least one tested condition is considered to have improved characteristic compared with the parent alkaline protease.According to purpose of the present invention, in some embodiments of the present invention, for adapting to the requirement of daily chemical washing industry, the improved characteristic is to increase stability.In some embodiments of the present invention, the improved characteristic is the thermostability of increase, for example, the high temperature stability of increase.In some embodiments of the present invention, the improved characteristic is the thermostability of increase and the laundry detergent stability of increase, for example, the laundry detergent tolerance of increase and the high temperature stability of increase.

[0037] Compared to the parent alkaline protease, some alkaline protease mutants of the present invention have higher enzymatic activity; increased stability at 50°C; and enhanced stability in a formulation containing laundry detergent after heat storage at 37°C. It can be understood that compared to the parent alkaline protease, the alkaline protease mutants of the present invention have at least one advantage of higher enzymatic activity, higher heat resistance, or laundry detergent tolerance.

[0038] Parent or parent alkaline protease: The term "parent" or "parent alkaline protease" refers to the alkaline protease having the amino acid sequence shown in SEQ ID NO:1.

[0039] Variant, mutant: The terms "variant" and "mutant" mean a polypeptide having alkaline protease activity comprising a mutation (i.e., substitution, insertion and / or deletion) at one or more (e.g., several) positions relative to the position of the parent alkaline protease shown in SEQ ID NO: 1. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. The mutant of the present invention has at least 20%, e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the alkaline protease activity of SEQ ID NO: 1.

[0040] In a preferred embodiment, corresponding to SEQ ID NO: 1, the alkaline protease variant of the present invention comprises the mutation T37V, wherein the alkaline protease variant has a sequence identity of at least 97% and less than 100% to SEQ ID NO: 1, and wherein the variant has alkaline protease activity. Therefore, the present invention relates to such variants, which have a sequence identity of at least 97%, at least 97.2%, at least 97.4%, at least 97.6%, at least 97.8%, at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% but less than 100% to the polypeptide of SEQ ID NO: 1.

[0041] In a preferred embodiment, corresponding to SEQ ID NO: 1, the alkaline protease variant of the present invention comprises the mutation T37V, wherein the alkaline protease variant has a sequence identity of at least 97% and less than 100% with SEQ ID NO: 1, wherein the variant has alkaline protease activity, and, wherein, compared with the parent alkaline protease having the amino acid sequence as shown in SEQ ID NO: 1, the alkaline protease mutant further comprises at least one of the following mutations: G113V, S182P, G223A, K229W, S253D, N255W, Q200Y, S210F, G116M, N255D. Thus, the present invention relates to such variants having at least 97%, at least 97.2%, at least 97.4%, at least 97.6%, at least 97.8%, at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.

[0042] Coding sequence: The term "coding sequence" or "coding region" means a polynucleotide sequence that specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence can be the sequence of genomic DNA, cDNA, synthetic polynucleotides, and / or recombinant polynucleotides.

[0043] Expression: The term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be measured, for example, to detect increased expression by techniques known in the art, such as measuring the levels of mRNA and / or translated polypeptide.

[0044] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

[0045] When describing mutants, the accepted IUPAC single-letter or three-letter amino acid abbreviations are used for ease of reference.

[0046] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. For example, a substitution of threonine at position 37 with valine is represented as "Thr37Val" or "T37V."

[0047] Example 1: Construction and expression of alkaline enzyme and its mutants

[0048] 1.1 Materials and Reagents

[0049] 1.1.1 Strains and vectors:

[0050] The expression strains containing the alkaline protease apr gene and its mutants, Escherichia coli TOP10, Bacillus subtilis WB600, vector pBE-S, antibiotics, kanamycin, and ampicillin were all purchased from Shanghai Shenggong Company.

[0051] 1.1.2 Enzymes and kits:

[0052] Ultra-fidelity 2× Master Mix PCR polymerase and restriction endonucleases were purchased from New England Biolabs, and plasmid extraction and purification kits were purchased from Shanghai Bioengineering Corporation.

[0053] 1.1.3 Culture medium:

[0054] Escherichia coli culture medium was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB+Amp medium was LB medium with ampicillin added to a final concentration of 100 μg / mL. Bacillus culture medium was TB medium (11.8 g / L tryptone, 23.6 g / L yeast extract, 9.4 g / L K₂HPO₄, 2.2 g / L KH₂PO₄). TB+Kan medium was TB medium with kanamycin added to a final concentration of 20 μg / mL.

[0055] 1.1.4 Chemical reagents:

[0056] Casein was purchased from Sinopharm Chemical Reagent Co., Ltd., and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0057] 1.2 Alkaline protease activity assay

[0058] Alkaline protease activity was determined using the Folin method as specified in the national standard "Alkaline Protease Preparation GB / T 23527-2009." Under certain temperature and pH conditions, the protease hydrolyzes the casein substrate to produce phenolic amino acids (such as tyrosine and tryptophan). Under alkaline conditions, the Folin reagent is reduced to form molybdenum blue and tungsten blue. The absorbance of the solution is measured at a wavelength of 680 nm using a spectrophotometer. The enzyme activity is proportional to the absorbance, and the enzymatic activity of the product can be calculated from this.

[0059] Protease activity is expressed in protease activity units, which is defined as 1 g of solid enzyme powder (or 1 mL of liquid enzyme) hydrolyzing casein for 1 min to produce 1 μg of tyrosine under certain temperature and pH conditions, which is 1 enzyme activity unit and is expressed in U / g (U / mL).

[0060] For gene mutations, the parent sequence and species are disclosed, and subsequent site numbering is based on the parent sequence. The amino acid sequence of the mutated alkaline protease (apr) gene of Bacillus lentus is shown in SEQ ID NO.1.

[0061] 1.3 Expression of alkaline protease and its mutants

[0062] 1.3.1 Alkaline protease apr gene synthesis and vector construction

[0063] The amino acid sequence of the mutated alkaline protease (apr) gene of Bacillus lentus is shown in SEQ ID NO. 1 (denoted as the parent alkaline protease).

[0064] NdeI and SalI restriction sites were introduced at the 5' and 3' ends of the alkaline protease apr gene, respectively, and ligated to the pUC57-amp vector. pUC57-apr was inoculated into LB+Amp medium and cultured overnight. The plasmid was extracted and digested with NdeI and SalI. The target gene fragment was recovered by gel cutting and ligated to the expression vector pBE-S to obtain the expression vector pBE-apr.

[0065] 1.3.2 Mutation Introduction

[0066] Using the aforementioned pBE-apr as a template, PCR was used to introduce the first mutation site, T37V, into the parent alkaline protease apr gene. After digestion with DpnI (DpnI endonuclease), the target fragment was recovered by gel excision. The decomposed product was transformed into Escherichia coli TOP10 competent cells using a chemical conversion heat shock method. Recombinant transformants were verified by bacterial liquid PCR. Plasmids from the verified transformants were extracted and sequenced to identify the corresponding mutants, resulting in the expression vector pBE-apr-M1. Chemical transformation was then performed into WB600 chemical transformation competent cells to obtain the Bacillus recombinant transformant APR-M1.

[0067] Using the above-mentioned pBE-apr-M1 as a template, PCR was used to introduce multiple sites, such as the second, third, or fourth, into the parent alkaline protease apr gene. After digestion with DpnI (DpnI endonuclease), the target fragment was recovered by gel excision. The decomposed product was transformed into Escherichia coli TOP10 competent cells using a chemical conversion heat shock method. The recombinant transformants were verified by bacterial liquid PCR. The plasmids of the verified transformants were extracted and sequenced to identify the corresponding mutants and obtain the corresponding expression vectors. Finally, chemical transformation was used to transform into WB600 chemical transformation competent cells to obtain the corresponding Bacillus recombinant transformants APR-Mx (x is sorted in order).

[0068] 1.3.3 Shake flask fermentation mutant strains

[0069] Use a toothpick to pick the recombinant transformants obtained in step 1.3.2 one by one into a 250 mL shake flask. Add 40 mL of TB medium to each shake flask and incubate at 37°C, 220 rpm, 85% humidity for about 48 hours. Centrifuge and collect the supernatant. Use affinity chromatography to purify the parent alkaline protease and mutants respectively to obtain enzyme solutions of each alkaline protease mutant. The mutation sites of each alkaline protease mutant are shown in Table 1.

[0070] Table 1

[0071]

[0072]

[0073] Example 2: Enzyme activity of alkaline protease mutants

[0074] The enzyme activity was tested at 40°C and pH 10.5. The relative enzyme activity of each alkaline protease mutant was calculated using the parent alkaline protease activity as a control. The results are shown in Table 2.

[0075] Relative enzyme activity refers to the ratio of the enzyme activity of a certain enzyme to the enzyme activity of the parent alkaline protease under certain conditions. The calculation formula for relative enzyme activity is as follows: relative enzyme activity = (enzyme activity of the tested mutant / enzyme activity of the parent alkaline protease) × 100%.

[0076] Table 2

[0077]

[0078]

[0079] As shown in Table 2, the enzyme activity of each alkaline protease mutant in the examples of the present invention is higher than that of the parent alkaline protease, with the highest being 26% higher.

[0080] Example 3: Detection of the Optimal Reaction pH of Alkaline Protease Mutants

[0081] Under the condition of temperature of 40°C, the enzymatic activity of each alkaline protease was measured at pH values ​​of 7.5, pH value of 9.0, pH value of 10.5, and pH value of 12, respectively. The results are shown in Table 3.

[0082] The alkaline protease activity measured at pH 10.5 was used as a control to calculate the relative enzyme activities of the mutant enzymes under different pH conditions.

[0083] Relative enzyme activity refers to the ratio of an enzyme's activity under certain conditions to the enzyme's activity under optimal reaction conditions. The formula for calculating relative enzyme activity is as follows: Relative enzyme activity = (activity of the enzyme being tested / activity of the enzyme under optimal reaction conditions) × 100%. The specific results are shown in Table 3.

[0084] Table 3

[0085]

[0086]

[0087] As shown in Table 3, under alkaline conditions, at pH 10.5, the alkaline protease mutants provided by the present invention exhibited the best enzymatic activity compared to the parent alkaline protease. Furthermore, at pH 12, the alkaline protease mutants provided by the present invention maintained over 88% of their enzymatic activity, which was higher than that of the parent alkaline protease.

[0088] Example 4: Detection of the Optimal Reaction Temperature of Alkaline Protease Mutants

[0089] The enzymatic activities of the parent alkaline protease and the alkaline protease mutants were measured at pH 10.5 at 30°C, 40°C, 50°C, 60°C, and 70°C. The relative enzymatic activities of the enzymes at different temperatures were calculated using the alkaline protease activity measured at 40°C as a control according to the national standard "Alkaline Protease Preparations GB / T 23527-2009."

[0090] Relative enzyme activity refers to the ratio of an enzyme's activity under certain conditions to the enzyme's activity under optimal reaction conditions. The formula for calculating relative enzyme activity is as follows: Relative enzyme activity = (activity of the enzyme being tested / activity of the enzyme under optimal reaction conditions) × 100%. The specific test results are shown in Table 4.

[0091] Table 4

[0092]

[0093] As can be seen from Table 4, the optimal reaction temperature of the alkaline protease mutant provided by the present invention and the parent protease is 60°C.

[0094] In summary, the optimal reaction pH of the alkaline protease mutant provided by the present invention is 10.5, and the optimal reaction temperature is 60° C. This performance gives it an outstanding advantage in the washing industry.

[0095] Example 5: Heat resistance of alkaline protease mutants

[0096] The purified parent alkaline protease and alkaline protease mutant enzyme solutions were heat treated at 50°C for 24 hours, cooled on ice, and then tested for enzyme activity at 40°C and pH 10.5. The enzyme activity retention rate was calculated using the following formula: enzyme activity retention rate = (enzyme activity after heat storage / enzyme activity before heat storage) × 100%. The enzyme activity before heat storage was measured at 40°C and pH 10.5. The heat resistance test results are shown in Tables 5 and Figure 1 shown.

[0097] Table 5

[0098]

[0099] From Table 5 and Figure 1 It can be seen that after the alkaline protease mutant was treated at 50°C for 24 hours, its enzyme activity retention rate was significantly higher than that of the parent alkaline protease, and its heat resistance was significantly improved.

[0100] Example 6: Detergency stability test after 4 weeks of heat storage at 37°C in commercial laundry detergents AF

[0101] Purified parent alkaline protease and alkaline protease mutant enzyme solutions were added to commercially available laundry detergent formulations in a specific ratio and mixed thoroughly to achieve an initial alkaline protease activity of 560 u / mL in each formulation (tested at 40°C, pH 10.5). Samples of the laundry detergent supplemented with the parent alkaline protease and alkaline protease mutant were frozen at -18°C and designated as the week 0 sample. The remaining laundry detergent was then placed in a 37°C oven for 4 weeks.

[0102] Based on the GB / T 13174-2021 standard and JB-02 protein-stained cloth, initial and four-week heat storage values ​​were measured. The removal ratio of a commercial laundry detergent containing the present alkaline protease mutant relative to the original commercial laundry detergent was calculated. The heat storage stability of the parent alkaline protease and the alkaline protease mutant in a commercial laundry detergent formulation was investigated by comparing the initial and four-week heat storage removal ratios. The test results are shown in Tables 6 and 7.

[0103] Table 6. Decontamination ratios of parent alkaline protease and alkaline protease mutants in commercial laundry detergents AC

[0104]

[0105]

[0106] Table 7. Decontamination ratios of parent alkaline protease and alkaline protease mutants in commercial laundry detergent DF

[0107]

[0108]

[0109] From the results in Tables 6 and 7, it can be seen that after 4 weeks of hot storage, the commercial laundry detergent formula containing the alkaline protease mutant of the present invention still has a cleaning ratio greater than 1, performing better than the original commercial detergent and having good laundry detergent tolerance.

[0110] In summary, compared with the parent alkaline protease, the alkaline protease mutants provided by the embodiments of the present invention have higher enzymatic activity, and at least one of higher heat resistance and laundry detergent stability; and most alkaline protease mutants also have excellent heat resistance and laundry detergent tolerance, and higher catalytic activity on proteins, which can better meet the needs of the daily chemical washing industry.

[0111] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. An alkaline protease mutant, characterized in that It has alkaline protease activity; compared with the parent alkaline protease with the amino acid sequence shown in SEQ ID NO: 1, the alkaline protease mutant includes the mutation T37V, and the alkaline protease variant has a sequence identity of at least 97% and less than 100% with SEQ ID NO:

1.

2. The alkaline protease mutant according to claim 1, characterized in that Compared with the parent alkaline protease with the amino acid sequence shown in SEQ ID NO: 1, the alkaline protease mutant further includes at least one of the following mutations: G113V, G116M, S182P, Q200Y, S210F, G223A, K229W, S253D, N255D, and N255W.

3. The alkaline protease mutant according to claim 2, characterized in that Compared with the parent alkaline protease having the amino acid sequence shown in SEQ ID NO: 1, the alkaline protease mutant comprises at least one of mutations (1) to (20): (1)T37V(2)T37V+G113V, (3)T37V+G113V+S253D, (4)T37V+G113V+N255W, (5)T37V+G113V+N 255D, (6)T37V+G113V+S253D+N255D, (7)T37V+G113V+S253D+N255W, (8)T37V+G113V+G116M+ S253D+N255W, (9)T37V+G113V+S182P+S253D+N255W, (10)T37V+G113V+Q200Y+S253D+N255W, (11)T37V+G113V+S210F+S253D+N255W, (12)T37V+G113V+G223A+S253D+N255W, (13)T37V+G1 13V+K229W+S253D+N255W, (14)T37V+G113V+G116M+K229W+S253D+N255W, (15)T37V+G113V+ S182P+K229W+S253D+N255W, (16)T37V+G113V+Q200Y+K229W+S253D+N255W, (17)T37V+G113V +S210F+K229W+S253D+N255W, (18)T37V+G113V+G223A+K229W+S253D+N255W, (19)T37V+G113 V+G116M+G223A+K229W+S253D+N255W, (20)T37V+G113V+S182P+G223A+K229W+S253D+N255W.

4. The alkaline protease mutant according to any one of claims 1 to 3, characterized in that The alkaline protease mutant has improved properties including increased enzyme activity and / or increased thermal stability.

5. The alkaline protease mutant according to claim 4, characterized in that The increased thermostability includes increased stability compared to the parent alkaline protease after heat treatment at 50°C for 24 hours.

6. A nucleic acid molecule, characterized in that Containing the nucleotide fragments shown in (a) and / or (b): (a) a nucleotide fragment encoding the alkaline protease mutant according to any one of claims 1 to 5; (b) A nucleotide fragment that is the reverse complement of (a).

7. A recombinant expression vector, characterized in that: Comprising the nucleic acid molecule of claim 6.

8. A recombinant bacterium, characterized in that Comprising the nucleic acid molecule according to claim 6 or the recombinant expression vector according to claim 7.

9. An enzyme-containing composition, characterized in that The enzyme-containing composition comprises the alkaline protease mutant according to any one of claims 1 to 5.

10. Use of the alkaline protease mutant according to any one of claims 1 to 5 in detergents.

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