CN118256474A - Alkaline protease mutant and application thereof - Google Patents

Abstract

The invention belongs to the technical field of protein engineering and provides an alkaline protease mutant and application thereof in liquid detergents. The parent protease of the alkaline protease mutant is a protease of bacillus circulans, and the alkaline protease mutant comprises the following amino acid substitutions: D208S/R210G/S268G/Q350R/N353D/V373I/E376F, wherein the position corresponds to the amino acid sequence of SEQ ID NO:1, and a polypeptide having an amino acid position. The invention can better retain activity under extreme conditions than the parent protease when used as a detergent, and can be used at higher temperature and in stronger alkaline environment, so that the protease can be better applied to the industrial field, in particular to the detergent industry. The application of the mutant is beneficial to reducing the production cost of the alkaline protease and lays a foundation for better adapting to industrial production.

Description

Alkaline protease mutant and application thereof

Technical Field

The invention belongs to the technical field of protein engineering, and particularly relates to an alkaline protease mutant and application thereof.

Background

Alkaline proteases (AlkalineProtease) belong to the class of serine proteolytic enzymes, serine proteolytic enzymes are enzymes with an active site serine (ec 3.4.21) that initiates the hydrolysis of the peptide bonds of proteins, mainly subtilisins (ec 3.4.21.62), which enzymes belong to the S8 peptidase family of the MEROPS classification scheme, members of the peptidase S8 family having in their amino acid sequence catalytic triplets with the enzyme catalytic active sites Asp, his and Ser. The alkaline protease hydrolyzes protein peptide bonds, ester bonds and amide bonds under neutral to alkaline conditions, and the optimal pH is generally 8-11. The enzyme species was found in the pig pancreas at the earliest. Alkaline proteases are widely found in microorganisms, plants and animals. The main application fields of alkaline protease include the fields of light industry such as detergents, feed, medicines, leather, soybean processing, breweries, meat tenderization, waste management, photography and diagnosis. The microorganism producing alkaline protease is mainly separated from alkaline environments such as saline-alkali lakes, deep sea, sand and the like. In recent decades, with the continuous isolation and purification of new alkaline protease producing strains, the research of alkaline proteases has been greatly progressed. To date, bacillus, actinomycetes (Actinomyces) and fungi have all been reported to produce alkaline proteases. The strains currently used for industrial production are mainly Bacillus licheniformis, bacillus subtilis, bacillus alkalophilus, bacillus amyloliquefaciens, etc. (Tekinetal.PolJ. Microbiol,2017, 66 (1): 39-56). Alkaline protease alone accounts for 25% of the global enzyme market (Mikkelsen MLal. Foodand chemical Toxicology, 2015:07-21). The protease has a great proportion in the enzyme industry, the sales amount of the protease in the global enzyme preparation market reaches 60%, and the alkaline protease accounts for 35%.

Alkaline proteases are the enzyme preparations that were first used in detergent products. Protein macromolecular substances such as blood, milk, egg, fruit juice, sweat stain, coffee and the like in stains have poor water solubility, common surfactants and other builders are difficult to remove, and alkaline protease can decompose the protein macromolecular substances into small molecular peptide bonds which are easy to dissolve in water and then into amino acids, so that the protein macromolecular substances can be easily washed out. Alkaline proteases in detergent products gradually lose enzymatic activity from enzymatic hydrolysis in the absence of stabilizers and inhibitors. To improve the stability of proteases in detergents, novelin has developed a highly potent protease inhibitor 4-FPBA (4-formyl-phenyl-boronicacid) for use in stabilizing detergent alkaline protease products, and subsequently has focused on developing inhibitors based on peptide aldehydes.

Alternatively, the amino acid change has the property: altering the physicochemical properties of the polypeptide. For example, amino acid changes may improve the thermostability of the polypeptide, change substrate specificity, change the pH optimum, and the like. Essential amino acids in polypeptides can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunninghametal. Science1989, 244.4908:1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the protease activity of the resulting mutant molecules is tested to identify amino acid residues that are critical to the activity of the molecule, see Hilton et al, results of research (Hilton et al, journal of biological chemistry,1996, 271.9:4699-4708.). The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by nuclear magnetic resonance crystallography (crystallography), electron diffraction, or photoaffinity labeling, along with mutation of the putative contact site (contractsite) amino acid, reference to Devosetal et al (Devosetal. Science,1992, 255.5042:306-312.). Iterative saturation mutagenesis is also a good method for efficient screening of protease mutations, reference (Reetzetal. Nature protocols,2007, 2.4:891-903), efficient protein engineering of alcohol dehydrogenases using iterative saturation mutagenesis (Sun, zhoutong, et al, ACSCatalysis,2016, 6.3:1598-1605.). For BPN (SEQ ID NO: 2), a catalytic triplet comprising amino acids S221, H64 and D32 is necessary for the protease activity of the enzyme.

More and more commercial proteases are protein engineered variants of naturally occurring wild-type proteases, such as Everlase, relase, ovozyme, polar zyme, liquanase, liquanaseUlta and Kannase (novelian (Novozymesa/s)), purafast, purafoctOXP, FN3, FN4 and Eraser, [ Excellase ] (jenergide international (GenencorInternational, inc.)). The current wild-type alkaline proteases cannot meet the washing requirements under different conditions, the performance of alkaline proteases is to be further improved, the washing conditions such as temperature and pH are changed with time, and many stains are still difficult to completely remove under the traditional washing conditions, and furthermore, the conditions in washing can lead to enzyme inactivation (such as pH, temperature or chelation instability), thereby leading to loss of washing performance during the washing cycle. Thus, there is still a great demand for alkaline proteases which have high washing stability and good washing performance.

The market demand of the alkaline protease in China is about 15 hundred million yuan, and the main reasons are that the production capacity of the alkaline protease production strains in our country is poor, the fermentation activity of the enzyme is low, the specific activity of the enzyme is low and the washing application effect is poor. Therefore, protein engineering and screening of alkaline protease with high activity, high stability and high titer and construction of high-yield engineering bacteria become research hotspots at home and abroad.

The catalytic activity, acid-base stability, heat stability, substrate specificity and expression titer of the enzyme can be effectively improved by protein engineering means (JohannesTWet. Curr. Microbiol,2006, 9:261-267). The protein engineering quality is improved and applied to the function improvement of the enzyme, opens up a brand-new way, and has great success in the fields of industry, agriculture, medicine and the like.

Disclosure of Invention

In view of the above, the present invention provides an alkaline protease mutant, which aims to obtain mutant proteins, improve alkali resistance, surfactant resistance and stability of the mutant proteins, and enable the mutant proteins to be better suitable for the detergent industry. The invention provides protein engineering modification and mutant of alkaline protease mutant for washing, which can improve the enzyme activity of alkaline protease in washing liquid under alkaline pH condition, thereby improving the washing effect of the detergent, laying a foundation for better adapting to industrial production, and being beneficial to the wide application of alkaline protease in washing field.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention is realized by the following technical scheme:

A mutant alkaline protease for washing, wherein the parent protease of the mutant alkaline protease is alkaline protease AP of bacillus circulans (Bacilluscirculans), genBank is SPU21234.1, and corresponds to amino acid sequence seq id no:1. the invention relates to an alkaline protease mutant, which corresponds to the amino acid sequence SEQ ID NO:2 comprising an amino acid sequence having at least 90% identity to seq id No. 1 and comprising a substitution of an amino acid at a position selected from the group consisting of: 208, 210, 268, 350, 353, 373, 376.

In some embodiments of the invention, the amino acid sequence of the mutant has at least 91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identity as compared to SEQ ID NO. 2.

In some more specific embodiments, the amino acid sequence of the mutant has at least 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or at least 99.9% identity as compared to seq id No. 2.

The invention also provides a liquid detergent composition comprising the protease mutant for washing.

The liquid detergents prepared according to the present invention use the MGDA and STPP standards in combination.

The invention also provides a preparation method of the alkaline protease mutant, which comprises the following steps:

Step 1: obtaining a DNA molecule encoding an alkaline protease mutant comprising an amino acid sequence having at least 90% identity to seq id No. 1.

Step 2: fusing the DNA molecule obtained in the step 1 with an expression vector, constructing a recombinant expression vector, and transforming a host cell;

Step 3: inducing host cells containing the recombinant expression vector to express the fusion protein, and separating and purifying the expressed fusion protein.

In some embodiments of the invention, the alkaline protease mutants described in step 1 comprise amino acid substitutions in the group consisting of: D208S, R210G, S268G, Q350R, N353D, V373I, E376F.

In some embodiments of the invention, the host cell described in step 2 is bacillus subtilis.

In some embodiments of the invention, the host cell described in step 2 is Bacillus licheniformis (Bacilluslicheniformis).

The invention also provides application of the alkaline protease mutant in liquid detergents.

The invention is based on alkaline protease PB92 and provides a combined mutant comprising 7 mutation sites in D208S, R210G, S268G, Q350R, N353D, V373I, E376F, which alkaline protease mutant is named JKX02. Alkaline protease mutant JKX02 has better retention activity than the parent protease under extreme conditions when used as a detergent, and can be used at higher temperatures and in a more alkaline environment, tests show that: on the premise of not adding any protective agent and stabilizer, the alkaline protease and the mutant thereof are kept at 50 ℃ for half an hour, so that the residual activity of the alkaline protease mutant JKX is obviously higher than that of the wild type, even if the alkaline protease is kept for a longer time, the residual activity of the mutant is always higher than that of the wild type. Without any protective and stabilizing agents, alkaline protease and mutants thereof were incubated at different pH values for 1 hour, and it was evident that JKX mutants (D208S, R210G, S268G, Q350R, N353D, V373I, E376F) had higher residual activity than wild-type enzyme at pH values above 9. On the premise of not adding any protective agent and stabilizer, the alkaline protease and the mutant JKX thereof have the same addition amount, and the alkaline protease mutant JKX has better stability compared with the wild type when being added in an MGDA and STPP washing system, and the good performance of the alkaline protease mutant JKX is beneficial to expanding the application range of the alkaline protease, thereby laying a foundation for better adapting to industrial production.

Detailed Description

The method of the present invention will be further described with reference to examples, in which the experimental method without specific conditions are not specified, and may be performed under conventional conditions, such as those described in molecular cloning Experimental guidelines written in J.Sambucus (Sambrook, 2001), et al, or under conditions recommended by the manufacturer. The present invention may be better understood and appreciated by those skilled in the art by reference to examples. The method of practicing the invention should not be limited to the specific method steps described in the examples of the invention.

The following examples are set forth to provide a better illustration of the present invention and are intended to provide a better understanding and appreciation of the invention by those skilled in the art. The protection of the invention and the scope of the claims are not limited to the cases provided.

Labeling of alkaline protease mutants: "amino acid substituted at the original amino acid position" is used to denote the mutated amino acid in the alkaline protease mutant. As shown in S259K, the amino acid at position 259 is replaced by lys (K) from Ser (S) of the original alkaline protease, and the number at the position corresponds to the number in SEQ ID No.1 of the accessory sequence Listing.

For the culture medium related in the embodiment of the invention, the specific formula is as follows:

LB liquid medium: 1% of tryptone, 0.5% of yeast powder and 1% of NaCl;

LB plate: 1% of tryptone, 0.5% of yeast powder, 1% of NaCl and 2% of agar;

solution a:0.4g of yeast extract, 0.08g of casein hydrolysate, was dissolved in 40mL of water.

Solution B:5g of glucose, dissolved in 10mL of water.

Solution C:4.8gKH ₂PO₄,11.2gK₂HPO₄,0.16gMgSO₄•7H₂ O,0.8g trisodium citrate, 1.6g (NH ₄)₂SO₄, dissolved in 200mL water).

Solution D:0.9gMnCl ₂•4H₂ O,1.415g of sodium ,0.68gFeSO₄•7H₂O,13.45mgCuCl₂•2H₂O,23.5mgZnSO₄•7H2O,20.2mgCoCl₂•6H₂O,12.6mg permanganate of boric acid, 0.855g of sodium tartrate, in 500mL of water.

Solution E:2.16g MgCl2.6H2O in 20mL water.

Solution F:147mgCaCl2 in 20mL of water.

Solution G:36.5g sorbitol, dissolved in 100mL water.

GM i: 10mL of solution A,1.5mL of solution B,25mL of solution C,100uL of solution D,25mL of solution G, and sterilized water was added to 100mL.

GMII:98mLGM I, 1mL of solution E,1mL of solution F, and mixing.

GM iii: 9mLGM II, 1mL glycerol.

Seed culture medium: yeast extract 0.5%, tryptone 0.5%, glucose 1%, K ₂HPO₄ 1.8.8%;

fermentation medium: 1-2% of yeast powder, 2-5% of bean cake powder, 5-10% of maltodextrin, 0.1-0.5% of sodium citrate and 0.5-2% of CaCl ₂0.1~0.5%,MgSO₄0.1~0.5%,K₂HPO₄.

The method for measuring the enzyme activity of the alkaline protease in the implementation of the invention can adopt the following method:

method for measuring enzyme activity of alkaline protease:

The method for measuring the enzyme activity of alkaline protease comprises the following steps: reference is made to the GB/T23527-2009 annex B Fulin method, the specific reaction procedure being as follows.

The protease hydrolyzes casein substrate under certain temperature and pH condition to generate amino acid containing phenolic group (such as tyrosine, tryptophan, etc.), and in alkaline condition, folin reagent (Folin) is reduced to generate molybdenum blue and tungsten blue, and spectrophotometry is used to measure absorbance of solution at 680 nm. The enzyme activity is directly proportional to the absorbance, whereby the enzyme activity of the product can be calculated. The protease activity is defined as 1g of solid enzyme powder (or 1ml of liquid enzyme) and 1 mu g of tyrosine is produced by hydrolyzing casein for 1min under the conditions of a certain temperature and a certain pH value, namely 1 enzyme activity unit is expressed as mu/g (mu/ml).

Reagents and solutions

(1) Forskolin (Folin) reagent (Fu Linshui =1:2); (2) 42.4g/L sodium carbonate solution; (3) 0.5mol/L sodium hydroxide solution; (4) boric acid buffer (pH 10.5); (5) 10.0g/L casein solution; (6) 100. Mu.g/mL and 1mg/mL of L-tyrosine standard solution; (7) 6.54% trichloroacetic acid.

Enzyme activity assay

(1) And (3) preparing a standard curve: l-tyrosine standard solutions were prepared at concentrations of 0. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL, 40. Mu.g/mL and 50. Mu.g/mL, respectively. Taking 1.00ml of standard solution respectively, adding 5.00ml of 0.4mol/L sodium carbonate solution and 1.00ml of forskolin reagent use solution respectively, oscillating uniformly, placing in a water bath at 40 ℃ for color development for 20min, taking out a cuvette with wavelength of 680nm and 10mm by using a spectrophotometer, and measuring the absorbance of the cuvette respectively by taking a0 tube without tyrosine as a blank. The absorbance a is taken as the ordinate and the concentration C of tyrosine is taken as the abscissa, and a standard curve is drawn (this line should pass through the zero point).

(2) Enzyme activity assay

Taking a proper amount of pre-diluted enzyme solution, adding an equal volume of 40 ℃ preheated 10% casein, and reacting for 10min at 40 ℃; then trichloroacetic acid (concentration 6.54%) was added in the same volume as the reaction system, and after mixing, the mixture was allowed to stand at room temperature for 10 minutes to terminate the reaction. Taking out 1ml of the terminated reaction solution, adding 5ml of 42.4g/L sodium carbonate solution, then adding 1ml of Folin (Folin) reagent, and performing color reaction at 40 ℃ for 20min; the OD608 value was finally determined.

(3) Calculation of

The enzyme activity of the final dilutions of the samples was read from the standard curve in μ/mL. The enzyme activity of the sample was calculated according to the following formula:

X=A×K×4/10×n=2/5×A×K×n

wherein: x-enzyme Activity of sample (mu/g or mu/ml)

Average absorbance for parallel test of A-sample

K-absorption constant

4-Total volume of reagents (ml)

10-Reaction time 10min, 1min

N-dilution factor.

Method II for measuring enzyme activity of alkaline protease:

Proteolytic activity can be determined by a method employing a Suc-AAPF-PNA substrate. Suc-AAPF-PNA is an abbreviation for N succinyl-alanine-proline-phenylalanine-p-nitroaniline, and it is a blocking peptide which can be cleaved by endoprotease, after cleavage, a free PNA molecule is released, and it has a yellow colour and can therefore be measured by visible spectrophotometry at a wavelength of 405 nm. Suc-AAPF-PNA substrates were manufactured by Baheng (Bachem) (catalog number L1400 dissolved in DMSO).

The protease sample to be analyzed was diluted in residual activity buffer (100 mM Tris pH 8.6). The assay was performed by transferring 30. Mu.l of diluted enzyme sample to a 96 well microtiter plate and adding 70. Mu.l of substrate working solution (0.72 mg/mI in 100mMTrispH 86). The solution was mixed at room temperature and absorbance was measured every 20 seconds over 5 minutes at 0D405 nm.

The slope of the time-dependent absorption curve (absorbance per minute) is directly proportional to the activity of the protease in question under a given set of conditions. The protease sample should be diluted to a level where the slope is linear.

Example 1: construction and expression of alkaline protease wild type JKX and mutant JKX 02: construction of alkaline protease wild-type JKX and mutant JKX02 of the invention can be constructed and expressed by methods well known to those skilled in the art.

The amino acid sequence of the alkaline protease wild JKX is SEQ ID NO.1, and the optimized nucleic acid sequence SEQ ID NO. 3 is obtained according to the amino acid sequence of SEQ ID NO. 1. The amino acid sequence of the alkaline protease mutant JKX is SEQ ID NO. 2, and the optimized nucleic acid sequence SEQ ID NO. 4 is obtained according to the amino acid sequence of SEQ ID NO. 2, and SEQ ID NO. 3 and SEQ ID NO. 4 are synthesized by the division of biological engineering (Shanghai) Co. SEQ ID NO. 3 and SEQ ID NO. 4 are respectively connected to a pBE2R vector by using a GibsonAssembly method, and are transferred into escherichia coli DH5 alpha by a heat shock method, and the plasmids are sequenced and determined to obtain alkaline protease recombinant plasmids pBE2R-AP (JKX 01) and pBE2R-AP (JKX).

Recombinant plasmids pBE2R-AP (JKX) and pBE2R-AP (JKX 02) with correct sequence are respectively transferred into competent cells WB600, and the specific transformation process is as follows: picking a WB600 single colony growing on an LB plate by using a gun head, and culturing for 12h in 2mLGM I; the overnight cultured bacterial liquid is added into 98mLGM I and cultured for about 4 hours at 37 ℃ and 200 rpm; 10mL of bacterial liquid is added into 90mLGMII, and the bacterial liquid is cultured for about 1.5 hours at 37 ℃ and 200 rpm; the thalli is subjected to ice water bath for 30min, is subjected to centrifugation at 4000rpm and is subjected to centrifugation at 4 ℃ for 30min, and the supernatant is removed; adding 10mLGM III, and mixing to obtain competent cell WB600. Then, 5 mu LpBE R-AP (JKX 02) plasmid was added to 500. Mu.L of competent cells, and the competent cells were directly subjected to shaking culture at 37℃and 200rpm for 1.5 hours, low-speed centrifugation for 3 minutes, and a part of the supernatant was discarded, and the mixture was uniformly spread on a skim milk powder medium plate containing 40. Mu.g/mL kanamycin, and cultured in a constant temperature incubator at 37℃for 12 hours. The single colonies on the next day plates were recombinant strains, designated WB600/pBE2R-AP (JKX) and WB600/pBE2R-AP (JKX 01), respectively. The recombinant engineering bacteria are inoculated in 5mLLB liquid culture medium (peptone 1%, naCl1% and yeast powder 0.5%), and cultured under shaking at 37 deg.C and 200rpm for 12h, and the bacterial solutions are respectively transferred into fermentation enzyme-producing culture medium according to 2% bacterial grafting quantity, and cultured under shaking at 37 deg.C and 200rpm for 84h.

Example 2: separation and purification of alkaline protease: after the fermentation was completed, the fermentation broth was centrifuged at 13000r/min for 15min, and then the supernatant was filtered on a positive pressure filter with a 0.22 μm membrane to remove residual Bacillus subtilis. The supernatant was used for electrophoresis detection and subsequent enzyme activity detection. The electrophoresis detection method comprises the following steps: 100% trichloroacetic acid (1 kg trichloroacetic acid in 454ml water) was added to the protein sample to give a final trichloroacetic acid concentration of 13%, and the mixture was left on ice for 30 minutes after mixing. 15000g, centrifuging at 4℃for 15 minutes, discarding the supernatant, and drying it by inversion to obtain a protein precipitate. Tris-HCl buffer (50 mM Tris-HCl,100mMNaCl,pH8) was added for resuspension, followed by electrophoresis detection according to SDS-PAGE method. SDS-PAGE shows the results as a single band protein sample.

Example 3: thermal stability and pH stability analysis of alkaline protease mutants: the enzyme activity of alkaline protease JKX and its mutant JKX02 was measured, and the protein concentration of wild-type alkaline protease JKX and its mutant JKX02 was 0.2mg/ml, and the protein buffer was 50mM Tris-HCl,100mMNaCl,pH8.0. The enzyme activity measurement method was carried out according to method I or method II, and the temperature was kept at 50℃for 2.5 hours, and samples were taken at intervals of 0.5 hour to measure the enzyme activity. The test results are shown in Table 1.

TABLE 1 residual protease Activity of wild-type alkaline protease and mutant JKX02 at 50℃for various times

Without any protective and stabilizing agents, the residual activity of the wild-type alkaline protease JKX and its mutant JKX02 was significantly higher than that of the wild-type JKX01 as evident from the presence of the mutant JKX02 at 50℃for half an hour, and the residual activity of the mutant JKX02 was still higher than that of the wild-type JKX01 even if incubated for a longer period of time.

Example 4: comparison of pH stability of wild-type alkaline protease JKX, alkaline protease mutant JKX: a series of pH gradient buffer solutions ：Na₂HPO₄-NaH₂PO₄（pH6.0-7.0）、Tris-HCl（pH8.0-9.0）、Gly-NaOH（pH10.0-12.0）, with a concentration of 0.2M were prepared, and enzyme solutions (with a concentration of 0.2 mg/ml) were stored at 25℃in a series of pH gradient buffer systems, and the enzyme activities were measured by referring to GB/T23527-2009 annex B Fulin method, and the results are shown in Table 2.

TABLE 2 Activity of wild-type alkaline protease and mutant at different pH conditions

Without any protective and stabilizing agents, the wild type alkaline protease JKX and its mutant JKX02 were incubated at different pH values for 1 hour, and it was evident that the residual activity of mutant JKX02 was higher than that of wild type JKX01 after pH values were greater than 9.

Example 5: activity determination of alkaline protease mutants in liquid detergents:

the liquid detergent formulations were prepared as shown in table 3.

Table 3 liquid detergent formulations

Both detergents were dissolved in 50 mches buffer (N-cyclohexyl-2-aminoethanesulfonic acid) to ensure that the pH was maintained at 10.0 during the experiment and after addition of the protease sample.

10. Mu.l of a protease solution (0.2 mg/ml) was mixed with 190. Mu.l of a standard detergent solution in a 1.5mlEP tube, and the enzyme activity was measured by referring to GB/T23527-2009 annex B furin method, and the results are shown in Table 4.

Table 4 protease and mutant JKX02 thereof in stability data in STPP and MGDA Standard washes

On the premise of not adding any protective agent and stabilizing agent, the same additive amount of the wild type alkaline protease JKX and the mutant JKX thereof is added into MGDA and STPP washing systems, and in the two washing systems, the alkaline protease mutant JKX02 has better stability compared with the wild type alkaline protease JKX.

The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, and all equivalent structural changes made by the content of the present invention or direct/indirect application in other related technical fields are included in the scope of the present invention.

Claims (6)

1. An alkaline protease mutant characterized in that: the parent protease of the alkaline protease mutant is alkaline protease of bacillus circulans, and the alkaline protease mutant comprises the following amino acid substitutions: D208S, R210G, S268G, Q350R, N353D, V373I, E376F, wherein the position corresponds to the amino acid sequence SEQ ID NO: 1, and a polypeptide having an amino acid position.

2. The alkaline protease mutant of claim 1, wherein: the alkaline protease mutants also comprise a d208s+r210g+s268g+q350r+n353d+v373i+e376F substitution combination.

3. Alkaline protease mutant according to any one of claims 1 to 2, characterized in that: the parent protease is identical to the amino acid sequence of SEQ ID NO: 1. an amino acid sequence having at least 95% sequence identity.

4. Alkaline protease mutants according to any of claims 1-2, characterized in that: the parent protease has an amino acid sequence represented by SEQ ID NO 2.

5. The alkaline protease mutant according to claim 4, wherein: the parent protease is identical to the amino acid sequence of SEQ ID NO:2 having an amino acid sequence with at least 95% sequence identity.

6. A liquid detergent composition characterized in that: comprising the alkaline protease mutant of claim 1.