Alkaline protease mutant and use thereof

Abstract

Provided are an alkaline protease mutant and a use thereof. Compared with a parent alkaline protease having the amino acid sequence shown in SEQ ID NO:1, the alkaline protease mutant has a disulfide bond site pair mutation, the disulfide bond site pair having the following characteristics: i. it is not within the active site region of the alkaline protease mutant; ii. the difference in amino acid residue numbers is greater than or equal to 10; iii. the distance between the two cysteine SG atoms forming the disulfide bond is within a specified range; iv. the Chi3 angle satisfies 30°<Chi3<140° or -30°>Chi3>-140°. Compared with the parent alkaline protease, the alkaline protease mutant has significantly improved heat resistance and / or detergent tolerance, and exhibits better stain removal performance on protein-stained fabrics, thereby better meeting the needs of the household chemical and detergent industry.

Description

alkaline protease mutants and their applications Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to alkaline protease mutants and their applications. Background Technology

[0002] Enzyme molecular modification can be achieved through rational protein design or directed evolution techniques. By modifying natural enzymes, new enzymes with higher stability, activity, selectivity, and tolerance to extreme environments can be provided for industrial production. Rational design is one of the important methods in protein engineering. Based on a clear understanding of the structure, function, and molecular mechanisms of protein-related properties, changes at specific amino acid sites in the protein molecule are theoretically designed to obtain mutants with special properties. The challenge of this research lies in finding effective modification sites.

[0003] Currently, the number of protein sequences stored in the three major databases—Genbank, EMBL, and DDBJ—is growing exponentially, while the growth rate of protein 3D structure information recorded in the PDB (Protein Data Bank) database lags far behind. Obtaining protein structures experimentally is extremely difficult; therefore, using homology modeling to obtain protein 3D structures has become a commonly used bioinformatics method for modern biologists. Currently, commonly used protein homology modeling programs include Swiss-Model, CPHmodel, SDSC1, 3D-jigsaw, InsightII, sybyl, COMPOSER, Modeller, AlphaFold2, and others.

[0004] Following the determination of protein three-dimensional structures, increasingly sophisticated molecular simulation techniques provide strong support for understanding the correlation between three-dimensional structure and protein function. Molecular simulation techniques can be used to simulate various dynamic behaviors of molecules, glassy molecular structures, characteristics of molecular motion, protein folding and unfolding, and so on. Common molecular force fields include Amber (Assisted Model Building with Energy Refinement) and Charmm (Chemistry at HARvard Molecular Mechanics).

[0005] Amber is used for computational simulations of biological macromolecules such as proteins, nucleic acids, and sugars. AMBER provides two parts: a set of molecular mechanical force fields for simulating biomolecules, and programs for molecular simulation, including source code and demonstrations.

[0006] CHARMM is a widely recognized and applied molecular simulation program used for simulating biological macromolecules, including molecular dynamics, energy minimization, and Monte Carlo simulations. The program utilizes the CHARMM force field, which provides users with empirical energy calculations, such as thermodynamic free energy and folding free energy, for various small and large molecules (including proteins, nucleic acids, and sugars).

[0007] Subtilisin (EC number 3.4.21.62), first obtained from Bacillus subtilis, belongs to the S8 peptidase family. Its amino acid sequence contains a catalytic triplet in the order Asp, His, and Ser. It is also known as a serine protease or alkaline protease.

[0008] Alkaline proteases are widely used in detergents, leather tanning, silk processing, animal feed, pharmaceuticals, and food industries, possessing significant industrial and economic value and representing the largest category of industrial enzymes. Alkaline proteases are currently the best-selling detergent additives on the market, significantly improving detergent cleaning power, particularly effective against protein-based stains such as blood, sweat, milk, and oil. However, maintaining high enzyme concentration and stability, especially in detergents, remains a crucial technical challenge in alkaline protease development. Therefore, modifying alkaline proteases using genetic engineering to develop those with improved high-temperature resistance and / or detergent resistance is of great significance to the daily chemical detergent industry.

[0009] Summary of the Invention

[0010] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes an alkaline protease mutant and its applications. Using a computer-aided method, based on the spatial structure and functional requirements of the parental alkaline protease, this invention predicts the disulfide bond sites formed in the alkaline protease structure. Finally, it analyzes the impact of mutations in amino acid sites capable of forming disulfide bonds on the formation of hydrogen bonds and salt bridges within the enzyme molecule, determining the sites that introduce disulfide bonds into the alkaline protease protein structure. The alkaline protease mutant provided by this invention exhibits good high-temperature resistance and / or detergent tolerance, better meeting the needs of the daily chemical detergent industry.

[0011] This invention provides an alkaline protease mutant.

[0012] Specifically, the alkaline protease mutant possesses alkaline protease activity. Compared to the parental alkaline protease with the amino acid sequence shown in SEQ ID NO:1, the alkaline protease mutant exhibits a disulfide bond pair mutation, which has the following characteristics:

[0013] i. Not at the active site of the alkaline protease mutant Within the range;

[0014] ii. The difference in amino acid site numbers of the disulfide bond site pair is greater than or equal to 10;

[0015] iii. The distance between the SG atoms of the two cysteines forming the disulfide bond is within ;

[0016] iv. The Chi3 angle is 30° < Chi3 < 140° or -30° > Chi3 > -140°.

[0017] In some embodiments of the present invention, the difference in amino acid site numbers of the disulfide bond site pair of the alkaline protease mutant is greater than 20; in some embodiments of the present invention, the difference in amino acid site numbers is greater than 40; in some embodiments of the present invention, the difference in amino acid site numbers is greater than 250.

[0018] In some embodiments of the present invention, the distance between the SG atoms of the two cysteines forming the disulfide bond of the alkaline protease mutant is within ; in some embodiments of the present invention, the distance between the SG atoms of the two cysteines forming the disulfide bond of the alkaline protease mutant is within ;

[0019] Compared with the parental alkaline protease of the amino acid sequence shown in SEQ ID NO: 1, the alkaline protease mutant has improved characteristics, and the improved characteristics include increased thermal stability and / or increased detergent tolerance.

[0020] In some embodiments of the present invention, the increased thermal stability includes that the alkaline protease mutant has increased stability at 50°C, and / or, the alkaline protease mutant has enhanced stability after heat storage at 37°C under detergent-containing conditions.

[0021] In some embodiments of the present invention, the Chi3 angle of the disulfide bond site pair is 30° < Chi3 < 140° or the Chi3 angle is -30° > Chi3 > -140°.

[0022] In some embodiments of the present invention, compared with the parental alkaline protease of the amino acid sequence shown in SEQ ID NO: 1, the alkaline protease mutant has a disulfide bond site pair mutation shown in any one of (1) to (80):

[0023] (1)V11C,H220C;(2)A13C,A264C;(3)A13C,V82C;(4)A16C,L227C;(5)L21C,L227C;(6)G23C,A226C;(7)S24C,S85C;(8)V26C,K229C;(9)V26C,A226C;(10)V26C,H118C;(11)V28C,A226C;(12)V28C,V119C;(13)V28C,A86C;(14)A29C,A120C;(15)A29C,M117C;(16)A29C,A112C;(17)A29C,Y89C;(18)I35C,A90C;(19)S36C,P204C;(20)H38C,S206C;(21)D40C,G78C;(22)D40C,A72C;(23)R44C,E87C;(24)G46C,A90C;(25)F49C,Q107C;(26)V50C,S104C;(27)T56C,A90C;(28)G68C,S201C;(29)T69C,S201C;(30)A71C,A86C;(31)A72C,T202C;(32)N74C,P84C;(33)V82C,G223C;(34)A83C,G23C;(35)A86C,A226C;(36)A86C,G23C;(37)V102C,L133C;(38)A106C,T132C;(39)G108C,F49C;(40)L109C,A140C;(41)G113C,A140C;(42)V119C,A222C;(43)N121C,A222C;(44)L122C,A149C; (45)G125C,G152C;(46)N138C,N167C;(47)L146C,N237C;(48)V147C,A168C;(49)V148C,A222C;(50)V148C,M169C;(51)A149C,A168C;(52)S151C,Q185C;(53)G157C,G187C;(54)P162C,L133C;(55)A170C,D191C;(56)V171C,V221C;(57)G172C,S184C;(58)A173C,G196C;(59)A173C,S184C;(60)T174C,V193C;(61)D175C,V197C;(62)Y186C,L256C;(63)I192C,V262C;(64)I192C,G258C;(65)V193C,G172C;(66)A194C,V262C;(67)A194C,H220C; (68)Q200C,S210C; (69)V221C,V262C; (70)A224C,A264C; (71)A225C,V119C; (72)A225C,I240C; (73)V228C,I240C ;(74)K229C,I240C;(75)K245C,S259C;(76)A248C,S259C;(77)G258C,L190C;(78)G260C,S250C;(79)N263C,T249C;(80)A264C,V11C. ;

[0024] In some embodiments of the present invention, the alkaline protease mutant retains ≥16% of its enzyme activity after heat treatment at 50°C for 24 hours.

[0025] The present invention also provides a nucleic acid molecule.

[0026] Specifically, a nucleic acid molecule that encodes the aforementioned alkaline protease mutant.

[0027] The present invention also provides a recombinant expression vector.

[0028] Specifically, a recombinant expression vector contains the aforementioned nucleic acid molecules.

[0029] In some embodiments of the present invention, the vector of the recombinant expression vector is a plasmid; preferably, the plasmid includes the pBE-S plasmid.

[0030] The present invention also provides a recombinant bacterium.

[0031] Specifically, a recombinant bacterium contains the aforementioned nucleic acid molecules or recombinant expression vectors.

[0032] In some embodiments of the present invention, the recombinant bacteria are selected from Escherichia coli cells or Bacillus cells.

[0033] The present invention also provides applications of the above-mentioned alkaline protease mutant.

[0034] Specifically, the application of the aforementioned alkaline protease mutant in detergents.

[0035] The present invention also provides a detergent.

[0036] Specifically, a detergent comprising the aforementioned alkaline protease mutant.

[0037] More specifically, the detergent, such as a household or industrial detergent, is used to remove protein stains. In some embodiments of the invention, the detergent retains ≥6% of its enzyme activity after being heat-stored at 37°C for 4 weeks.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] Compared to the parental alkaline protease, the alkaline protease mutant provided by this invention exhibits superior heat resistance, detergent tolerance, and stability, and demonstrates better detergency on protein-soaked fabrics, thus better meeting the needs of the daily chemical detergent industry. Specifically, after treatment at 50°C for 24 hours, the enzyme activity retention rate of the alkaline protease mutant provided by this invention is significantly higher than that of the parental alkaline protease, indicating a significant improvement in its heat resistance. After heat storage in a constant temperature oven at 37°C for 4 weeks, its stability in detergents is also significantly improved. After 4 weeks of heat storage, commercially available detergent formulations containing the alkaline protease mutant of this invention show significantly higher detergency ratios than those containing the parental alkaline protease mutant, and the detergency ratio remains greater than 1, demonstrating superior performance compared to standard detergents and exhibiting excellent heat storage stability. Attached Figure Description

[0040] Figure 1 is a schematic diagram of the Chi3 angle of the present invention;

[0041] Figure 2 shows the homology comparison between the parental alkaline protease and the PDB ID:1 GCI sequence;

[0042] Figure 3 shows the heat resistance test results of the alkaline protease mutant in Example 5;

[0043] Figure 4 shows the stability test results of the alkaline protease mutant in CM-1 detergent in Example 6. Detailed Implementation

[0044] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0045] Unless otherwise specified, the biological materials, reagents, or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods. Molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0046] definition

[0047] Alkaline protease: The term "alkaline protease" refers to an enzyme that, under alkaline conditions, can cleave peptide bonds within a protein molecule, breaking it down into smaller polypeptides and amino acids. Alkaline protease activity can be determined using methods known in the art (e.g., the Folin method in GB / T 23527-2009, "Protein Preparations").

[0048] According to the present invention, variants exhibiting improved properties under at least one test condition are considered to have improved properties compared to the parental alkaline protease. In accordance with the objectives of the present invention, in some embodiments, to meet the requirements of the daily chemical detergent industry, the improved properties include increased stability. In some embodiments of the present invention, the improved properties include increased thermal stability, for example, increased high-temperature stability. In some embodiments of the present invention, the improved properties include increased thermal stability and increased detergent stability, for example, increased detergent tolerance and increased high-temperature stability.

[0049] Compared to the parental alkaline protease, some alkaline protease mutants of the present invention exhibit increased stability at 50°C, and / or, in detergent-containing formulations, also exhibit enhanced stability after heat storage at 50°C. It is understood that, compared to the parental alkaline protease, the alkaline protease mutants of the present invention possess at least one advantage in heat resistance or detergent tolerance.

[0050] Parental or parental alkaline protease: The term “parental” or “parental alkaline protease” refers to a parental alkaline protease with the amino acid sequence shown in SEQ ID NO:1.

[0051] Variant, Mutant: The terms "variant" and "mutant" refer to a polypeptide with alkaline protease activity that contains mutations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the parental alkaline protease shown in SEQ ID NO:1. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The mutants of the present invention have at least 20%, for example 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%, at least 99%, or at least 100% of the alkaline protease activity of the mature polypeptide of SEQ ID NO:1.

[0052] Coding sequence: The term "coding sequence" or "coding region" refers to a polynucleotide sequence that specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are generally determined by a read frame, which typically begins with an ATG start codon or an alternative start codon (such as GTG and TTG) and ends with a stop codon (such as TAA, TAG, and TGA). Coding sequences can be sequences of genomic DNA, cDNA, synthetic polynucleotides, and / or recombinant polynucleotides.

[0053] Control sequences: The term "control sequence" refers to the nucleic acid sequence essential for peptide expression. Control sequences for the polynucleotides encoding the peptide can be native or exogenous, and can be native or exogenous to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, and transcription terminator sequences. These control sequences can provide multiple linkers for the purpose of introducing specific restriction sites that facilitate the linking of control sequences to the coding regions of the polynucleotides encoding the peptide.

[0054] 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—using techniques known in the art, such as measuring the level of mRNA and / or translated polypeptide.

[0055] Expression vector: The term "expression vector" refers to a straight or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operatively linked to a control sequence that provides for its expression.

[0056] For the purposes described herein, the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 1970, 48, 443-453) was used to determine the degree of sequence identity between two amino acid sequences. This algorithm is implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., Trends Genet. [Trends in Genetics] 2000, 16, 276-277) (preferably version 3.0.0 or later). Optional parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of “longest identity” marked by Needle (obtained using the non-simplified (-nobrief) option) was used as the identity percentage and calculated as follows:

[0057] (identical residues × 100) / (length of reference sequence - total number of vacancies in alignment).

[0058] Variant Nomenclature Rules: For the purposes of this invention, the polypeptide disclosed in SEQ ID NO:1 is used to determine the corresponding amino acid residues in other alkaline proteases. The amino acid sequence of the alkaline protease mutant is compared with the polypeptide disclosed in SEQ ID NO:1, and the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453) implemented in the Niedleman program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) is used to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO:1.

[0059] When describing mutants, the accepted IUPAC single-letter or three-letter amino acid abbreviations are used for ease of reference.

[0060] Substitution: For amino acid substitutions, use the following nomenclature: original amino acid, position, substituted amino acid. For example, replacing valine at position 11 with cysteine ​​is represented as "Val11Cys" or "V11C".

[0061] Amino acid site number difference: According to the present invention, "amino acid site number difference" refers to the difference between the number values ​​of two amino acid sites, used to express the distance between the two amino acids in the sequence, that is, to express how many amino acids are between the two amino acids. In the embodiments of the present invention, the difference between the number values ​​of the disulfide bond site and the amino acid site is greater than or equal to 10, which means that the difference between the amino acids of the disulfide bond site pair is greater than or equal to 10 amino acids. For example, for the disulfide bond site pair V11C, A264C, the difference between the amino acids of this disulfide bond site pair is 253 amino acids.

[0062] Cysteine ​​SG atom: This refers to the cysteine ​​gamma-sulfur atom. The thiol groups of two cysteine ​​residues on the peptide chain undergo oxidation, forming a covalent bond, i.e., a disulfide bond, between the gamma-sulfur atoms. In some embodiments of this invention, the distance between the two cysteine ​​SG atoms forming the disulfide bond is... Within; in some embodiments of the invention, the distance between the two cysteine ​​SG atoms forming the disulfide bond is within; Within; in some embodiments of the invention, the distance between the two cysteine ​​SG atoms forming the disulfide bond is within; Within; in some embodiments of the invention, the distance between the two cysteine ​​SG atoms forming the disulfide bond is within; within; in some embodiments of the present invention, the distance between the SG atoms of the two cysteines forming the disulfide bond is within within; in some embodiments of the present invention, the distance between the SG atoms of the two cysteines forming the disulfide bond is within within; in some embodiments of the present invention, the distance between the SG atoms of the two cysteines forming the disulfide bond is within within; in some embodiments of the present invention, the distance between the SG atoms of the two cysteines forming the disulfide bond is within within.

[0063] Chi3 angle: The abbreviation "χ3", where "χ" is the 22nd letter of the Greek alphabet. The Chi3 angle is the dihedral angle defined by the Ciβ-Si-Sj-Cjβ torsion, as shown in Figure 1. In the embodiments of the present invention, the Chi3 angle of the disulfide bond site pair is 30° < Chi3 < 140° or -30° > Chi3 > -140°. In some embodiments of the present invention, the Chi3 angle of the disulfide bond site pair is 30° < Chi3 < 140° or the Chi3 angle is -30° > Chi3 > -140°.

[0064] There are mutations in the disulfide bond site pairs of the alkaline protease mutant of the present invention, and its thermal stability has been improved to a certain extent. In some embodiments of the present invention, after the alkaline protease mutant is heat-treated at 50°C for 24 hours, the enzyme activity retention rate ≥ 16%. In some embodiments of the present invention, after the alkaline protease mutant is heat-treated at 50°C for 24 hours, the enzyme activity retention rate ≥ 20%. In some embodiments of the present invention, after the alkaline protease mutant is heat-treated at 50°C for 24 hours, the enzyme activity retention rate ≥ 30%. In some embodiments of the present invention, after the alkaline protease mutant is heat-treated at 50°C for 24 hours, the enzyme activity retention rate ≥ 40%. In some embodiments of the present invention, after the alkaline protease mutant is heat-treated at 50°C for 24 hours, the enzyme activity retention rate ≥ 50%.

[0065] Based on the purpose of the present invention, the alkaline protease mutant provided by the present invention can be applied to detergents. In some embodiments of the present invention, after the alkaline protease mutant provided by the present invention is added to the detergent, the enzyme activity retention rate of the detergent after heat storage at 37°C for 4 weeks ≥ 6%; in some embodiments of the present invention, the enzyme activity retention rate ≥ 10%; in some embodiments of the present invention, the enzyme activity retention rate ≥ 20%; in some embodiments of the present invention, the enzyme activity retention rate ≥ 30%; in some embodiments of the present invention, the enzyme activity retention rate ≥ 40%; in some embodiments of the present invention, the enzyme activity retention rate ≥ 50%; in some embodiments of the present invention, the enzyme activity retention rate ≥ 60%; in some embodiments of the present invention, the enzyme activity retention rate ≥ 70%.

[0066] Example 1: Rational Design of Alkaline Protease

[0067] 1.1 Homologous Modeling

[0068] The parental alkaline protease of this invention (SEQ ID NO:1) has the lowest proportion of essential cysteine ​​residues for forming disulfide bonds, containing only one, thus possessing potential for disulfide bond design. Sequence searching and homology comparison using a BLAST server in the GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank / ) showed that an alkaline protease (PDB ID:1GCI) also derived from Bacillus lentus has 98.51% homology with the alkaline protease sequence of this invention. As shown in Figure 2, the parental alkaline protease of this invention (SEQ ID NO:1) has 98.51% sequence homology with PDB ID:1GCI, confirming that the key catalytic active site is also a catalytic ternary group composed of Asp32, His62, and Ser215. A key feature of the disulfide bond design in this invention is that the disulfide bond site pair is not present in the active site of the alkaline protease mutant. Within a certain range, thus avoiding the formation of disulfide bonds from affecting the substrate catalytic activity of alkaline proteases.

[0069] Homology modeling is based on the principle that similar sequences have similar protein three-dimensional structures. This method uses SWISS-MODEL to obtain the parental alkaline protease sequence structure of the present invention. SEQ_01 (SEQ ID NO:1) is used as the sequence input, and 1GCI is used as the template for modeling. After the operation, the GMQE score is 0.96, which shows high modeling accuracy and can be used for further analysis.

[0070] 1.2 Assessment of the rationality of structural conformation

[0071] The structural model obtained from the aforementioned steps was evaluated for the rationality of protein structure conformation using the SAVES v6.0 (https: / / saves.mbi.ucla.edu / ) scoring program and presented in the form of a Ramachandran plot. Generally speaking, if the proportion of amino acid residues falling within the allowed region and the maximally allowed region in the whole protein is higher than 90%, it can be considered that the conformation of this model conforms to the rules of stereochemistry. A model with more than 90% of the residues in the most favorable region is considered to be of high quality, while a reliable model should have more than 80% of the residues. The evaluation results are as follows: The overall structural quality score of the structural model constructed by SWISS-MODEL is 90.98. The Ramachandran plot shows that 91.8% of the residues in the predicted model are in the most favorable region. The results show that the homologous model constructed by SWISS-MODEL has a very high precision and a comprehensive and intuitive reasonable conformation. Therefore, the model predicted by SWISS-MODEL can be selected for subsequent experiments.

[0072] 1.3 Disulfide bond design

[0073] In order to discover reasonable and effective disulfide bond pairs and improve the thermostability of alkaline protease, this method uses a deep learning algorithm model and combines set conditions to screen disulfide bond pairs: a) The disulfide bond site pairs are not within the active region range; b) The amino acid sequence difference of the disulfide bond site pairs is greater than or equal to 10; c) The distance between the SG atoms of cysteine after mutation of the disulfide bond site pairs is within ; d) The Chi3 angle after mutation of the disulfide bond site pairs is between -140 < Chi3 < -30 or 30 < Chi3 < 140; The final summary calculation results are shown in Table 1. A total of 80 pairs of disulfide bond candidate mutation libraries were screened based on the above conditions and can be used for experimental verification;

[0074] Table 1

[0075] Example 2: Construction and expression of alkaline protease and its variants

[0076] 2.1 Materials and reagents

[0077] 2.1.1 Strains and vectors:

[0078] Expression strains containing the alkaline protease apr gene and its variants, Escherichia coli TOP10, Bacillus subtilis WB600, vector pBE-S, antibiotics, kanamycin, and ampicillin were all purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0079] 2.1.2 Enzymes and kits:

[0080] Ultra-fidelity 2×Master Mix PCR polymerase, restriction endonucleases, etc. were purchased from NEB Corporation, and plasmid extraction kits and purification kits were purchased from Shanghai Sangon Biotech Co., Ltd.

[0081] 2.1.3 Culture medium:

[0082] Escherichia coli was cultured on 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 was cultured on 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.

[0083] 2.1.4 Chemical reagents:

[0084] Casein was purchased from Sinopharm Chemical Reagent Co., Ltd., and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0085] 2.2 Alkaline protease activity assay method

[0086] The activity of alkaline protease was determined using the Folin-Ciocalteu method as specified in the national standard GB / T 23527-2009, "Alkaline Protease Preparations". Under specific temperature and pH conditions, the protease hydrolyzes casein substrates to produce amino acids containing phenolic groups (such as tyrosine and tryptophan). Under alkaline conditions, the Folin-Ciocalteu reagent is reduced to produce molybdenum blue and tungsten blue. The absorbance of the solution was measured at 680 nm using a spectrophotometer. Enzyme activity is proportional to absorbance, and the enzyme activity of the product can be calculated from this.

[0087] Protease activity is expressed in protease activity units, defined as the amount of 1 μg of tyrosine produced by hydrolyzing casein in 1 minute under certain temperature and pH conditions from 1 g of solid enzyme powder (or 1 mL of liquid enzyme), which is expressed as U / g (U / mL).

[0088] In cases involving gene mutations, the parental sequence and its species are disclosed, and subsequent site numbering is based on the parental sequence: the alkaline protease (apr) gene of the mutated Bacillus lentus, whose amino acid sequence is shown in SEQ ID NO.1.

[0089] 2.2 Expression of alkaline proteases and their variants

[0090] 2.2.1 Synthesis of alkaline protease apr gene and vector construction

[0091] The mutated alkaline protease (apr) gene of Bacillus lentus, the amino acid sequence of which is shown in SEQ ID NO.1 (denoted as parental alkaline protease).

[0092] NdeI and SalI restriction sites were introduced at the 5' and 3' ends of the alkaline protease apr gene, respectively, and ligated into the pUC57-amp vector. pUC57-amp was seeded into LBA medium and cultured overnight. The plasmid was extracted, digested with NdeI and SalI, and the target gene fragment was recovered by gel excision and ligated into the expression vector pBE-S to obtain the expression vector pBE-amp.

[0093] 2.2.2 Introduction of Mutual Changes in Disulfide Bonds

[0094] Using pBE-apr as a template, the first cysteine ​​was introduced into the alkaline protease apr gene using PCR. After digestion with DpnI (DpnI restriction enzyme), the target fragment was recovered by gel excision. The product was then transformed into *E. coli* TOP10 competent cells using a chemical transformation and heat shock method. Recombinant transformants were verified by colony PCR, and plasmids from correctly verified transformants were extracted and sequenced to identify the corresponding mutants, resulting in the expression vector pBE-apr-M1-1. Using pBE-apr-M1-1 as a template, the second cysteine ​​was introduced into the alkaline protease apr gene using PCR. After digestion with DpnI (DpnI restriction enzyme), the target fragment was recovered by gel excision. The product was then transformed into *E. coli* TOP10 competent cells using a chemical transformation and heat shock method. Recombinant transformants were verified by colony PCR, and plasmids from correctly verified transformants were extracted and sequenced to identify the corresponding mutants, resulting in the expression vector pBE-apr-M1 containing a cysteine ​​mutation. Bacillus recombinant transformants were obtained by chemical transformation into WB600 chemically transformed competent cells.

[0095] 2.2.3 Shake-flask fermentation of mutant strains containing disulfide bonds

[0096] Using toothpicks, each recombinant transformant obtained in step 2 was picked up and transferred to a 250 mL shake flask. 40 mL of TB medium was added to each shake flask. The flasks were incubated at 37°C, 220 rpm, and 85% humidity for about 48 hours. The supernatant was collected by centrifugation, and the parental alkaline protease gene and the mutant were purified by affinity chromatography to obtain enzyme solutions of each alkaline protease mutant.

[0097] Example 3: Determination of the optimal reaction pH for alkaline protease mutants

[0098] The enzyme activity of alkaline protease was measured at a temperature of 40℃ and at pH values ​​of 6.5, 7.5, 9.0, 10.5 and 12. The enzyme activity at the optimal reaction pH of each enzyme was then counted as 100%, and the relative enzyme activity of the corresponding enzyme at different pH values ​​was calculated. The results are shown in Table 2.

[0099] Relative enzyme activity refers to the ratio of the activity of a certain enzyme under specific conditions to its 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%.

[0100] Table 2

[0101] Table 2 shows that the optimal reaction pH for most alkaline protease mutants is 10.5, which is consistent with the optimal reaction pH of the parental alkaline protease. Furthermore, most alkaline protease mutants retain higher enzyme activity compared to the parental alkaline protease at pH 12. Simultaneously, most alkaline protease mutants also exhibit high enzyme activity at pH 6.5. This indicates that these alkaline protease mutants possess good catalytic efficiency not only under alkaline conditions but also under neutral conditions, making them better suited to the application conditions in the daily chemical detergent industry.

[0102] Example 4: Determination of the optimal reaction temperature for alkaline protease mutants

[0103] Under a pH of 10.5, the enzyme activities of the parental alkaline protease and the alkaline protease mutant were measured at 30℃, 40℃, 50℃, 60℃, 70℃ and 80℃, respectively. The enzyme activity at the optimal reaction temperature of each enzyme was then counted as 100%, and the relative enzyme activity of the corresponding enzyme at different temperatures was calculated. The results are shown in Table 3.

[0104] Relative enzyme activity refers to the ratio of the activity of a certain enzyme under specific conditions to the activity of the enzyme 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 the optimal reaction conditions) × 100%.

[0105] Table 3

[0106] Table 3 shows that the optimal reaction temperature for some alkaline protease mutants is 60℃, while the optimal reaction temperature for the parental alkaline protease is 50℃. This indicates that some alkaline protease mutants can also exhibit high enzyme activity under high temperature conditions.

[0107] Example 5: Thermostability of Alkaline Protease Mutants

[0108] The purified parental alkaline protease and alkaline protease mutant enzyme solutions were heat-treated at 50°C for 24 hours, cooled on ice, and then enzyme activity was detected 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 treatment / Enzyme activity before heat treatment) × 100%. The heat resistance test results are shown in Table 4 and Figure 3. In Figure 3, the parental alkaline protease is denoted as the parent, and the alkaline protease mutants 1-80 are abbreviated as SEQ NO: 2-81 (the same below).

[0109] Table 4

[0110] As shown in Table 4 and Figure 3, the alkaline protease mutant provided by this invention, after treatment at 50°C for 24 hours, exhibits an enzyme activity retention rate ≥16%, reaching as high as 59%; while the parental alkaline protease is completely inactivated at this time. The enzyme activity retention rate of the alkaline protease mutant provided by this invention is significantly higher than that of the parental alkaline protease, and its heat resistance is significantly improved.

[0111] Example 6: Stability test of parental alkaline protease and alkaline protease mutant in commercial detergents

[0112] Purified parental alkaline protease and alkaline protease mutant enzyme solutions were added to commercial detergent (CM-1) at a certain ratio and mixed thoroughly to ensure an initial alkaline protease activity of 560 u / mL in both CM-1 samples. Then, CM-1 samples containing both parental alkaline protease and alkaline protease mutant were stored at -18°C as week 0 samples. The remaining detergent was stored in a 37°C oven for 4 weeks. Using Megazyme Protazyme AK Tablets (T-PRAK-1000T, 1000 tablets per pack, repeatability error less than 5%), the protease activity in each group of week 0 and week 4 samples was tested, and the enzyme activity retention rate was calculated. Enzyme activity retention rate = (protease activity of week 4 sample / protease activity of week 0 sample) × 100%.

[0113] The CM-1 detergent formula consists of sodium dodecylbenzenesulfonate (5%), sodium ethoxylated alkyl sulfate (8%), fatty alcohol polyoxyethylene ether-9 (5%), cocamidopropyl betaine (2%), sodium citrate (1%), Kathon (0.1%), sodium chloride (1%), and water (balance).

[0114] The results are shown in Table 5 and Figure 4.

[0115] Table 5. Stability test results of parental alkaline protease and alkaline protease mutant in CM-1 detergent.

[0116] As shown in Table 5 and Figure 4, the alkaline protease mutant provided by this invention, after being stored in CM-1 detergent and in a constant temperature oven at 37°C for 4 weeks, retains ≥6% of its enzyme activity, reaching as high as 86%; while the parental alkaline protease is completely inactivated at this time. The stability of the alkaline protease mutant provided by this invention in CM-1 detergent is significantly improved.

[0117] Example 7: Stain removal stability test in commercially available detergent AH after 4 weeks of heat storage at 37°C

[0118] The purified parental alkaline protease and alkaline protease mutant enzyme solutions were added to commercially available detergent formulations in a specific ratio and mixed thoroughly to ensure that the initial alkaline protease activity in each detergent formulation was 560 u / mL. Detergent samples containing both parental alkaline protease and alkaline protease mutant were frozen at -18°C as week 0 samples. The remaining detergent was stored in a 37°C oven for 4 weeks.

[0119] Referring to GB / T 13174-2021 standard, based on JB-02 protein-soaked fabric, the detergency was tested initially and after 4 weeks of heat storage, and the corresponding detergency ratio was obtained (according to GB / T 13174-2021 standard, detergency ratio = detergency value of sample % / detergency value of standard detergent %). By comparing the detergency ratios after the initial test and after 4 weeks of heat storage, the heat storage stability of the parental alkaline protease and the alkaline protease mutant in commercially available detergent formulations was investigated.

[0120] The results are shown in Tables 6 and 7.

[0121] Table 6. Stain removal ratios of parental alkaline protease and alkaline protease mutant in commercially available detergent AD.

[0122] Table 7. Stain removal ratios of parental alkaline protease and alkaline protease mutant in commercially available detergent EH.

[0123] The results in Tables 6 and 7 show that after 4 weeks of heat storage, the commercially available detergent formulations containing the alkaline protease mutant of this invention have higher detergency ratios than those containing the parental alkaline protease mutant, and the detergency ratio is still greater than 1, which is better than the standard detergent and has good heat storage stability.

[0124] In summary, compared with the parental alkaline protease, the alkaline protease mutant provided in this invention has excellent heat resistance, detergent tolerance, and stability, and higher catalytic activity for proteins, thus better meeting the needs of the daily chemical detergent industry.

[0125] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An alkaline protease mutant, characterized in that, Compared with the parental alkaline protease of the amino acid sequence shown in SEQ ID NO:1, the alkaline protease mutant has mutations in disulfide bond sites pairs, and the disulfide bond sites pairs have the following characteristics: i. Not at the active site of the alkaline protease mutant within the range; ii. The difference in amino acid site numbers is greater than or equal to 10; iii. The distance between the two cysteine ​​SG atoms forming a disulfide bond is within; iv. The Chi3 angle is 30° < Chi3 < 140° or -30° > Chi3 > -140°.

2. The alkaline protease mutant according to claim 1, characterized in that, Compared with the parental alkaline protease of the amino acid sequence shown in SEQ ID NO:1, it has improved properties, and the improved properties include increased thermal stability and / or increased thermal storage stability.

3. The alkaline protease mutant according to claim 2, characterized in that, The thermal storage stability includes the thermal storage stability of the alkaline protease mutant in detergent products.

4. The alkaline protease mutant according to claim 1, characterized in that, Compared with the parental alkaline protease of the amino acid sequence shown in SEQ ID NO:1, the alkaline protease mutant has any one of the following mutations in disulfide bond sites pairs: (1) V11C, H220C; (2) A13C, A264C; (3) A13C, V82C; (4) A16C, L227C; (5) L21C, L227C; (6) G23C, A226C; (7) S24C, S85C; (8) V26C, K229C; (9) V26C, A226C; (10) V26C, H118C; (11) V28C, A226C; (12) V28C, V119C; (13) V28C, A86C; (14) A29C, A120C; (15) A29C, M117C; (16) A29C, A112C; (17) A29C, Y89C; (18) I35C, A90C; (19) S36C, P204C; (20) H38C, S206C; (21) D40C, G78C; (22) D40C, A72C; (23) R44C, E87C; (24) G46C, A90C; (25) F49C, Q107C; (26) V50C, S104C; (27) T56C, A90C; (28) G68C, S201C; (29) T69C, S201C; (30) A71C, A86C; (31) A72C, T202C; (32) N74C, P84C; (33) V82C, G223C; (34) A83C, G23C; (35) A86C, A226C; (36) A86C, G23C; (37) V102C, L133C; (38) A106C, T132C; (39) G108C, F49C; (40) L109C, A140C; (41) G113C, A140C; (42) V119C, A222C; (43) N121C, A222C; (44) L122C, A149C; (45) G125C, G152C; (46) N138C, N167C; (47)L146C,N237C; (48)V147C,A168C; (49)V148C,A222C; (50)V148C,M169C; (51)A149C,A168C; (52)S151C,Q185C; (53)G157C,G187C; (54)P162C,L133C; (55)A170C,D191C; (56)V171C,V221C; (57)G172C,S184C; (58)A173C,G196C; (59)A173C,S184C; (60)T174C,V193C; (61)D175C,V197C; (62)Y186C,L256C; (63)I192C,V262C; (64)I192C,G258C; (65)V193C,G172C; (66)A194C,V262C; (67)A194C,H220C; (68)Q200C,S210C; (69)V221C,V262C; (70)A224C,A264C; (71)A225C,V119C; (72)A225C,I240C; (73)V228C,I240C; (74)K229C,I240C; (75)K245C,S259C; (76)A248C,S259C; (77)G258C,L190C; (78)G260C,S250C; (79)N263C,T249C; (80)A264C,V11C.

5. The alkaline protease mutant according to claim 1, characterized in that, The alkaline protease mutant retained ≥16% of its enzyme activity after heat treatment at 50°C for 24 hours.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the alkaline protease mutant according to any one of claims 1 to 5.

7. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 6.

8. A recombinant bacterium, characterized in that, It comprises the nucleic acid molecule of claim 6 or the recombinant expression vector of claim 7.

9. A detergent, characterized in that, The detergent contains the alkaline protease mutant according to any one of claims 1 to 5.

10. The detergent according to claim 9, characterized in that, After being stored at 37°C for 4 weeks, the enzyme activity retention rate of the detergent is ≥6%.

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