Alkaline protease mutant and application thereof

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and discloses an alkaline protease mutant and application thereof. Compared with parent alkaline protease with an amino acid sequence as shown in SEQ ID NO: 1, the alkaline protease mutant has disulfide key site pair mutation, and the disulfide key site pair has the following characteristics: i, the disulfide key site pair is not in the range of an active site # imgabs0 # of the alkaline protease mutant; ii, the difference value of amino acid site numbers is greater than or equal to 10; iii, the distance between two cysteine SG atoms forming a disulfide bond is within # imgabs 1 #; iv, the angle of Chi3 is 30 degrees lt; chi3lt; chi3lt; 140 DEG or-30 DEG gt; chi3gt; chi3gt; and-140 DEG C. Compared with the parent alkaline protease, the alkaline protease mutant has the advantages that the heat resistance and / or detergent tolerance are / is obviously improved, the decontamination effect on protein dirty cloth is better, and the requirements of the daily chemical washing industry can be better met.

Description

Technical Field

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

[0002] Enzyme molecular modification can be achieved through protein rational design or directed evolution. By molecularly modifying natural enzymes, new enzymes with enhanced stability, activity, selectivity, and tolerance to extreme environments can be developed for industrial production. Rational design is a key approach in protein engineering. After clarifying the structure, function, and molecular mechanisms of protein-related properties, specific amino acid sites within the protein molecule are theoretically designed to alter their activity, thereby generating mutants with specialized properties. The challenge of this research lies in identifying effective modification sites.

[0003] Currently, the number of protein sequences stored in the three major databases of Genbank, EMBL, and DDBJ is growing exponentially, while the growth rate of the number of protein three-dimensional structure information recorded in the PDB (Protein DataBank) database lags far behind. However, it is extremely difficult to obtain protein structure through experimental methods. Therefore, using homology modeling technology to obtain the three-dimensional structure of proteins has become a bioinformatics method commonly used by modern biologists. Currently, commonly used protein homology modeling programs include Swiss-Model, CPHmodel, SDSC1, 3D-jigsaw, InsightII, sybyl, COMPOSER, Modeller, AlphaFold2, etc.

[0004] Following the determination of protein three-dimensional structures, increasingly sophisticated molecular simulation techniques have provided powerful support for understanding the relationship between 3D structure and protein function. Molecular simulation techniques can be used to simulate various dynamic molecular behaviors, glassy molecular structures, molecular motion characteristics, and protein folding and unfolding. 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 biomolecules such as proteins, nucleic acids, and sugars. AMBER provides two components: a set of molecular mechanics force fields for simulating biomolecules, and a program for molecular simulations, including source code and demonstrations.

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

[0007] 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 called serine protease or alkaline protease.

[0008] Alkaline proteases are widely used in detergents, leather, silk, feed, medicine, and food, possessing significant industrial and economic value. They represent the largest proportion of industrial enzymes. Currently a best-selling detergent additive, alkaline proteases significantly enhance 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 heat and / or detergent resistance is of great significance to the daily chemical detergent industry. Summary of the Invention

[0009] 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 proposes an alkaline protease mutant and its application. The present invention uses a computer-assisted method to predict the disulfide bond sites generated in the alkaline protease structure based on the spatial structure and functional requirements of the parent alkaline protease. Finally, the effect of the mutation of the amino acid site capable of forming disulfide bonds on the formation of hydrogen bonds and salt bridges within the enzyme molecule is analyzed, and the site for introducing disulfide bonds into the alkaline protease protein structure is determined. The alkaline protease mutant provided by the present invention has good high temperature resistance and / or detergent tolerance, and can better meet the needs of the daily chemical washing industry.

[0010] The present invention provides an alkaline protease mutant.

[0011] 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 has a disulfide bond site pair mutation, and the disulfide bond site pair has the following characteristics:

[0012] i. Not in the active site of the alkaline protease mutant within the scope;

[0013] ii. The difference in amino acid site numbers is greater than or equal to 10;

[0014] iii. The distance between the SG atoms of the two cysteines forming a disulfide bond is within ;

[0015] iv. The Chi3 angle is 30° < Chi3 < 140° or -30° > Chi3 > -140°.

[0016] SEQ ID NO:1:

[0017] AQSVPWGIWRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLPASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVYSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTDLYGSGLVNAEAATR.

[0018] In some embodiments of the present invention, the difference in amino acid site numbers of the disulfide bond site pairs 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.

[0019] In some embodiments of the present invention, the distance between the SG atoms of the two cysteines forming a disulfide bond in the alkaline protease mutant is within ; in some embodiments of the present invention, the distance between the SG atoms of the two cysteines forming a disulfide bond in the alkaline protease mutant is within ;

[0020] Compared with the parental alkaline protease of the amino acid sequence shown in SEQ ID NO:1, the alkaline protease mutant has improved properties, and the improved properties include increased thermal stability and / or increased detergent tolerance.

[0021] 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.

[0022] 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°.

[0023] In some embodiments of the present invention, compared with the parental alkaline protease having 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):

[0024] (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. ;

[0025] In some embodiments of the present invention, the alkaline protease mutant has an enzyme activity retention rate of ≥16% after heat treatment at 50° C. for 24 hours.

[0026] The present invention also provides a nucleic acid molecule.

[0027] Specifically, a nucleic acid molecule, which encodes the above alkaline protease mutant.

[0028] The present invention also provides a recombinant expression vector.

[0029] Specifically, a recombinant expression vector comprises the above nucleic acid molecule.

[0030] 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.

[0031] The present invention also provides a recombinant bacterium.

[0032] Specifically, a recombinant bacterium comprises the above nucleic acid molecule or recombinant expression vector.

[0033] In some embodiments of the present invention, the recombinant bacteria are selected from Escherichia coli cells or Bacillus cells.

[0034] The present invention also provides the application of the alkaline protease mutant.

[0035] Specifically, the use of the alkaline protease mutant in detergents.

[0036] The present invention also provides a detergent.

[0037] Specifically, a detergent comprising the above alkaline protease mutant.

[0038] More specifically, the detergent is used, for example, in household or industrial detergents, to remove protein stains. In some embodiments of the present invention, the detergent has an enzyme activity retention rate of ≥6% after heat storage at 37°C for 4 weeks.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] Compared with the parent alkaline protease, the alkaline protease mutant provided by the present invention has excellent heat resistance, laundry detergent tolerance, and stability, and has a better decontamination effect on protein-stained cloth, which can better meet the needs of the daily chemical washing industry. Specifically, after the alkaline protease mutant provided by the present invention is treated at 50°C for 24 hours, the enzyme activity retention rate is significantly higher than that of the parent alkaline protease, and its heat resistance is significantly improved; after heat storage in a constant temperature oven at 37°C for 4 weeks, the stability in the detergent is also significantly improved; after heat storage for 4 weeks, the commercially available detergent formula added with the alkaline protease mutant of the present invention has a significantly higher decontamination ratio than the commercially available detergent formula added with the parent alkaline protease mutant, and the decontamination ratio is still greater than 1, which is better than the standard detergent and has good heat storage stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the Chi3 angle of the present invention;

[0042] Figure 2 It is a sequence homology comparison diagram between the parent alkaline protease and PDB ID: 1GCI;

[0043] Figure 3 Graph showing the heat resistance test results of the alkaline protease mutant in Example 5;

[0044] Figure 4 This is a graph showing the stability test results of the alkaline protease mutant in CM-1 detergent in Example 6. DETAILED DESCRIPTION

[0045] 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.

[0046] 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.

[0047] definition

[0048] 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").

[0049] 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 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 detergent stability of increase, for example, the detergent tolerance of increase and the high temperature stability of increase.

[0050] Compared to the parent alkaline protease, some alkaline protease mutants of the present invention have increased stability at 50°C and / or, in a detergent-containing formulation, also have enhanced stability after heat storage at 50°C. It will be appreciated that, compared to the parent alkaline protease, the alkaline protease mutants of the present invention have at least one advantage in heat resistance or detergent tolerance.

[0051] Parent or parent alkaline protease: The term "parent" or "parent alkaline protease" refers to the parent alkaline protease having the amino acid sequence shown in SEQ ID NO:1.

[0052] 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%, at least 99% or at least 100% of the alkaline protease activity of the mature polypeptide of SEQ ID NO: 1.

[0053] 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.

[0054] Control sequences: The term "control sequence" refers to nucleic acid sequences necessary for expression of a polypeptide. Control sequences may be native or foreign to the polynucleotide encoding the polypeptide, and may be native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter sequence, a signal peptide sequence, and a transcription terminator sequence. These control sequences may be provided with a plurality of linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences to the coding region of the polynucleotide encoding the polypeptide.

[0055] 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—e.g., to detect increased expression—by techniques known in the art, such as measuring the levels of mRNA and / or translated polypeptide.

[0056] Expression vector: The term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

[0057] For purposes described herein, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, J.Mol.Biol. [Journal of Molecular Biology] 1970,48,443-453), as implemented in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite), the people such as Rice, TrendsGenet. [Genetics Trend] 2000,16,276-277) (preferred 3.0.0 version or later) Needle program. The optional parameters employed are gap opening penalty 10, gap extension penalty 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output (using non-simplified (-nobrief) option to obtain) of the "longest identity" mark of Needle is used as percent identity and is calculated as follows:

[0058] (identical residues x 100) / (length of reference sequence - total number of gaps in the alignment).

[0059] Variant nomenclature convention: For the purposes of the present 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 aligned with the polypeptide disclosed in SEQ ID NO: 1, and the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman 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).

[0060] When describing mutants, the accepted IUPAC single-letter or three-letter amino acid abbreviations are used for ease of reference.

[0061] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. For example, a substitution of valine at position 11 with cysteine ​​is represented as "Val11Cys" or "V11C".

[0062] Amino acid site number difference: According to the present invention, the "amino acid site number difference" refers to the difference between two amino acid site number values, which is used to express the distance between two amino acids in a sequence, that is, to express how many amino acids are there between two amino acids. In the embodiments of the present invention, the amino acid site number difference of the disulfide bond site pair is greater than or equal to 10, which means that there are more than or equal to 10 amino acids between the disulfide bond site pair. For example, for the disulfide bond site pair V11C, A264C, there are 253 amino acids between the amino acids of this disulfide bond site pair.

[0063] Cysteine SG atom: That is, the cysteine gamma sulfur atom. The sulfhydryl groups of two cysteine residues on the peptide chain undergo an oxidation reaction, causing a covalent bond to form between the gamma sulfur atoms, that is, a disulfide bond. In some embodiments of the present invention, the distance between the two cysteine SG atoms forming the disulfide bond is within ; In some embodiments of the present invention, the distance between the two cysteine SG atoms forming the disulfide bond is within ; In some embodiments of the present invention, the distance between the two cysteine SG atoms forming the disulfide bond is within ; In some embodiments of the present invention, the distance between the two cysteine SG atoms forming the disulfide bond is within ; In some embodiments of the present invention, the distance between the two cysteine SG atoms forming the disulfide bond is within ; In some embodiments of the present invention, the distance between the two cysteine SG atoms forming the disulfide bond is within ; In some embodiments of the present invention, the distance between the two cysteine SG atoms forming the disulfide bond is within ; In some embodiments of the present invention, the distance between the two cysteine SG atoms forming the disulfide bond is within ; In some embodiments of the present invention, the distance between the two cysteine SG atoms forming the disulfide bond is within

[0064] Chi3 angle: Abbreviation "χ3", "χ" is the 22nd letter in 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°.

[0065] The alkaline protease mutant of the present invention has a mutation at a disulfide bond site, and its thermal stability is improved to a certain extent. In some embodiments of the present invention, after heat treatment at 50°C for 24 hours, the enzyme activity retention rate of the alkaline protease mutant is ≥16%. In some embodiments of the present invention, after heat treatment at 50°C for 24 hours, the enzyme activity retention rate of the alkaline protease mutant is ≥20%. In some embodiments of the present invention, after heat treatment at 50°C for 24 hours, the enzyme activity retention rate of the alkaline protease mutant is ≥30%. In some embodiments of the present invention, after heat treatment at 50°C for 24 hours, the enzyme activity retention rate of the alkaline protease mutant is ≥40%. In some embodiments of the present invention, after heat treatment at 50°C for 24 hours, the enzyme activity retention rate of the alkaline protease mutant is ≥50%.

[0066] Based on the purposes of the present invention, the alkaline protease mutant provided by the present invention can be used in detergents. In some embodiments of the present invention, after the alkaline protease mutant provided by the present invention is added to the detergent, the detergent retains an enzyme activity of ≥6% after heat storage at 37°C for 4 weeks; in some embodiments of the present invention, the enzyme activity retention rate is ≥10%; in some embodiments of the present invention, the enzyme activity retention rate is ≥20%; in some embodiments of the present invention, the enzyme activity retention rate is ≥30%; in some embodiments of the present invention, the enzyme activity retention rate is ≥40%; in some embodiments of the present invention, the enzyme activity retention rate is ≥50%; in some embodiments of the present invention, the enzyme activity retention rate is ≥60%; in some embodiments of the present invention, the enzyme activity retention rate is ≥70%.

[0067] Example 1: Rational design of alkaline protease

[0068] 1.1 Homology modeling

[0069] The amino acid sequence of the parent alkaline protease of the present invention (SEQ ID NO: 1) has the lowest percentage of essential cysteine ​​residues required for disulfide bond formation, with only one residue, suggesting potential for disulfide bond design. Sequence searches and homology comparisons using the BLAST server in the GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank / ) revealed that an alkaline protease also derived from Bacillus lentus (PDB ID: 1GCI) shares 98.51% homology with the alkaline protease sequence of the present invention. Figure 2 As shown, the parent alkaline protease of the present invention (SEQ ID NO: 1) has 98.51% sequence homology with PDB ID: 1GCI, and it can be determined that the key catalytic active site is also a catalytic triad consisting of Asp32, His62 and Ser215. A major feature of the disulfide bond design of the present invention is that the disulfide bond site is not in the active site of the alkaline protease mutant. within a certain range, thus avoiding the influence of the formation of disulfide bonds on the substrate catalytic activity of alkaline protease.

[0070] Homology modeling is based on the principle that similar sequences have similar three-dimensional protein structures. In this method, SWISS-MODEL is used for homology modeling to obtain the sequence structure of the parental alkaline protease 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 is completed, the GMQE score is 0.96, indicating high modeling accuracy and allowing for further analysis.

[0071] 1.2 Structural conformation rationality assessment

[0072] The structural model obtained in the previous step is evaluated for the rationality of the protein structural conformation using the SAVES v6.0 (https: / / saves.mbi.ucla.edu / ) scoring program and presented in the form of a Ramachandran plot. Generally, if the proportion of amino acid residues falling within the allowed and maximally allowed regions in the whole protein is higher than 90%, it can be considered that the conformation of the 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, and 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, and the Ramachandran plot shows that 91.8% of the residues of the predicted model are in the most favorable region. The results show that the homology model constructed by SWISS-MODEL has very high precision and a comprehensive and intuitive reasonable conformation. Therefore, the model predicted by SWISS-MODEL can be selected for subsequent experiments.

[0073] 1.3 Disulfide bond design

[0074] 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 between the disulfide bond site pairs is greater than or equal to 10; c) the distance between the SG atoms of cysteine after mutation at the disulfide bond site pairs is within ; d) the Chi3 angle after mutation at 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 are screened based on the above conditions and can be used for experimental verification;

[0075] Table 1

[0076]

[0077]

[0078]

[0079] Example 2: Construction and expression of alkaline protease and its variants

[0080] 2.1 Materials and reagents

[0081] 2.1.1 Strains and vectors:

[0082] The 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 Shanghai Shenggong Company.

[0083] 2.1.2 Enzymes and kits:

[0084] 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.

[0085] 2.1.3 Culture medium:

[0086] Escherichia coli culture medium was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB+Amp medium was LB medium supplemented with ampicillin 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 supplemented with kanamycin to a final concentration of 20 μg / mL.

[0087] 2.1.4 Chemical reagents:

[0088] Casein was purchased from Sinopharm Chemical Reagent Co., Ltd., and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0089] 2.2 Alkaline protease activity assay

[0090] 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.

[0091] 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).

[0092] For gene mutations, the parent sequence and species are disclosed, and subsequent site numbering is based on the parent sequence: the mutated alkaline protease (apr) gene of Bacillus lentus, whose amino acid sequence is shown in SEQ ID NO.1.

[0093] 2.2 Expression of alkaline protease and its variants

[0094] 2.2.1 Alkaline protease apr gene synthesis and vector construction

[0095] 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).

[0096] 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 LBA 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.

[0097] 2.2.2 Introduction of disulfide bond mutations

[0098] Using the above-mentioned pBE-apr as a template, the first cysteine ​​was introduced into the alkaline protease apr gene by PCR. After digestion with DpnI (DpnI endonuclease), the target fragment was recovered by gel excision. The decomposition product was transformed into E. coli TOP10 competent cells by chemical conversion and heat shock. Recombinant transformants were verified by culture PCR. The plasmids of the verified transformants were extracted and sequenced to identify the corresponding mutants, thereby obtaining 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 by PCR. After digestion with DpnI (DpnI endonuclease), the target fragment was recovered by gel excision. The decomposition product was transformed into E. coli TOP10 competent cells by chemical conversion and heat shock. Recombinant transformants were verified by culture PCR. The plasmids of the verified transformants were extracted and sequenced to identify the corresponding mutants, thereby obtaining the expression vector pBE-apr-M1 containing a pair of cysteine ​​mutations. The recombinant Bacillus transformants were obtained by chemical transformation into WB600 chemical transformation competent cells.

[0099] 2.2.3 Shake flask fermentation of disulfide bond-containing mutant strains

[0100] Use a toothpick to pick up the recombinant transformants obtained in step 2 one by one into a 250 mL shake flask. Add 40 mL of TB medium to each shake flask and culture at 37°C, 220 rpm, 85% humidity for about 48 hours. Centrifuge and collect the supernatant. Use affinity chromatography purification to purify the parent alkaline protease gene and mutants respectively to obtain enzyme solutions of each alkaline protease mutant.

[0101] Example 3: Detection of the Optimal Reaction pH of Alkaline Protease Mutants

[0102] At a temperature of 40°C, the enzyme activity of alkaline protease was measured at pH 6.5, pH 7.5, pH 9.0, pH 10.5, and pH 12, respectively. The enzyme activity at the optimal reaction pH of each enzyme was taken as 100%, and the relative enzyme activity of the corresponding enzyme at different pH values ​​was calculated. The results are shown in Table 2.

[0103] 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%.

[0104] Table 2

[0105]

[0106]

[0107]

[0108] As shown in Table 2, the optimal reaction pH for most alkaline protease mutants is 10.5, which is consistent with the optimal reaction pH of the parent alkaline protease. Furthermore, most alkaline protease mutants retain relatively high enzymatic activity at pH 12, compared to the parent alkaline protease. Furthermore, most alkaline protease mutants also exhibit high enzymatic activity at pH 6.5. This indicates that these alkaline protease mutants exhibit good catalytic efficiency not only under alkaline conditions but also under neutral conditions, better meeting the application requirements of the daily chemical detergent industry.

[0109] Example 4: Detection of the Optimal Reaction Temperature of Alkaline Protease Mutants

[0110] Under the condition of pH 10.5, the enzyme activities of the parent alkaline protease and the alkaline protease mutants were measured at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C, respectively. The enzyme activity at the optimal reaction temperature of each enzyme was calculated as 100%, and the relative enzyme activities of the corresponding enzymes at different temperatures were calculated. The results are shown in Table 3.

[0111] Relative enzyme activity refers to the ratio of the activity of an enzyme under certain 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%.

[0112] Table 3

[0113]

[0114]

[0115]

[0116]

[0117] As shown in Table 3, the optimal reaction temperature of some alkaline protease mutants is 60°C, while the optimal reaction temperature of the parent alkaline protease is 50°C. This indicates that some alkaline protease mutants can also have higher enzyme activity under high temperature conditions.

[0118] Example 5: Heat resistance of alkaline protease mutants

[0119] 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 heat resistance test results are shown in Tables 4 and Figure 3 shown. Figure 3 The parent alkaline protease is denoted as the parent, and the alkaline protease mutants 1 to 80 are abbreviated as SEQ NOs: 2 to 81, respectively (the same below).

[0120] Table 4

[0121]

[0122]

[0123]

[0124] From Table 4 and Figure 3 It can be seen that after treatment at 50°C for 24 hours, the alkaline protease mutant provided by the present invention retained an activity of ≥16%, reaching 59%; at this time, the parent alkaline protease was completely inactivated. The alkaline protease mutant provided by the present invention had an activity retention rate significantly higher than that of the parent alkaline protease, and its heat resistance was significantly improved.

[0125] Example 6: Stability test of parent alkaline protease and alkaline protease mutants in commercial detergents

[0126] Purified parent alkaline protease and alkaline protease mutant enzyme solutions were added to a commercial detergent (CM-1) in a specific ratio and mixed thoroughly to achieve an initial alkaline protease activity of 560 u / mL in CM-1. Samples of CM-1 supplemented with the parent alkaline protease and the alkaline protease mutant were then frozen at -18°C and used as the 0-week sample. The remaining detergent was stored in a 37°C oven for 4 weeks. Protazyme AK Tablets (T-PRAK-1000T, packaged in 1000 tablets, with a repeatability error rate of less than 5%) from Megazyme were used. The protease activity in the 0-week sample and the 4-week sample in each group was tested, and the enzyme activity retention rate was calculated. Enzyme activity retention rate = (protease activity of the 4-week sample / protease activity of the 0-week sample) × 100%.

[0127] The CM-1 detergent formula is sodium dodecylbenzene sulfonate (5%), sodium ethoxylated alkyl sulfate (8%), fatty alcohol polyoxyethylene ether-9 (5%), cocamidopropyl betaine (2%), sodium citrate (1%), kasonon (0.1%), sodium chloride (1%), and water (balance).

[0128] The results are shown in Table 5 and Figure 4 shown.

[0129] Table 5 Stability test results of parent alkaline protease and alkaline protease mutants in CM-1 detergent

[0130]

[0131]

[0132]

[0133] From Table 5 and Figure 4 The alkaline protease mutant provided by the present invention retained ≥6% of its enzyme activity after heat storage in a 37°C constant-temperature oven for four weeks in CM-1 detergent, reaching a high of 86%. At this time, the parent alkaline protease was completely inactivated. This significantly improved stability of the alkaline protease mutant provided by the present invention in CM-1 detergent.

[0134] Example 7: Detergent Stability Test at 37°C for 4 Weeks in Commercially Available Detergents A and H

[0135] Purified parent alkaline protease and alkaline protease mutant enzyme solutions were added to commercially available detergent formulations in a specific ratio and mixed thoroughly to achieve an initial alkaline protease activity of 560 u / mL in each detergent formulation. Detergent samples supplemented with the parent alkaline protease and alkaline protease mutant were frozen at -18°C and designated as the week 0 sample. The remaining detergent was stored in a 37°C oven for 4 weeks.

[0136] Based on the GB / T 13174-2021 standard, detergency was tested on JB-02 protein-stained cloth at initial and after four weeks of heat storage, and the corresponding removal ratios were calculated (according to GB / T 13174-2021, removal ratio = sample removal value (%) / standard detergent removal value (%)). By comparing the initial and after four weeks of heat storage removal ratios, the heat storage stability of the parent alkaline protease and the alkaline protease mutants in commercial detergent formulations was investigated.

[0137] The results are shown in Tables 6 and 7.

[0138] Table 6 Decontamination ratios of parent alkaline protease and alkaline protease mutants in commercial detergents AD

[0139]

[0140]

[0141]

[0142]

[0143] Table 7 Decontamination ratios of parent alkaline protease and alkaline protease mutants in commercial detergent EH

[0144]

[0145]

[0146]

[0147]

[0148] The results in Tables 6 and 7 indicate that after four weeks of heat storage, the commercially available detergent formulations containing the alkaline protease mutants of the present invention exhibited higher detergency ratios than the commercially available detergent formulations containing the parent alkaline protease mutants. Furthermore, the detergency ratios were still greater than 1, outperforming standard detergents and demonstrating excellent heat storage stability.

[0149] In summary, compared with the parent alkaline protease, the alkaline protease mutant provided by the embodiment of the present invention has excellent heat resistance, detergent tolerance, stability, and higher catalytic activity on proteins, and can better meet the needs of the daily chemical washing industry.

[0150] 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 Compared with the parental alkaline protease with 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 in the active site of the alkaline protease mutant within the scope; ii. The difference in amino acid site numbers is greater than or equal to 10; iii. The distance between the two cysteine ​​SG atoms that form the 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 with 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 heat storage stability.

3. The alkaline protease mutant according to claim 2, characterized in that The heat storage stability includes the heat 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 with the amino acid sequence shown in SEQ ID NO:1, the alkaline protease mutant has any 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 After the alkaline protease mutant is heat-treated at 50° C. for 24 hours, the enzyme activity retention rate is ≥16%.

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

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

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