High-stability alkaline protease mutant and application thereof in liquid detergent
阅读说明：本技术 一种稳定性高的碱性蛋白酶突变体及其在液体洗涤剂中的应用 (High-stability alkaline protease mutant and application thereof in liquid detergent ) 是由 汪小杰 赵吉斌 张艺达 于 2021-10-05 设计创作，主要内容包括：本发明属于碱性蛋白酶的蛋白质工程改造技术领域,提供一种稳定性高的洗涤用碱性蛋白酶突变体及其在液体洗涤剂中的应用,所述碱性蛋白酶突变体JD109(SEQ ID NO：2)的亲本蛋白酶为枯草芽孢杆菌PB92的碱性蛋白酶或者诺维信的碱性蛋白酶产品Savinase(SEQ ID NO：1)。所述碱性蛋白酶突变包含以下氨基酸的置换：T22A,N42R,S85N,V199L,Q200L,Y203W,S253D,N255W,L256E,其中所述位置对应于氨基酸序列SEQ ID NO：1的多肽的氨基酸位置(根据SEQ ID NO：1进行的编号)。本发明的碱性蛋白酶突变体适合于在清洁或者洗涤剂组合物中使用。本发明的碱性蛋白酶突变体在碱性条件下的酶活比亲本蛋白酶Savinase有显著的提高,同时在不同洗涤剂中的稳定性也有所提高。(The invention belongs to the technical field of protein engineering modification of alkaline protease, and provides a washing alkaline protease mutant with high stability and application thereof in a liquid detergent, wherein the parent protease of the alkaline protease mutant JD109 (SEQ ID NO:2) is alkaline protease of bacillus subtilis PB92 or an alkaline protease product Savinase (SEQ ID NO: 1) of novacin. The alkaline protease mutation comprises the substitution of the following amino acids: T22A, N42R, S85N, V199L, Q200L, Y203W, S253D, N255W, L256E, wherein the positions correspond to the amino acid sequence SEQ ID NO:1 (numbering according to SEQ ID NO: 1). The alkaline protease mutants of the present invention are suitable for use in cleaning or detergent compositions. The alkaline protease mutant of the invention has obviously improved enzyme activity under alkaline conditions compared with parent protease Savinase, and simultaneously has improved stability in different detergents.)
1. A high-stability alkaline protease mutant characterized by: the parent protease of the high-stability alkaline protease mutant is protease of bacillus subtilis PB92, and the high-stability alkaline protease mutant comprises the following amino acid substitutions: T22A, N42R, S85N, V199L, Q200L, Y203W, S253D, N255W, L256E, wherein the positions correspond to the amino acid sequence SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.
2. The highly stable alkaline protease mutant according to claim 1, which is characterized in that: the alkaline protease mutant with high stability also comprises a T22A + N42R + S85N + V199L + Q200L + Y203W + S253D + N255W + L256E substitution combination.
3. The highly stable alkaline protease mutant according to claim 1, which comprises: the parent protease is compared with the amino acid sequence SEQ ID NO:1 has at least 95% sequence identity.
4. The highly stable alkaline protease mutant according to claim 1, which comprises: the parent protease has the amino acid sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.
5. The highly stable alkaline protease mutant according to claim 4, wherein: the parent protease is compared with the amino acid sequence SEQ ID NO:2 having at least 95% sequence identity.
6. A liquid detergent composition characterized by: the alkaline protease mutant with high stability as claimed in claim 1 to 5.
Technical Field
The invention belongs to the technical field of protein engineering modification, and particularly relates to an alkaline protease mutant with high stability and application thereof in a liquid detergent.
Background
Alkaline proteases (alkalineproteases) belong to the group of serine proteolytic enzymes, which are enzymes having an active site serine which initiates hydrolysis of protein peptide bonds (ec 3.4.21), mainly the subtilisin group (ec 3.4.21.62), which belongs to the S8 peptidase family of the MEROPS classification scheme, members of the peptidase S8 family having in their amino acid sequence a catalytic triad which catalyzes the active sites Asp, His and Ser. The alkaline protease hydrolyzes protein peptide bonds, ester bonds and amido bonds under neutral to alkaline conditions, and the optimal action pH of the alkaline protease is generally 8-11. This enzyme species was first found in the pancreas of pigs. Alkaline proteases are widely found in microorganisms, plants and animals. The main application fields of the alkaline protease comprise light industrial fields of detergents, feeds, medicines, leather, soybean processing, breweries, meat tenderization, waste management, photography, diagnosis and the like. The microorganism producing the alkaline protease is mainly separated from alkaline environments such as saline-alkali lakes, deep sea, sand lands and the like. In recent decades, the research on alkaline proteases has been greatly developed with the continuous isolation and purification of new alkaline protease producing strains. So far, Bacillus (Bacillus), Actinomyces (Actinomyces) and fungi have been reported to produce alkaline proteases. The strains currently used for industrial production mainly comprise bacillus licheniformis, bacillus subtilis, bacillus alkalophilus, bacillus amyloliquefaciens and the like (TekinNet. PolJ. Microbiol, 2017, 66(1): 39-56). Alkaline protease alone accounts for 25% of the global enzyme market (mikkelsen mlet. foodandchemibiology, 2015: 07-21). It accounts for a great proportion of the enzyme industry, with protease sales reaching 60% and alkaline protease accounting for 35% of the global enzyme preparation market.
Alkaline proteases were the first enzyme preparations to be used in detergent products. The water solubility of protein macromolecular substances such as blood, milk, eggs, fruit juice, sweat stain, coffee and the like in stains is poor, common surfactants and other builders are difficult to remove, and alkaline protease can decompose the protein macromolecular substances into small molecular peptide bonds which are easy to dissolve in water and then into amino acid, so that the protein macromolecular substances can be easily washed away. Alkaline proteases in detergent products undergo autoenzymatic degradation without stabilizers and inhibitors and gradually lose enzyme activity. To improve the stability of proteases in detergents, Novitin developed a highly potent protease inhibitor 4-FPBA (4-formyl-phenyl-boronicacid) for stabilizing detergent alkaline protease products, and subsequently focused on developing peptide aldehyde-based inhibitors.
Alternatively, amino acid changes have the property that: altering the physicochemical properties of the polypeptide. For example, amino acid changes can improve the thermostability, change substrate specificity, change the pH optimum, etc. of a polypeptide. Essential amino acids in polypeptides can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunninghametal. Science1989, 244.4908: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the resulting mutant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule, in reference to Hilton et al research results (Hilton et al, journal of biological chemistry, 1996, 271.9: 4699-4708.). The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by nuclear magnetic resonance crystallography (crystallography), electron diffraction, or light affinity labeling, as well as by mutation of putative contact site (connected) amino acids, referenced by Devos et al (revolute. science, 1992, 255.5042: 306-. Iterative saturation mutagenesis is also a good method for efficient screening of protease mutations, reference (Reetzetal. Nature protocols, 2007, 2.4: 891-. For BPN (SEQ ID NO:2), the catalytic triad comprising amino acids S221, H64 and D32 is essential for the protease activity of the enzyme.
Increasingly, commercial proteases are protein engineered variants of naturally occurring wild type proteases, for example Everlase, Relase, Ovozyme, Polarzyme, Liquanase, Liquasse Ulta and Kannase (Novozymes), Purafast, PurafactOXP, FN3, FN4 and Eraser @, excelase @, (Genenerg International, Inc.)). The current wild-type alkaline proteases do not meet the washing requirements under different conditions, the performance of the alkaline proteases needs to be further improved, the washing conditions such as temperature and pH vary with time, and many stains are still difficult to completely remove under traditional washing conditions, furthermore, the conditions in washing can lead to enzyme inactivation (e.g. pH, temperature or chelation instability) leading to loss of washing performance during the washing cycle. Therefore, there is still a great demand for alkaline proteases having high washing stability and good washing performance.
The market demand of alkaline protease in China is about 15 billion yuan, which is mainly monopolized by enzyme preparations of Macrocephalin and DuPont, and the main reasons are that the production capacity of alkaline protease production strains in China is poor, the fermentation activity of the enzyme is low, the specific activity of the enzyme is low and the washing application effect is poor. Therefore, the protein engineering transformation and screening of the alkaline protease with high activity, high stability and high titer and the construction of the high-yield engineering bacteria are hot research at home and abroad.
The catalytic activity, acid-base stability, thermal stability, substrate specificity and expression titer of the enzyme can be effectively improved by means of protein engineering (Johannes TWet. Curr. Microbiol, 2006, 9: 261-267). The protein engineering quality modification opens up a new way for the function improvement and the application of the enzyme, and has great success in the fields of industry, agriculture, medicine and the like.
Disclosure of Invention
In view of the above, the invention provides an alkaline protease mutant with high stability, and aims to obtain a mutant protein, improve the alkali resistance, surfactant resistance and stability of the mutant protein, and enable the mutant protein to be better suitable for the detergent industry. The invention provides protein engineering quality modification of an alkaline protease mutant with high stability and the mutant thereof, so that the enzyme activity of the alkaline protease in a washing liquid under an alkaline pH condition is improved, and the washing effect of a detergent is improved. And lays a foundation for better adapting to industrial production. Thereby being beneficial to the wide application of the alkaline protease in the washing field.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention is realized by the following technical scheme: a high-stability alkaline protease mutant, wherein the parent protease of the high-stability alkaline protease mutant is alkaline protease PB92 of Bacillus alcalophilus (Bacillus alcalophilus), and the amino acid sequence corresponds to SEQ ID NO: 1. the amino acid sequence of Lederbergialentus alkaline protease Subtilisin Savinase has 99.7% identity (N85S, 268 of the 269 amino acids identical according to the numbering made according to SEQ ID NO: 1) compared to SEQ ID NO:1, the amino acid sequence of Alkalihalobacillus clausii alkaline protease has 98.9% identity (N85S, S253N, N255K, 266/269 amino acids according to the numbering made according to SEQ ID NO: 1); an amino acid sequence of Bacillus clausii KSM-K16 having 98.5% identity to alkaline protease (N85S, A224V, S250G, S253N, numbering according to SEQ ID NO:1, amino acid 265/269); an amino acid sequence of Bacillus circulans alkaline protease with 97.7% identity (N85S, S97D, S99R, S101A, V102I, numbering according to SEQ ID NO:1, amino acid 263/269); the amino acid sequence of Alkalihalobacillus clausii TH2019 alkaline protease has 96.6% identity (N85S, A224V, S250G, S253N, T254P, S259N, G260R, A266T, T268R, numbering according to SEQ ID NO:1, amino acids 260/269).
The invention provides an alkaline protease mutant with high stability, which comprises an amino acid sequence with at least 90% of identity with SEQ ID NO. 1 and comprises amino acid substitution at a position selected from the following group compared with SEQ ID NO. 1: 22, 42, 85, 199, 200, 203, 253, 255, 256.
In some embodiments of the invention, the amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to seq id No. 2.
In some more specific embodiments, the amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity compared to seq id No. 2.
A liquid detergent composition comprising the protease mutant with high stability.
The liquid detergent compositions prepared according to the present invention use the MGDA and STPP standards.
The invention also provides a preparation method of the alkaline protease mutant with high stability, which comprises the following steps:
step 1: obtaining a DNA molecule encoding a highly stable mutant of alkaline protease comprising an amino acid sequence having at least 90% identity to SEQ ID NO. 1.
Step 2: fusing the DNA molecule obtained in the step 1 with an expression vector to construct a recombinant expression vector and transform a host cell;
and step 3: inducing host cell containing recombinant expression vector to express fusion protein, and separating and purifying the expressed fusion protein.
In some embodiments of the invention, the high stability alkaline protease mutant of step 1 comprises a substitution of an amino acid in the group consisting of: T22A, N42R, S85N, V199L, Q200L, Y203W, S253D, N255W, L256E.
In some embodiments of the invention, the host cell of step 2 is bacillus subtilis.
In some embodiments of the invention, the host cell of step 2 is bacillus licheniformis (bacillus licheniformis).
The invention also provides the application of the alkaline protease mutant with high stability in washing.
The invention provides a combined mutant containing 9 mutation sites in T22A, N42R, S85N, V199L, Q200L, Y203W, S253D, N255W and L256E based on alkaline protease PB92, and the alkaline protease mutant with high stability is named as JD 118. Mutant JD118 retains activity better than the parent protease in extreme conditions when used as a detergent and can be used at higher temperatures and in more alkaline environments, and experiments have shown that: on the premise of not adding any protective agent and stabilizing agent, the alkaline protease and the mutant thereof are insulated for half an hour at 50 ℃, so that the alkaline protease mutant JD118 with high stability is obviously higher than the wild strain in residual activity, and the residual activity of the mutant is higher than that of the wild strain all the time even if the alkaline protease is insulated for a longer time. Without adding any protective agent and stabilizer, the alkaline protease and its mutant were incubated at different pH for 1 hour, and it was found that the residual activity of JD118 mutant (T22A, N42R, S85N, V199L, Q200L, Y203W, S253D, N255W, L256E) was higher than that of the wild enzyme after pH was greater than 9. On the premise of not adding any protective agent and stabilizer, the alkaline protease and the mutant JD118 thereof have the same addition amount, and are added into MGDA and STPP washing systems, the alkaline protease mutant JD118 with high stability has better stability compared with wild type, and the good performance of the alkaline protease mutant JD118 with high stability is beneficial to expanding the application range of the alkaline protease, thereby laying a foundation for better adapting to industrial production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is an alignment chart of amino acid sequences of alkaline protease subtilisin savinase (SEQ ID NO: 1) and alkaline protease variant JD118 (SEQ ID NO:2) with high stability.
FIG. 2: SDS-PAGE electrophoresis of different fermentation times of alkaline protease subtilisin savinase (SEQ ID NO: 1).
FIG. 3: SDS-PAGE electrophoresis images of protein of alkaline protease mutant JD118 (SEQ ID NO:2) with high stability at different fermentation times.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The process of the present invention is further illustrated below with reference to examples, in which experimental procedures not specifying specific conditions may be performed under conventional conditions, such as those described in molecular cloning, a laboratory manual written by J. Sambruk (Sambrook, 2001), etc., or according to conditions recommended by the manufacturer. The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the methods of practicing the present invention should not be limited to the specific method steps described in the examples of the present invention.
The following examples are included to better illustrate and explain the present disclosure, and to enable others skilled in the art to better understand and understand the present disclosure with the aid of the examples. However, the protection of the invention and the scope of the claims are not limited to the examples provided.
Labeling of alkaline protease mutants with high stability: "amino acid substituted at the original amino acid position" is used to indicate a mutated amino acid in the alkaline protease mutant having high stability. If shown in S259K, the amino acid at position 259 is replaced by lys (K) from Ser (S) of the original alkaline protease, and the position numbers correspond to those in the attached sequence list SEQ ID NO: 1.
In the present invention, the nomenclature used for defining amino acid positions is based on the amino acid sequence of the alkaline protease of Bacillus deposited at Genbank under the accession number PB92, which is given in the sequence listing as SEQ ID NO:1 (amino acids 1-269 of SEQ ID NO: 1). Thus, in this context, the bases seq id no:1, starting from a1 (Ala 1) and ending at R269 (Arg 269). SEQ ID NO:1 serves as a standard for position numbering and thus as a basis for naming.
The culture medium involved in the examples of the present invention has the following specific formulation:
LB liquid medium: tryptone 1%, yeast powder 0.5%, NaCl 1%;
LB plate: tryptone 1%, yeast powder 0.5%, NaCl1%, agar 2%;
solution A: 0.4g yeast extract, 0.08g casein hydrolysate, dissolved in 40mL water.
Solution B: 5g glucose, dissolved in 10mL water.
Solution C: 4.8gKH2PO4, 11.2g K2HPO4, 0.16g MgSO4.7H2O, 0.8g trisodium citrate, 1.6g (NH4)2SO4, dissolved in 200mL of water.
Solution D: 0.9g of MnCl2.4H2O, 1.415g of boric acid, 0.68g of FeSO4.7H2O, 13.45mg of CuCl2.2H2O, 23.5mg of ZnSO4.7H2O, 20.2mg of CoCl2.6H2O, 12.6mg of sodium homomolybdate and 0.855g of sodium tartrate were dissolved in 500mL of water.
Solution E: 2.16g MgCl2.6H2O, dissolved in 20mL water.
Solution F: 147mg of CaCl2 was dissolved in 20mL of water.
Solution G: 36.5g sorbitol, dissolved in 100mL water.
GM I: 10mL of solution A, 1.5mL of solution B, 25mL of solution C, 100uL of solution D, 25mL of solution G, and sterile water to 100 mL.
GMII: 98mL of LGM I, 1mL of solution E and 1mL of solution F were mixed well.
GM III: 9mL of LGMII, 1mL of glycerol.
Seed culture medium: yeast extract powder 0.5%, tryptone 0.5%, glucose 1%, K2HPO41.8%；
Fermentation medium: 1-2% of yeast powder, 2-5% of bean cake powder, 5-10% of maltodextrin, 0.1-0.5% of sodium citrate, CaCl20.1~0.5%，MgSO40.1~0.5%，K2HPO40.5~2%。
The enzyme activity determination method of the alkaline protease in the implementation of the invention can adopt the following methods:
method I for measuring enzyme activity of alkaline protease:
the method for measuring the enzyme activity of the alkaline protease comprises the following steps: the method is carried out according to GB/T23527-2009 appendix B Folin method, and the specific reaction process is as follows.
Hydrolyzing casein substrate by protease under certain temperature and pH conditions to generate amino acids (such as tyrosine, tryptophan, etc.) containing phenolic group, reducing Folin reagent (Folin) under alkaline condition to generate molybdenum blue and tungsten blue, and measuring absorbance of the solution at 680nm wavelength by spectrophotometer. The enzyme activity is in direct proportion to the absorbance, so that the enzyme activity of the product can be calculated. The protease activity is defined, namely the protease activity is expressed by protease activity units, is defined as 1g of solid enzyme powder (or 1ml of liquid enzyme), and hydrolyzes casein for 1min under the conditions of certain temperature and pH value to generate 1 mu g of tyrosine, namely 1 enzyme activity unit, which is expressed by mu/g (mu/ml).
Reagents and solutions
(1) Folin (Folin) reagent (Folin: water = 1: 2); (2) 42.4g/L sodium carbonate solution; (3) 0.5mol/L sodium hydroxide solution; (4) boric acid buffer (ph 10.5); (5) 10.0g/L casein solution; (6) 100 mug/mL and 1mg/mL of L-tyrosine standard solution; (7) 6.54% trichloroacetic acid.
Enzyme activity assay
(1) And (3) standard curve preparation: preparing the L-tyrosine standard solution with the concentrations of 0 mu g/mL, 10 mu g/mL, 20 mu g/mL, 30 mu g/mL, 40 mu g/mL and 50 mu g/mL respectively. Respectively taking 1.00ml of each standard solution, adding 5.00ml of 0.4mol/L sodium carbonate solution and 1.00ml of the solution for Forlin reagent, oscillating uniformly, placing in a water bath at 40 ℃ for color development for 20min, taking out a cuvette with the wavelength of 680nm and the thickness of 10mm by using a spectrophotometer, and respectively measuring the absorbance of the cuvette by using a 0 tube without tyrosine as a blank. The absorbance A is plotted on the ordinate and the concentration C of tyrosine on the abscissa, to form a standard curve (the line should pass through zero).
(2) Enzyme activity assay
Taking a proper amount of pre-diluted enzyme solution, adding isovolumetric casein preheated by 10% at 40 ℃, and reacting for 10min at 40 ℃; then trichloroacetic acid (the concentration is 6.54 percent) which is equal to the volume of the reaction system is added into the mixture, the mixture is uniformly mixed, and then the mixture is kept stand for 10min at room temperature to stop the reaction. 1ml of the reaction solution which is terminated is taken out, then 5ml of 42.4g/L sodium carbonate solution is added, 1ml of Folin reagent is added, and color reaction is carried out for 20min at 40 ℃; finally, the OD608 values were determined.
(3) Computing
The enzyme activity of the final dilution of the sample was read from the standard curve in μ/mL. The enzyme activity of the sample was calculated according to the following formula:
X=A×K×4/10×n=2/5×A×K×n
in the formula: x-enzyme Activity of the sample (μ/g or μ/ml)
A-average absorbance of parallel test of samples
K-absorption constant
4-Total volume of reaction reagents (ml)
10-reaction time 10min, in terms of 1min
n is the dilution multiple.
Method II for determining enzyme activity of alkaline protease:
proteolytic activity can be determined by methods employing Suc-AAPF-PNA substrates. Suc-AAPF-PNA is an abbreviation for N succinyl-alanine-proline-phenylalanine-p-nitroanilide, and it is a blocking peptide that can be cleaved by endoproteases, after cleavage, a free PNA molecule is released, and it has a yellow color and can therefore be measured by visible spectrophotometry at a wavelength of 405 nm. Suc-AAPF-PNA substrates were manufactured by Bahent (Bachem) (Cat. L1400 dissolved in DMSO).
The protease sample to be analyzed was diluted in residual activity buffer (100mM Tris pH8.6). The assay was performed by transferring 30. mu.l of the diluted enzyme sample to a 96-well microtiter plate and adding 70. mu.l of substrate working solution (0.72 mg/ml in 100mM Tris pH 86). The solution was mixed at room temperature and the absorption was measured at 0D405nm over 5 minutes every 20 seconds.
The slope of the time-dependent absorption curve (absorbance per minute) under a given set of conditions is directly proportional to the activity of the protease in question. The protease sample should be diluted to a level where the slope is linear.
Example 1: construction and expression of alkaline protease mutant JD118 with high stability: the highly stable alkaline protease mutant of the present invention can be constructed and expressed by methods well known to those skilled in the art.
The amino acid sequence of the alkaline protease mutant JD118 with high stability is SEQIDNO: 2. The optimized nucleic acid sequence of SEQ ID NO. 3 is obtained according to the amino acid sequence of SEQ ID NO. 2, and SEQ ID NO. 3 is synthesized by the company of Biotechnology engineering (Shanghai). The alkaline protease recombinant plasmid pBE2R-AP (JD118) was obtained by ligating SEQ ID NO:3 to the vector pBE2R by the Gibsonassembly method, transferring into E.coli DH 5. alpha. by the heat shock method, and sequencing the plasmid.
The correctly sequenced recombinant plasmid pBE2R-AP (JD118) was transferred into competent cell WB600 by the following specific transformation process: picking WB600 single colonies growing on an LB plate by using a gun head, putting the single colonies into 2 mLGMII, and culturing for 12 h; adding overnight cultured bacterial liquid into 98mLGM I, and culturing at 37 ℃ and 200rpm for about 4 h; adding 10mL of bacterial liquid into 90mLGMII, and culturing at 37 ℃ and 200rpm for about 1.5 h; centrifuging the thallus in ice water bath for 30min at 4000rpm and 4 ℃ for 30min, and removing the supernatant; adding 10mLGM III, and mixing to obtain competent cell WB 600. Then, 5. mu.L of plasmid pBE2R-AP (JD118) was added to 500. mu.L of competent cells, the competent cells were directly subjected to shake culture at 37 ℃ and 200rpm for 1.5 hours, centrifuged at low speed for 3 minutes, and the supernatant was discarded and spread uniformly on a plate of skimmed milk powder medium containing 40. mu.g/mL kanamycin, and cultured in a 37 ℃ incubator for 12 hours. A single colony on the next plate is the recombinant strain WB600/pBE2R-AP (JD118) containing the alkaline protease mutant AP (JD118) with high stability. The alkaline protease mutant bacillus subtilis recombinant engineering bacteria with high stability are inoculated in a 5mLLB liquid culture medium (peptone 1%, NaCl1%, yeast powder 0.5%), subjected to shake culture at 37 ℃ and 200rpm for 12h, and respectively inoculated in a fermentation enzyme production culture medium according to 2% inoculation amount, and subjected to shake culture at 37 ℃ and 200rpm for 84 h.
Example 2: separation and purification of alkaline protease mutants with high stability: after the fermentation is finished, the fermentation liquid is centrifuged at 13000r/min for 15min, and then the supernatant is filtered on a positive pressure filter by a 0.22 mu m membrane to remove the residual bacillus subtilis. The supernatant is used for electrophoresis detection and subsequent enzyme activity detection. The electrophoresis detection method comprises the following steps: 100% trichloroacetic acid (1 kg trichloroacetic acid in 454ml water) was added to the protein sample to give a final concentration of trichloroacetic acid of 13%, mixed and then placed on ice for 30 minutes. 15000g, centrifuge at 4 ℃ for 15 minutes, discard the supernatant, invert it and dry to obtain a protein precipitate. Tris-HCl buffer (50 mM Tris-HCl, 100mM NaCl, pH 8) was added for resuspension, followed by electrophoresis according to the SDS-PAGE method. SDS-PAGE showed a single band of protein samples.
Example 3: analysis of thermostability and pH stability of alkaline protease mutants having high stability: the enzyme activity of alkaline protease and its mutant JD118 was measured, the protein concentration of wild-type alkaline protease and its mutant JD118 was 0.2mg/ml, and the protein buffer was 50mM Tris-HCl, 100mM NaCl, pH 8.0. The enzyme activity determination method is carried out according to method I or method II, the temperature is kept at 50 ℃ for 2.5h, and samples are taken every 0.5h to determine the enzyme activity. The test results are shown in Table 1.
TABLE 1 residual protease activity of wild-type alkaline protease and mutant JD118 incubated at 50 ℃ for various periods of time
On the premise of not adding any protective agent and stabilizing agent, the wild type alkaline protease and the mutant JD118 thereof are incubated at 50 ℃ for half an hour, so that the residual activity of the mutant JD118 is obviously higher than that of the wild bacteria, and the residual activity of the mutant JD118 is always higher than that of the wild bacteria even if the wild bacteria are incubated for a longer time.
Example 4: comparison of the stability of wild-type alkaline protease and of the high-stability alkaline protease mutant JD118 pH: preparing a series of buffer solutions with pH gradient and concentration of 0.2M: na (Na)2HPO4-NaH2PO4(pH6.0-7.0), Tris-HCl (pH8.0-9.0) and Gly-NaOH (pH10.0-12.0), respectively preserving enzyme solution (with the concentration of 0.2 mg/ml) in a series of pH gradient buffer solution systems at 25 ℃ for lh, and determining the enzyme activity by referring to GB/T23527-2009 appendix B Folin method, wherein the results are shown in Table 2.
TABLE 2 Activity of wild-type alkaline protease and mutant at different pH conditions
Without any addition of protective and stabilizing agents, wild-type alkaline protease and its mutant JD118 were incubated at different pH values for 1 hour, it was evident that the residual activity of mutant JD118 was higher than that of wild-type enzyme after pH value was greater than 9.
Example 5: determination of activity of alkaline protease mutants with high stability in liquid detergent:
liquid detergent formulations were prepared as shown in table 3.
TABLE 3 liquid detergent formulations
Both detergents were dissolved in 50mM CHES buffer (N-cyclohexyl-2-aminoethanesulfonic acid) to ensure that the pH was maintained at 10.0 during the experiment and after addition of the protease sample.
10 mul of protease solution with the concentration of 0.2mg/ml and 190 mul of standard detergent solution are mixed in a 1.5ml EP tube, and the enzyme activity is determined according to the appendix B Folin method of GB/T23527-2009, and the result is shown in Table 4.
TABLE 4 protease and its mutant JD118 stability data in STPP and MGDA standard washes
Under the premise of not adding any protective agent and stabilizing agent, the same addition amount of wild alkaline protease and the mutant JD118 thereof are added into MGDA and STPP washing systems, and in the two washing systems, the alkaline protease mutant JD118 with high stability has better stability compared with the wild alkaline protease.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Shanghai robust and sturdy Kaixing Biotech Co., Ltd
<120> alkaline protease mutant with high stability and application thereof in liquid detergent
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 269
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Ala Gly Ser Val Pro Thr Gly Ile Ser Ala Val Gly Ala Pro Ala Ala
1 5 10 15
His Ala Ala Gly Leu Thr Gly Ser Gly Val Leu Val Ala Val Leu Ala
20 25 30
Thr Gly Ile Ser Thr His Pro Ala Leu Ala Ile Ala Gly Gly Ala Ser
35 40 45
Pro Val Pro Gly Gly Pro Ser Thr Gly Ala Gly Ala Gly His Gly Thr
50 55 60
His Val Ala Gly Thr Ile Ala Ala Leu Ala Ala Ser Ile Gly Val Leu
65 70 75 80
Gly Val Ala Pro Ser Ala Gly Leu Thr Ala Val Leu Val Leu Gly Ala
85 90 95
Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gly Gly Leu Gly Thr Ala
100 105 110
Gly Ala Ala Gly Met His Val Ala Ala Leu Ser Leu Gly Ser Pro Ser
115 120 125
Pro Ser Ala Thr Leu Gly Gly Ala Val Ala Ser Ala Thr Ser Ala Gly
130 135 140
Val Leu Val Val Ala Ala Ser Gly Ala Ser Gly Ala Gly Ser Ile Ser
145 150 155 160
Thr Pro Ala Ala Thr Ala Ala Ala Met Ala Val Gly Ala Thr Ala Gly
165 170 175
Ala Ala Ala Ala Ala Ser Pro Ser Gly Thr Gly Ala Gly Leu Ala Ile
180 185 190
Val Ala Pro Gly Val Ala Val Gly Ser Thr Thr Pro Gly Ser Thr Thr
195 200 205
Ala Ser Leu Ala Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala
210 215 220
Ala Ala Leu Val Leu Gly Leu Ala Pro Ser Thr Ser Ala Val Gly Ile
225 230 235 240
Ala Ala His Leu Leu Ala Thr Ala Thr Ser Leu Gly Ser Thr Ala Leu
245 250 255
Thr Gly Ser Gly Leu Val Ala Ala Gly Ala Ala Thr Ala
260 265
<210> 2
<211> 269
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
Ala Gly Ser Val Pro Thr Gly Ile Ser Ala Val Gly Ala Pro Ala Ala
1 5 10 15
His Ala Ala Gly Leu Ala Gly Ser Gly Val Leu Val Ala Val Leu Ala
20 25 30
Thr Gly Ile Ser Thr His Pro Ala Leu Ala Ile Ala Gly Gly Ala Ser
35 40 45
Pro Val Pro Gly Gly Pro Ser Thr Gly Ala Gly Ala Gly His Gly Thr
50 55 60
His Val Ala Gly Thr Ile Ala Ala Leu Ala Ala Ser Ile Gly Val Leu
65 70 75 80
Gly Val Ala Pro Ala Ala Gly Leu Thr Ala Val Leu Val Leu Gly Ala
85 90 95
Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gly Gly Leu Gly Thr Ala
100 105 110
Gly Ala Ala Gly Met His Val Ala Ala Leu Ser Leu Gly Ser Pro Ser
115 120 125
Pro Ser Ala Thr Leu Gly Gly Ala Val Ala Ser Ala Thr Ser Ala Gly
130 135 140
Val Leu Val Val Ala Ala Ser Gly Ala Ser Gly Ala Gly Ser Ile Ser
145 150 155 160
Thr Pro Ala Ala Thr Ala Ala Ala Met Ala Val Gly Ala Thr Ala Gly
165 170 175
Ala Ala Ala Ala Ala Ser Pro Ser Gly Thr Gly Ala Gly Leu Ala Ile
180 185 190
Val Ala Pro Gly Val Ala Leu Leu Ser Thr Thr Pro Gly Ser Thr Thr
195 200 205
Ala Ser Leu Ala Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala
210 215 220
Ala Ala Leu Val Leu Gly Leu Ala Pro Ser Thr Ser Ala Val Gly Ile
225 230 235 240
Ala Ala His Leu Leu Ala Thr Ala Thr Ser Leu Gly Ala Thr Thr Gly
245 250 255
Thr Gly Ser Gly Leu Val Ala Ala Gly Ala Ala Thr Ala
260 265
<210> 3
<211> 807
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
gcacaatcag ttccgtgggg catttcaaga gttcaagcac cggcagcaca taatcgcgga 60
ctggcaggct caggcgttaa agttgcagtt ctggatacag gcattagcac acatccggat 120
ctgagaatta gaggcggagc aagctttgtt cctggcgaac cgtcaacaca agatggcaat 180
ggccatggca cacatgttgc aggcacaatt gcagcactga ataattcaat tggcgttctg 240
ggcgttgcac cgaatgcaga actgtatgca gttaaagttc ttggcgcatc aggcagcggc 300
tcagtttcat caattgcaca aggcctggaa tgggcaggca ataatggcat gcatgttgca 360
aatctgtcac tgggctcacc gtcaccgtca gcaacactgg aacaagcagt taattcagca 420
acatcaagag gcgttcttgt tgttgcagca agcggcaatt caggcgcagg ctcaattagc 480
tatccggcaa gatatgcaaa tgcaatggca gttggcgcta cagatcaaaa taacaataga 540
gcgagcttta gccaatatgg cgcaggcctg gatattgttg cacctggcgt taatctgctg 600
tcaacatggc ctggctcaac atatgcatca ctgaatggca catcaatggc aacaccgcat 660
gtcgcaggcg cagcagcact ggttaaacag aaaaatccgt catggtcaaa tgtccagatt 720
cgcaatcatc tgaaaaatac agcaacaagc ctgggcgata catgggaata tggctcagga 780
cttgttaatg cagaagcagc gacaaga 807