Acidic protease mutant and its application

Abstract

The present invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically discloses an acidic protease mutant and its application. The present invention is based on genomic DNA from Aspergillus niger, obtains the parent acidic protease gene sequence by PCR amplification, performs random mutation by error-prone PCR, and screens and obtains acidic protease mutants and recombinant strains with improved enzyme activity. After the transformation, under the optimal conditions, the enzyme activity of the acidic protease mutant is 251.64U / mL, which is 23.43% higher than that of the parent acidic protease (203.64U / mL). At the same time, it is not easy to be inactivated during the reaction process, has higher stability, can tolerate strong acid and high salt environment, and shows a higher conversion rate in enzymatic hydrolysis of collagen, and has good application prospects in leather softening.

Description

Technical Field

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

[0002] Protease is one of the three major industrial enzymes, accounting for about 60% of the enzyme market share, and is widely used in food, medicine, washing and feed. Acidic protease is an enzyme that can hydrolyze protein in an environment with a pH value of 2.5-5.0. Acidic protease has a wide range of sources and can be produced by animals, plants and microorganisms. Currently, most commercialized acidic proteases are derived from microorganisms, mainly Aspergillus oryzae, Aspergillus niger and Aspergillus usami. The optimum pH of fungal acidic protease is around 3.0. A large number of research results show that the suitable temperature of acidic protease is generally between 30-50°C, and the optimum action temperature is around 40°C. It has better stability below 50°C. As the acidity decreases and the temperature increases, the enzyme activity decreases, which in turn affects the catalytic efficiency.

[0003] Leather softening is an important step in the leather industry. It can improve comfort, improve hand feel, enhance durability, increase design flexibility, etc., which is of great significance to improving the quality and performance of leather. Acidic proteases soften leather by hydrolyzing proteins in raw hides, especially collagen. In the softening process, microwave radiation is usually used to improve the softening efficiency and speed up the softening process, but microwave irradiation will reduce the activity of acidic proteases, thereby affecting the maximum speed of the enzymatic reaction. In addition, the deashing, softening and pickling processes are usually combined in the leather production process, which requires acidic proteases to have strong acid resistance and enzyme activity stability, and can also adapt to other process conditions in the integrated process, such as high temperature, high sodium chloride concentration, etc. Therefore, improving the enzymatic activity, stability and acid resistance of acidic proteases is of great significance to the leather industry.

[0004] Protein engineering is to change or design a new enzyme primary structure or amino acid sequence through gene recombination technology to obtain a conformationally stable enzyme. Protein engineering is the most fundamental method to improve the stability of enzyme activity. The activity, stability, selectivity and other characteristics of the enzyme are essentially related to the structure of the enzyme. The structure determines the properties. Changing the bacterial gene or amino acid sequence of the enzyme through protein engineering to obtain a more active and stable enzyme is the fundamental means to improve the stability of enzyme activity.

[0005] Based on this, the present invention is proposed. Summary of the invention

[0006] Based on the above reasons, the purpose of the present invention is to provide an acidic protease mutant, which is screened through a large number of mutations based on the parent acidic protease to ultimately obtain an acidic protease mutant with significantly improved enzyme activity and stability.

[0007] The present invention is achieved through the following technical solutions:

[0008] An acidic protease mutant, the amino acid sequence of the acidic protease mutant is shown as SEQ ID NO.1.

[0009] The present invention also provides an acidic protease mutant gene encoding the above-mentioned acidic protease mutant.

[0010] The present invention also provides a recombinant expression vector, which carries the gene of the acidic protease mutant.

[0011] The present invention also provides a recombinant host cell, wherein the recombinant host cell is a host cell transformed / transfected with the above-mentioned recombinant expression vector.

[0012] Preferably, the host cell is Pichia pastoris.

[0013] The invention also provides the application of the acidic protease mutant in the degradation of proteins and peptide chains.

[0014] Furthermore, the application is enzymatic hydrolysis of collagen.

[0015] The invention also provides the application of the acidic protease mutant in the textile and leather industry, food industry, feed industry, alcohol industry and medical field.

[0016] Furthermore, the method is used for softening leather in the textile and leather industry.

[0017] The present invention also provides an enzyme preparation, which comprises the above-mentioned acidic protease mutant.

[0018] The present invention has the following beneficial effects:

[0019] The present invention uses error-prone PCR technology to carry out directed evolution of the acid protease DHQT from Aspergillus niger in vitro, and screens and obtains the acid protease mutant DHQTM and its recombinant strain with improved enzyme activity. After the transformation, under the optimal conditions, the enzyme activity of the acid protease DHQTM is 251.64U / mL, which is 23.43% higher than that of the parent acid protease DHQT (203.64U / mL). At the same time, it is not easy to be inactivated during the reaction process, has higher stability, can tolerate strong acid and high salt environment, and shows a higher conversion rate in enzymatic hydrolysis of collagen, and has good application prospects in leather softening. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments.It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.Unspecified conditions in the embodiments are carried out according to the conditions of normal conditions or manufacturer recommendations.Reagents used or instruments that do not specify manufacturers are conventional products that can be purchased and obtained commercially.

[0021] The terms involved in the present invention are explained as follows:

[0022] 1. Parent protease: refers to the protease that can produce the mutant of the present invention after undergoing the mutation described in the present invention, namely the acidic protease DHQT.

[0023] 2. Protease mutant: refers to a mutated protease having one or more mutations in the sequence compared to the amino acid sequence of the parent protease. The mutant is expressed in a host cell by transferring the mutant gene into a plasmid.

[0024] 3. Host cell: refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with a vector containing a nucleic acid of the present invention; it covers any progeny of a parent cell that is not completely identical to the parent cell due to mutations that occur during replication.

[0025] 4. Protease activity is defined as: the amount of protease required to hydrolyze casein to produce 1 μg of tyrosine in 1 min at 25°C and pH = 3.0, which is 1 protease activity unit U.

[0026] 5. The identification of the acidic protease mutant uses "amino acid replaced by the parent amino acid position" to indicate the mutated amino acid in the acidic protease mutant. For example, N12F means that the amino acid at position 12 is replaced by F from N of the parent acidic protease DHQT, and the position number corresponds to the number in SEQ ID NO:4 in the attached sequence table.

[0027] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] The first embodiment of the present invention provides an acidic protease mutant, the amino acid sequence of the acidic protease mutant is shown in SEQ ID NO.1. The invention adopts error-prone PCR technology, utilizes PCR to amplify the acidic protease DHQT gene from Aspergillus niger, adjusts PCR reaction conditions, changes the mutation frequency in the amplification process, randomly mutates DNA fragments, constructs an acidic protease mutation library, connects the mutant gene fragments with an expression vector, transforms them into a host cell after recombination, and screens out mutants with significantly improved enzyme activity. It is known through sequencing that the acidic protease mutant DHQTM comprises 7 mutation sites compared with the parent acidic protease DHQT: N12F, Q113S, L151M, G191L, S207D, A243F, V311I, that is, asparagine, glutamine, leucine, glycine, serine, alanine and valine at the 12th, 113th, 151st, 191st, 207th, 243rd and 311th sites of the amino acid sequence of the acidic protease DHQT are mutated to phenylalanine, serine, methionine, leucine, aspartic acid, phenylalanine and isoleucine, respectively.

[0029] The embodiments of the present invention also provide the gene coding of the above-mentioned acidic protease mutant and a recombinant expression vector carrying the gene coding, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.2. The vector backbone of the recombinant expression vector described in the present invention can be selected by those skilled in the art according to the implementation needs, and can be any vector with expression ability disclosed or undisclosed in the art, including but not limited to eukaryotic vectors and prokaryotic vectors. Preferably, it is a pPIC9K plasmid, and the protease gene of the present invention is inserted into a suitable site of the expression vector so that its nucleotide sequence can be operably connected to the expression regulatory sequence.

[0030] The present invention also provides a host cell comprising the above-mentioned recombinant expression vector, wherein the host cell is Pichia pastoris. The base cell of the host cell can be any cell type with expression ability disclosed or undisclosed in the art, including but not limited to eukaryotic cells or prokaryotic cells; preferably Pichia pastoris, more preferably Pichia pastoris KM71.

[0031] The embodiment of the present invention also provides the application of the acidic protease mutant in the degradation of proteins and peptide chains; the application is enzymatic hydrolysis of collagen. The acidic protease mutant obtained by the present invention can effectively hydrolyze collagen and convert it into small-molecule collagen peptides, which is helpful for its application in different products; and because of the improved enzyme activity, it can work at a lower pH value and temperature, which helps to maintain the biological activity and functionality of collagen peptides; it can also improve the solubility of collagen, which is more conducive to the extraction and application of collagen; compared with traditional chemical methods, the use of acidic protease for collagen extraction and hydrolysis reduces the use and discharge of harmful chemicals and is more environmentally friendly.

[0032] The embodiment of the present invention also provides the application of the above acidic protease mutant in the textile leather industry, food industry, feed industry, alcohol industry and medical field. The enzyme activity, acid resistance and stability of the acidic protease obtained by the present invention are all improved. In the textile leather industry, it is suitable for leather softening and cleaning in a strong acid environment. The acidic protease can also help remove grease and fiber interstitial in raw hides, which can improve the cleanliness and quality of leather; in the food industry, it can be used to improve the taste and nutritional value of dairy products, flour products and meat products; in the feed industry, it can be used as an additive to promote the digestion and absorption of protein by animals and improve the utilization rate of feed; in the alcohol industry, it can improve the wine yield of raw materials on the one hand, and accelerate the fermentation speed and shorten the fermentation cycle on the other hand; in the medical field, it can be used to prepare biological drugs and diagnostic reagents, which is helpful for the treatment and diagnosis of diseases.

[0033] In some specific embodiments, the acidic protease mutants of the present invention are used in the textile leather industry to soften leather. Acidic proteases can effectively hydrolyze proteins in raw hides, especially collagen, to soften leather, which is essential for improving the softness and fullness of leather. At the same time, they can also remove dirt on the leather surface and residual skin dirt in the leather, especially excess residual fat, to improve product quality.

[0034] The present invention also provides an enzyme preparation comprising the acidic protease mutant.

[0035] The present invention will be described in more detail below through specific examples. Unless otherwise specified, in the following examples,

[0036] The culture medium and related determination methods used in the present invention are as follows:

[0037] 1. Preparation of culture medium

[0038] LB medium: 1% tryptone, 0.5% yeast powder, 1% NaCl, pH 7.2±0.2;

[0039] LB plate: 1% tryptone, 0.5% yeast powder, 1% NaCl, 2% agar powder, pH 7.2±0.2;

[0040] YPD medium: 1% yeast extract, 2% tryptone, 2% glucose;

[0041] YPD plate: 1% yeast extract, 2% tryptone, 2% glucose, 2% agar powder;

[0042] MD medium: 1.34% YNB, 10-5% biotin, 2% glucose;

[0043] MD plate: 1.34% YNB, 10-5% biotin, 2% glucose, 2% agar powder;

[0044] BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 4×10-5% Biotin, 1% glycerol (v / v), 100 mmol / L potassium phosphate buffer (pH 6.0);

[0045] BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 4×10-5% Biotin, 3% methanol, 100 mmol / L potassium phosphate buffer (pH 6.0).

[0046] 2. Protease activity determination method: refer to the Folin method for the determination of protease activity in the national standard "GB / T 23527.1-2023 Quality requirements for enzyme preparations Part 1: Protease preparations"; place the casein solution in a 40℃ constant temperature water bath for preheating for 5 minutes, take 1mL of protease solution (preheated at 40℃ for 2 minutes) and mix it with 1mL of casein substrate (10.0g / L, pH=3.0), react at 40℃ for 10 minutes, then add 2mL of trichloroacetic acid solution to terminate the reaction (0.4mol / L), centrifuge the suspension to take 1mL of supernatant, add 5mL of sodium carbonate solution (0.4mol / L) and 1mL of Folin solution for color development at 40℃ for 20 minutes, and then determine the protease activity according to the absorbance at 680nm.

[0047] The following will disclose specific embodiments for implementing the present application, as well as corresponding comparative examples to demonstrate the relevant technical effects of the present application.

[0048] Example 1 Obtaining the parent acidic protease gene

[0049] The protease gene sequence from Aspergillus niger was codon optimized to obtain the optimized parent acidic protease DHQT gene sequence (as shown in SEQ ID NO: 3) and its amino acid sequence (as shown in SEQ ID NO: 4). A biological company was then commissioned to synthesize the above gene sequence and amplify it by PCR. The PCR primers and reaction conditions are as follows:

[0050] DHQT upstream primer: 5'-GCGTCGACAGCAAAGGCAGCGCGGTG-3'

[0051] DHQT downstream primer: 5'-GCGTCGACCGCCTGCGCCGCAAAGC-3'

[0052] PCR reaction system: DNA template 1μL, 10μM upstream primer 2μL, 10μM downstream primer 2μL, 10× Buffer (with Mg 2+ )5μL, 10mM dNTP 1μL, Pfu DNA Polymerase 1μL, ddH2O up to50μL;

[0053] The amplification program was as follows: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 3 min, 20 cycles; extension at 72°C for 10 min;

[0054] The PCR amplification product was subjected to 1% (w / v) agarose gel electrophoresis, and the gel was cut and recovered to obtain the complete target gene fragment, which was stored in a -20°C refrigerator for later use. After being digested with Sal I, it was connected with the vector pPIC9K to obtain the recombinant plasmid pPIC9K-DHQT, which was then transformed into Escherichia coli JM109 and Pichia pastoris KM71.

[0055] Example 2 Construction of an acidic protease mutant library and screening of highly active acidic protease mutants

[0056] (1) Random mutagenesis based on error-prone PCR technology to construct an acid protease mutant library

[0057] The primers were designed to be the same as the upstream and downstream primers of the parent acidic protease DHQT, and error-prone PCR was performed using the parent acidic protease genome as a template.

[0058] The amplification reaction system is as follows: DNA template 1μL, 10μM upstream primer 2μL, 10μM downstream primer 2μL, 10×PCRBuffer 5.0μL, 0.2mM dATP 0.1μL, 0.2mM dGTP 0.1μL, 1mM dCTP 0.5μL, 1mM dTTP 0.5μL, rTaq DNA polymerase 0.5μL, 25mM MgCl2 (10mM) 20μL, 5mM MnCl2 (0.3mM) 3.0μL, ddH2O up to 50μL;

[0059] The amplification program was as follows: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s, annealing at 50°C for 30 s, extension at 72°C for 3 min, 30 cycles; extension at 72°C for 12 min;

[0060] The PCR amplification product was subjected to 1% agarose gel electrophoresis, and the PCR product was recovered using a small amount of DNA recovery kit to obtain the gene DHQT-Mx of the acid protease with random mutations (x is the number of the random mutation gene).

[0061] (2) Screening of highly active acidic protease mutants

[0062] The acid protease random mutation gene DHQT-Mx was connected to the expression vector pPIC9K to obtain a recombinant vector, which was transformed into Escherichia coli JM109 to extract its plasmid to obtain the recombinant plasmid pPIC9K-DHQT-Mx, and then the recombinant plasmid was transformed into the host cell Pichia pastoris KM71 by the competent cell method. After screening, a recombinant strain KM71 / pPIC9K-DHQT-M with the highest enzyme activity was obtained. The specific transformation and screening process is as follows:

[0063] Prepare Pichia competent medium, 100 mM lithium acetate, 0.6 mM sorbitol, 10 mM Tris-HCl, and adjust the final pH to 7.5; separately prepare 10 mM dithiothreitol and 1 M sorbitol;

[0064] 1) Streak Pichia pastoris KM71 on a YPD plate and culture at 30°C for 3-4 days until a single colony grows. Then pick a single colony and inoculate it into YPD liquid medium and culture it in seed liquid for 16-18 hours.

[0065] 2) Inoculate 250 mL shake flasks containing 50 mL YPD liquid medium at a 1% inoculum volume, control OD600 at 1.2-1.5, centrifuge and discard the supernatant, add 8 mL treatment solution (160 μL dithiocarbyl alcohol), stand at room temperature for 30 min, wash three times with 2.5 mL sorbitol, resuspend and mix with 1 M sorbitol, and then aliquot and store at -80°C;

[0066] 3) Prepare 4 g of the linear plasmid pPIC9K-DHQT-Mx to be transformed and the thawed competent system, mix well and place them on ice for 15 min together with the electroporation cup, and also place 1 M sorbitol on ice for precooling, put them into the electroporator for electroporation, the electroporation parameters are: 1500V, 25μF, 200 Ω, quickly add 200μL of 1M sorbitol solution to the electroporation cup, then add 800μL of MD medium, mix well and transfer to a 1.5mL EP tube, activate on a 30℃ shaker for 2h, centrifuge the bacterial suspension to remove the supernatant, add 80μL of MD medium to resuspend, spread on the MD plate, invert in a 30℃ incubator, and culture until a single colony appears to obtain a recombinant strain with transformants;

[0067] 4) Pick a single colony on the MD plate with transformants, spot it on the YPD plate according to the number, and place the YPD plate in a 30±2℃ incubator for 2-3 days until a single colony appears. Pick the transformants from the YPD plate according to the number and inoculate them into a centrifuge tube containing 3mL of BMGY medium, and culture them in a shaking incubator at 30±2℃ and 200rpm for 48h; centrifuge the bacterial solution cultured in the shaking incubator for 48h at 3000×g for 15min, remove the supernatant, add 1mL of BMMY medium containing 0.5% methanol to the centrifuge tube, and induce culture at 30±2℃ and 220rpm; after induced culture for 48h, centrifuge at 3000×g for 5min, take the supernatant for enzyme activity detection, and screen out transformants with high protease activity. Finally, a recombinant strain KM71 / pPIC9K-DHQT-M with significantly improved acid protease activity was screened.

[0068] (3) Determination of sequence

[0069] The acidic protease gene sequence was extracted from the recombinant strain obtained by screening and sequenced (Shanghai Biotechnology Co., Ltd.), and the coding gene was named DHQTM (as shown in SEQ ID NO: 2). Sequencing showed that the acidic protease mutant DHQTM contained 7 mutation sites compared with the parent acidic protease DHQT: N12F, Q113S, L151M, G191L, S207D, A243F, and V311I.

[0070] Example 3 Determination of enzymatic properties of acidic protease mutants

[0071] Preparation of enzyme solution: The recombinant strain KM71 / pPIC9K-DHQT-M and the parent acidic protease strain were streaked on a YPD plate and inverted at 30°C. The activated single colony was picked and placed in 5 mL of YPG medium, and cultured at 30°C and 220 r / min for 48 h. The inoculation amount was transferred to 50 mL of BMGY liquid medium until OD600 reached 2-6, and then transferred to 50 mL of BMMY liquid medium, and cultured at 30°C and 220 r / min. 0.5% methanol was added every 12 h to induce expression for 48 h. The fermentation broth was centrifuged at 4°C and 12000 r / min for 2 min, and the supernatant was taken as the acidic protease enzyme solution.

[0072] Optimum temperature test: The enzyme solutions of the parent protease DHQT and its acidic protease mutant DHQTM were taken and placed in water baths at 40°C, 45°C, 50°C, 55°C, 60°C, and 65°C, respectively, and the enzyme activity of each acidic protease was determined under pH 3.0. The results are shown in Table 1. The highest enzyme activity at the measured temperature is 100%, the optimum enzyme activity temperature of the parent acidic protease DHQT is 40°C, and 90% of the enzyme activity is maintained below 45°C. The optimum temperature of the acidic protease mutant DHQTM is 45°C, and 90% of the enzyme activity can be maintained at 50°C. At the optimum enzyme activity temperature, the mutant enzyme activity is 47.72 U / mL higher than that of the parent protease. It can be seen that the acidic protease mutant DHQTM of the present invention improves the temperature tolerance.

[0073] Optimum pH test: The enzyme solution of the parent protease DHQT and its acidic protease mutant DHQTM was taken, and the enzymatic activity of each acidic protease was measured at 45°C under different pH values ​​of 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0. The results are shown in Table 1. The highest enzyme activity at the measured pH is 100%. The optimal pH of the parent acidic protease DHQT and the acidic protease mutant DHQTM is 3.0, but the enzyme activity of the mutant is higher in a strong acid environment.

[0074] Table 1 Results of enzymatic properties determination of acidic protease mutant enzymes

[0075] .

[0076] Example 4 Stability determination of acidic protease mutants

[0077] Temperature stability determination: The enzyme solutions of the parent protease DHQT and its acidic protease mutant DHQTM were placed in a water bath at 40°C, 50°C, and 60°C for 2 hours, and the residual enzyme activity was calculated with the enzyme activity without heat preservation treatment as 100%. The results are shown in Table 2. The overall stability of the acidic protease mutant DHQTM is better than that of the parent acidic protease DHQT. When kept at 60°C for 2 hours, its residual enzyme activity exceeded 50%, which was 16.84% higher than that of the parent acidic protease DHQT.

[0078] pH stability determination: The enzyme solutions of the parent protease DHQT and its acidic protease mutant DHQTM were stored at pH 1.0, 2.0, and 3.0 for 48 hours, and the residual enzyme activity was calculated with the enzyme activity without storage treatment as 100%. The results are shown in Table 2. The acidic protease mutant DHQTM has higher stability in a strong acid environment. When stored at room temperature for 48 hours at pH 1.0, its residual enzyme activity is 67.12%, which is 11.89% higher than that of the parent acidic protease DHQT.

[0079] Salt concentration stability determination: The enzyme solutions of the parent protease DHQT and its acidic protease mutant DHQTM were stored at room temperature for 48 hours at sodium chloride concentrations of 20, 25, and 30%, respectively. The residual enzyme activity was calculated with the enzyme activity without storage treatment as 100%. The results are shown in Table 2. The acidic protease mutant DHQTM showed higher stability in high-concentration salt solutions. When stored at room temperature for 48 hours at a sodium chloride concentration of 30%, its residual enzyme activity was 33.49%, which was 8.35% higher than that of the parent acidic protease DHQT.

[0080] In summary, it can be seen that the acidic protease mutant DHQTM of the present invention is less likely to be inactivated during a long reaction period than the parent acidic protease DHQT, has higher temperature and pH stability, and can tolerate strong acid and high-concentration salt solutions.

[0081] Table 2 Stability test results of acidic protease mutants

[0082] .

[0083] Example 5 Detection of collagen hydrolysate

[0084] Weigh 10 g of sheepskin collagen, dissolve it in deionized water and make it up to 250 mL, then adjust its pH to 3.0 with 2 mol / L HCl, first heat the solution in a water bath to 45 °C, then add the corresponding amount of acidic protease mutant DHQTM according to the enzyme-substrate ratio of 3000 U / g, inactivate the enzyme in a boiling water bath for 15 minutes after 4 h of enzymolysis, quickly cool to room temperature, centrifuge at 4000 r / min for 15 min, take the supernatant and use ultrafiltration membrane with molecular weight cutoff range of 10 kDa, 6 kDa and 2 kDa to separate the enzymatic hydrolyzate into 4 components with different molecular weight ranges, namely, components with molecular weight greater than 10 kDa, components with molecular weight between 6 kDa and 1 wDa, components with molecular weight between 2 kDa and 6 kDa, and components with molecular weight less than 2 kDa, dry after separation, and calculate the mass percentage of components in each interval based on the dry peptide basis, assuming that the mass of all peptides is 100%. Comparative Example Sheepskin collagen was treated in the same manner, except that acidic protease DHQT was used for enzymatic hydrolysis. The test results are shown in Table 3.

[0085] Collagen is a macromolecular protein with a molecular weight of about 30 wDa. After hydrolysis of collagen, low molecular weight collagen peptides are obtained. The molecular weight distribution is generally below 10 kDa, ranging from several hundred Daltons to several thousand Daltons. The molecular weight of most collagen peptides is between 2-6 kDa. As can be seen from Table 3, compared with the enzymatic treatment with acidic protease DHQT, the molecular weight mass percentage of small molecular collagen peptides obtained after enzymatic treatment with acidic protease mutant DHQTM is significantly increased, especially the content of 2-6 kDa collagen peptides. It can be seen that the modified acidic protease mutant has a higher conversion rate for collagen.

[0086] Table 3

[0087] .

[0088] Example 6 Study on the performance of leather softened by acid protease

[0089] Take 1kg of naked sheepskin after deliming, clean it to remove impurities and dirt on the surface, put it into a drum, add about 5kg of water, adjust the temperature to 35℃, add 300IU / g of acidic protease mutant DHQTM, rotate for about 60 minutes, drain the liquid, wash it with water, and follow the conventional processing steps such as pickling, chrome tanning, retanning, fatliquoring and drying. Determine the softness of the obtained crust leather, observe the degree of grain damage and evaluate its overall sensory performance. Another group was softened by adding acidic protease DHQT, and the rest of the steps were the same. The softening properties of the two groups of crust leather were tested, and the results are shown in Table 4.

[0090] It can be seen from Table 4 that after softening treatment with the deliming acidic protease mutant enzyme preparation of the present invention, the obtained leather has higher softness, almost no grain damage and better overall sensory performance, indicating that the acidic protease mutant DHQTM obtained in the present application has good application prospects in leather softening.

[0091] Table 4 Crust leather softening performance test results

[0092] .

[0093] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. An acidic protease mutant, characterized in that The amino acid sequence of the acidic protease mutant is shown in SEQ ID NO.

1.

2. An acidic protease mutant gene, characterized in that: Encoding the acidic protease mutant according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector carries the gene according to claim 2.

4. A recombinant host cell, characterized in that The recombinant host cell is a host cell transformed / transfected with the recombinant expression vector according to claim 3.

5. The recombinant host cell according to claim 4, characterized in that The host cell is Pichia pastoris.

6. Use of the acidic protease mutant according to claim 1 in the degradation of proteins and peptide chains.

7. The use according to claim 6, characterized in that The application is enzymatic hydrolysis of collagen.

8. Application of the acidic protease mutant according to claim 1 in enzymatic protein hydrolysis in the textile and leather industry, food industry, feed industry, alcohol industry and medical field.

9. The use according to claim 8, characterized in that The invention is used in the textile and leather industry to soften leather.

10. An enzyme preparation, characterized in that The enzyme preparation comprises the acidic protease mutant according to claim 1.