Acid protease mutant, encoding gene therefor, and use thereof

Abstract

Provided are an acid protease mutant, an encoding gene therefor, and a use thereof, relating to the technical field of genetic engineering. Compared with a parent acid protease having an amino acid sequence as shown in SEQ ID NO: 1, the acid protease mutant comprises mutant S60V; and the acid protease mutant has at least 98% and less than 100% sequence identity with the parent acid protease. Compared with the parent acid protease, the provided acid protease mutant exhibits significantly improved thermal stability, and has an enzyme activity retention rate greater than 63% after being treated at 75°C for 3 minutes, with a maximum retention rate of up to 90.6%. The provided acid protease mutant facilitates a use of a protease in industrial production.

Description

Acidic protease mutants, their encoding genes, and applications Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to acidic protease mutants, their encoding genes, and their applications. Background Technology

[0002] Aspartic proteases (EC 3.4.23), also known as aspartic endopeptidases and aspartic acyl proteases, use activated water molecules bound to one or more catalytic aspartic residues to hydrolyze peptide bonds in polypeptide substrates. Unlike serine or cysteine ​​proteases, aspartic proteases do not form covalent intermediates during cleavage. Therefore, proteolysis is completed in one step. Typically, they have two highly conserved aspartic residues at their active site and are most active at acidic pH.

[0003] Therefore, aspartic proteases, also known as acidic proteases, are mainly derived from animal organs and microbial secretions. They are enzymes suitable for hydrolyzing proteins under acidic conditions (pH 2.0-5.0) and are widely used in food, animal husbandry, medicine, leather, and aquatic product processing. Currently, the properties of most acidic proteases are not ideal, with low enzyme activity. This not only leads to resource waste in industrial production and food processing but also limits their application range to some extent. The main reason is the difference between the enzyme's optimal operating conditions and the actual catalytic environment conditions (such as pH and temperature), which reduces the enzyme's catalytic efficiency and thus limits its industrial application.

[0004] The gastrointestinal tract of animals consists of a series of segments, each exhibiting a different pH environment. In monogastric animals such as pigs and poultry, as well as many types of fish, the stomach is potentially highly acidic with a pH as low as 2-3, while the intestine has a more neutral pH of around 6-7.5. Therefore, for proteases to function effectively in the digestive tract, they need to be stable in an acidic environment, able to survive in the gastric environment, and simultaneously be effectively active across a wide range of physiological pH levels within the animal's digestive tract. On the other hand, feed pelleting processes typically require high temperatures, which makes heat-labile proteases prone to inactivation and denaturation.

[0005] Most acidic proteases used in industrial production are fungal acidic proteases. The optimal pH for these enzymes is approximately 3.0; their activity decreases significantly as the pH increases. Furthermore, these enzymes have poor heat tolerance and are easily unstable at temperatures above 50°C, further limiting their applications. Therefore, there is an urgent need to provide acidic proteases with high thermal stability, which is of great significance for both scientific research and industrial applications.

[0006] Summary of the Invention

[0007] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes an acidic protease mutant, its encoding gene, and its applications. Compared to the parental protease, this acidic protease mutant exhibits significantly improved thermostability, which is beneficial for the application of the protease in industrial production.

[0008] This invention provides an acidic protease mutant.

[0009] Specifically, the acidic protease mutant, compared to the parental acidic protease with the amino acid sequence shown in SEQ ID NO: 1, includes the following mutation: S60V; the acidic protease mutant has at least 98% and less than 100% sequence identity with the parental acidic protease, for example, at least 99% sequence identity, and the acidic protease mutant has acidic protease activity.

[0010] Preferably, the acidic protease mutant has at least 99% and less than 100% sequence identity with the parental acidic protease. More preferably, the acidic protease mutant has at least 99.5% and less than 100% sequence identity with the parental acidic protease.

[0011] Preferably, the acidic protease mutant, compared to the parental acidic protease with the amino acid sequence shown in SEQ ID NO: 1, includes at least one of the following mutations (1)-(14):

[0012] (1)S60V, (2)S60V+K119M+A215P, (3)S60V+K119L+A215P, (4)S60V+S181K+A215P+Q294H, (5)S6 0V+K119L+S181K+A215P, (6)S60V+S181K+A215P+N286D, (7)S60V+K119L+N286D, (8)S60V+K119M , (9)S60V+K119M+A215P+Q294H, (10)S60V+S181K+N286D, (11)S60V+S181K+A215P+N286E+Q296H , (12)S60V+N82M+A215P+Q296H, (13)S60V+K119L+S181K+A215P, (14)S60V+N82M+S181K+A215P.

[0013] Preferably, the acidic protease mutant exhibits higher residual enzyme activity after treatment at 60-85°C for 1-5 minutes compared to the parental acidic protease. For example, it exhibits higher residual enzyme activity after treatment at 75°C for 3 minutes.

[0014] Preferably, the acidic protease mutant has an enzyme activity retention rate greater than 63% after treatment at 75°C for 3 minutes compared to the parental acidic protease.

[0015] The present invention also provides a nucleic acid molecule comprising the nucleotide fragments shown in (a) and / or (b):

[0016] (a) The nucleotide fragment encoding the above-mentioned acidic protease mutant;

[0017] (b) A nucleotide fragment that is the reverse complementary to (a).

[0018] The present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0019] The present invention also provides a recombinant cell comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector.

[0020] Preferably, the recombinant cells comprise bacterial or fungal cells.

[0021] More preferably, the fungal cells are Pichia pastoris cells.

[0022] This invention also provides a method for preparing the above-mentioned acidic protease mutant, comprising the following steps:

[0023] (1) Culture recombinant cells;

[0024] (2) Induce the recombinant cells to express the above-mentioned protease mutant.

[0025] The present invention also provides applications of the above-mentioned acidic protease mutant.

[0026] Specifically, the application of the above-mentioned acidic protease mutant in feed, wherein the above-mentioned acidic protease mutant can

[0027] (i) Used alone; or

[0028] (ii) Used in combination with direct-feed microorganisms; or

[0029] (iii) Used in conjunction with at least one other enzyme; or

[0030] (iv) Used in combination with direct-feed microorganisms and at least one other enzyme.

[0031] Preferably, the other enzymes are selected from phytase, α-amylase, galactanase, α-galactosidase, protease, lipase, phospholipase, amylase, phytase, amylopectinase, β-glucanase, cellulase, or xylanase.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] The acidic protease mutant provided by this invention, compared to the parental acidic protease with the amino acid sequence shown in SEQ ID NO: 1, includes at least the mutant S60V and has at least 98% and less than 100% sequence identity with the parental acidic protease. Compared to the parental protease, the acidic protease mutant provided by this invention exhibits significantly improved thermostability, with an enzyme activity retention rate greater than 63% and reaching a maximum of 90.6% after treatment at 75°C for 3 minutes. The acidic protease mutant provided by this invention is beneficial for the application of proteases in industrial production. Detailed Implementation

[0034] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0035] Unless otherwise specified, the biological materials, reagents, or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods. Molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0036] Variant, Mutant: The terms "variant" and "mutant" refer to a polypeptide with protease activity that contains mutations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the parental protease shown in SEQ ID NO:1. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The acidic protease mutant of the present invention has at least 20%, for example at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the protease activity of the mature polypeptide of SEQ ID NO:1.

[0037] Expression: The term "expression" includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be measured—for example, to detect increased expression—using techniques known in the art, such as measuring the level of mRNA and / or translated polypeptide.

[0038] Expression Vectors: The present invention also relates to recombinant expression vectors comprising a polynucleotide, a promoter, and transcription and translation termination signals of a protease mutant of the present invention. Multiple nucleotides and control sequences may be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is positioned in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0039] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0040] The term "direct-feed microorganisms" refers to a source of live (vibrant) naturally occurring microorganisms. Direct-feed microorganisms can include one or more such naturally occurring microorganisms, such as bacterial strains. Categories of direct-feed microorganisms include lactic acid bacteria, lactococci, streptococci, Bacillus, spirochetes, enterococci, Candida albicans, granulomas, propionibacteria, bifidobacteria, clostridium, and megasporidiids, and combinations thereof. Therefore, the term "direct-feed microorganisms" encompasses one or more of the following: direct-feeding bacteria, direct-feeding yeast, direct-feeding yeast, and combinations thereof.

[0041] Fermentable medium: The term "fermentable medium" or "fermentation medium" refers to a medium containing one or more sugars (e.g., two or more), such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides, wherein the medium can be partially converted (fermented) by host cells into a desired product. In some cases, the fermentation medium is derived from a natural source, such as sugarcane, starch, or cellulose; and may be derived from enzymatic hydrolysis pretreatment of such sources. The term fermentation medium is understood herein to refer to the medium prior to the addition of the fermenting organism.

[0042] Host Cells: The present invention also relates to recombinant host cells containing polynucleotides of the invention operably linked to one or more control sequences that direct the production of polypeptides of the invention. A construct or vector containing the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The choice of host cell will depend largely on the gene encoding the polypeptide and its origin.

[0043] The recombinant production of the protease mutant according to the present invention can be carried out in hosts known in the art. Suitable hosts may be selected from filamentous fungal strains, such as Aspergillus niger, Aspergillus sojae, and Aspergillus oryyzae. Suitable hosts may be selected from yeast strains, such as Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha.

[0044] In some embodiments of the present invention, the host cell may also be a prokaryotic cell, including any Gram-positive or Gram-negative bacteria. The bacterial host cell may be any Bacillus genus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceae, Bacillus coagulans, Bacillus sclerotiorum, Bacillus splenium, Bacillus tarda, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0045] The bacterial host cell can also be any Streptomyces cell, including but not limited to: Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces griseus, and Streptomyces lividans cells.

[0046] The cells expressing the protease mutant used in this invention are processed using any method known to those skilled in the art. In some embodiments of this invention, the protease mutant can be expressed in a bacterial host, or the protein can be secreted into the periplasm or extracellular space. When the protease mutant is secreted into the nutrient medium, it can be directly recovered from the medium. If the protease mutant is not secreted, it can be recovered from cell lysates.

[0047] The proteases of the present invention can be recovered using methods known in the art. For example, peptides can be recovered from fermentation media by conventional methods, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the entire fermentation broth containing the protease of the present invention can be recovered.

[0048] The expression organism is cultured according to standard fermentation methods in appropriate volumes. In a preferred embodiment, cells are grown in a fermenter, with growth conditions such as pH, temperature, oxygen, and / or nutrient supply optionally controlled. The first step of purification involves separating the cells from the supernatant using one or more of several techniques such as sedimentation, microfiltration, centrifugation, or flocculation. In a preferred embodiment, microfiltration is a suitable method. If expressed intracellularly, the cells are treated to release the protein from the intracellular space. These treatments may include, for example, pressurization, enzymatic stimulation, osmotic shock, freezing, sonication, or other treatments, to produce a cell extract, which may be further purified or may not be performed.

[0049] In some embodiments of the invention, after induction culture, the protease mutant is secreted into a supernatant. Further purification of the protein from the supernatant or concentrated supernatant can be performed using one or more methods including: extraction or fractionation methods such as ammonium sulfate or ethanol or acid precipitation, or chromatography methods including but not limited to ion exchange, hydrophobic interactions, hydroxyapatite, particle size fractionation by gel filtration, cellulose phosphate or lectin chromatography and affinity chromatography, or any combination thereof. In some preferred methods, the affinity-labeled protein is purified by affinity chromatography with a metal chelating agent to obtain a high-purity target protein. In other preferred embodiments, a high-purity target protein is obtained by HPLC purification.

[0050] In other embodiments of the invention, the supernatant, or the supernatant partially purified by ultrafiltration, or the supernatant concentrated and / or diafiltrated, is further dried by any of the following techniques: such as, but not limited to, spray drying, freeze drying, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyor drying, or any combination thereof.

[0051] In a further embodiment of the invention, the fermented cell suspension containing the expressed protease is dried as a whole using methods such as, but not limited to, fluidized bed drying, conveyor drying, spray drying, or drum drying or any combination thereof.

[0052] In this invention, the application of the protease mutant in the preparation of feed or food includes its use as a feed, food additive, or in the preparation of feed or food. Depending on the use and / or application mode and / or administration mode, the feed or food may be in solution, solid, or semi-solid form. In some embodiments of this invention, when the acidic protease mutant is used as a feed additive,

[0053] (i) Used alone; or

[0054] (ii) Used in combination with direct-feed microorganisms; or

[0055] (iii) Used in conjunction with at least one other enzyme; or

[0056] (iv) Used in combination with direct-feed microorganisms and at least one other enzyme.

[0057] Other enzymes may include, but are not limited to, phytase, α-amylase, galactanase, α-galactosidase, protease, lipase, phospholipase, amylase, phytase, amylopectinase, β-glucanase, cellulase, xylanase, etc.

[0058] At least one other enzyme may also comprise at least one selected from the group consisting of: acetylxylan esterase (EC 3.1.1.23), acylglycerol lipase (EC 3.1.1.72), α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), arabinofuranylase (EC 3.2.1.55), cellobiose hydrolase (EC 3.2.1.91), cellulase (EC 3.2.1.4), ferulic acid esterase (EC 3.1.1.73), galactanase (EC 3.2.1.89), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), β-glucanase (EC 3.2.1.6), β-glucosidase (EC 3.2.1.21), triacylglycerol lipase (EC 3.2.1.23), acetylxylan ester ... 3.1.1.3), lysophospholipase (EC 3.1.1.5), lysozyme (EC 3.2.1.17), α-mannosidase (EC 3.2.1.24), β-mannosidase (mannanase) (EC 3.2.1.25), phytase (EC 3.1.3.8, EC 3.1.3.26, EC 3.1.3.72), phospholipase A1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), phospholipase D (EC 3.1.4.4), protease (EC 3.4), amylopectinase (EC 3.2.1.41), pectin esterase (EC 3.1.1.11), xylanase (EC 3.2.1.8, EC 3.2.1.136), β-xylosidase (EC 3.1.1.3), lysophospholipase (EC 3.1.1.5), lysozyme (EC 3.2.1.17), α-mannosidase (EC 3.2.1.24), β-mannosidase (EC 3.2.1.25), phytase (EC 3.1.3.8, EC 3.1.3.26, EC 3.1.3.72), phospholipase A1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), phospholipase D (EC 3.1.4.4), protease (EC 3.4), amylopectinase (EC 3.2.1.41), pectin esterase (EC 3.1.1.11), xylanase (EC 3.2.1.8, EC 3.2.1. 3.2.1.37) or any combination thereof.

[0059] The term "activity" or "catalytic activity" quantitatively describes the conversion of a given substrate under specified reaction conditions. The term "specific activity" quantitatively describes the catalytic activity relative to the amount of enzyme under specified reaction conditions.

[0060] Example 1: Construction and expression of acidic protease mutants

[0061] Experimental materials and reagents:

[0062] 1. Strains and vectors

[0063] Escherichia coli strain Top10, Pichia pastoris GS115, vector pPIC9K, and antibiotic G418 were purchased from Invitrogen.

[0064] 2. Enzymes and kits

[0065] PCR enzymes, plasmid extraction kits, and gel purification kits were purchased from Shanghai Sangon Biotech Co., Ltd., and restriction endonucleases were purchased from NEB Corporation.

[0066] 3. Culture medium

[0067] E. coli culture medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB-Amp was LB medium with 100 μg / ml ampicillin. LB-Zeocin was LB medium with 25 μg / ml zeocin. Yeast culture medium was YPD (1% yeast extract, 2% peptone, 2% glucose). Yeast induction media were BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (v / v)) and BMMY (except that 0.5% methanol replaced glycerol, the other components were the same as BMGY). Basic salt medium for recombinant yeast fermentation: 5% diammonium hydrogen phosphate, 0.5% potassium dihydrogen phosphate, 1.5% magnesium sulfate heptahydrate, 1.95% potassium sulfate, 0.1% calcium sulfate, and 0.03% antifoaming agent. After autoclaving, 4.35 ml PTM1 was added per liter. PTM1 (trace salt solution): Copper sulfate 0.6%, potassium iodide 0.018%, manganese sulfate monohydrate 0.3%, sodium molybdate dihydrate 0.02%, boric acid 0.002%, cobalt chloride in running water 0.05%, zinc chloride 2%, ferric sulfate heptahydrate 6.5%, concentrated sulfuric acid 0.5%, biotin 0.02%.

[0068] 4. Chemical reagents:

[0069] Acidic protease standard, trichloroacetic acid, sodium carbonate, casein, tyrosine, Folin reagent, and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0070] 5. Acidic protease assay method

[0071] The activity of acidic protease was determined using the Folin-Ciocalteu reagent method (GBT23527-2009). The reaction was carried out at 40℃ and pH 3.0, and the absorbance of the reaction solution was measured at 680 nm. The enzyme activity of acidic protease was calculated based on the standard curve.

[0072] 6. Single-point mutations in acidic proteases

[0073] The amino acid sequence of the parental acidic protease PEP1 (derived from the thermophilic fungus Bisporasp.) of the acidic protease mutant of this invention is shown in SEQ ID NO:1. Using the parental recombinant vector pPIC9K-PEP1 as a template, mutations were performed at nine sites. The amplification primers are shown in Table 1.

[0074] Table 1

[0075] PCR amplification results were detected by agarose gel electrophoresis, and the target PCR product was purified and recovered. Template was digested with restriction endonuclease DpnI, and the digested product was transformed into *E. coli* Top10 competent cells using a chemical transformation heat shock method. Recombinant transformants were verified by colony PCR, and plasmids from verified transformants were extracted and sequenced to identify the corresponding mutants. The sequenced mutant plasmids were linearized with PmeI, the linear plasmid fragments were purified, and transformed into *Pichia pastoris* GS115 competent cells using electroporation. Selection was performed using YPD+G418 medium. Each yeast recombinant transformant was individually transferred to a 24-well plate with 1 mL of BMGY medium in each well. The plates were incubated at 30°C and 220 rpm for approximately 24 hours, followed by centrifugation to remove the supernatant. Then, 1.6 mL of BMGY medium was added for induction culture. After 24 hours of culture, the supernatant was collected by centrifugation, and 200 μL of the supernatant was transferred to each well for analysis of the heat resistance characteristics of acidic proteases.

[0076] Example 2: Determination of thermal stability of single-point mutants

[0077] (1) Test the fermentation enzyme activity of the parent acidic protease without high temperature treatment and each mutant. The enzyme activity of the parent acidic protease is counted as 100%, and the relative fermentation enzyme activity is calculated as follows: relative fermentation enzyme activity = fermentation enzyme activity of mutant / fermentation enzyme activity of parent × 100%.

[0078] (2) Test the parental acidic protease and the thermal stability of each mutant.

[0079] After treating the fermentation supernatant of 24-well plates at 75°C for 3 minutes, the residual enzyme activity was measured and the enzyme activity retention rate was calculated. The enzyme activity retention rate was calculated as: (residual enzyme activity after high-temperature treatment / enzyme activity before high-temperature treatment) × 100%.

[0080] The test results are shown in Table 2. The results indicate that the nine mutants significantly improved the thermostability of the protease. After heat treatment, the residual enzyme activity of the parental acidic protease was only 42.96%, while the thermostability of each mutant was improved to varying degrees, with residual enzyme activity ranging from 48.84% to 59.54% after heat treatment. S60V, N82M, S181K, and A215P showed significant improvements, with retention rates of 55.18%, 56.37%, 59.54%, and 58.85%, respectively. S60V, N82M, A215P, Q294H, N286D, and N286E exhibited higher relative fermentation enzyme activities.

[0081] Table 2

[0082] Example 3: Combinatorial Mutation of Acidic Proteases

[0083] To further enhance the heat resistance of acidic proteases, this invention employs combined mutations at sites that enhance heat resistance to screen for more heat-resistant mutants. Using pPIC9K-PEP1 as a template, multi-site combined mutations were performed. The combined mutation sites were S60V, N82M, K119L, K119M, S181K, A215P, N286D, N286E, and Q294H. These advantageous sites were combined using a multi-site mutation kit, and finally, mutants with improved characteristics were obtained through high-throughput screening, as detailed in Table 3.

[0084] Table 3. Preferred combination mutants and mutation sites of the acidic protease of the present invention.

[0085] According to the acidic protease activity and heat resistance assay methods in Example 2, the enzyme activity and heat resistance of each protease mutant in this invention were measured, and the enzyme activity retention rate and relative fermentation enzyme activity at 75℃-3min were calculated. The test results are shown in Table 4.

[0086] Table 4. Fermentation heat tolerance and relative fermentation enzyme activity of the combined mutants.

[0087] As shown in Table 4, the thermostability of the combined mutants PEP2-PEP14 based on the PEP1 parental acidic protease was significantly improved. Among them, the thermostability of mutants with mutations (2), (3), (5), (6), (7), (8), (9), (11), (12), and (13) reached over 70%, with the highest reaching 90.6%. The enzyme activity retention rates of PEP2, PEP3, and PEP12 remained above 80%. These results demonstrate that the present invention obtained mutants with high thermostability through site mutation.

[0088] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An acidic protease mutant, characterized in that, Compared to the parental acidic protease with the amino acid sequence shown in SEQ ID NO: 1, the acidic protease mutant includes the following mutation: S60V; the acidic protease mutant has at least 98% and less than 100% sequence identity with the parental acidic protease, and the acidic protease mutant has acidic protease activity.

2. The acidic protease mutant according to claim 1, characterized in that, The acidic protease mutant has at least 99% and less than 100% sequence identity with the parental acidic protease.

3. The acidic protease mutant according to claim 1 or 2, characterized in that, The acidic protease mutant, compared to the parental acidic protease with the amino acid sequence shown in SEQ ID NO: 1, includes at least one of the mutations (1)-(14): (1)S60V, (2)S60V+K119M+A215P, (3)S60V+K119L+A215P, (4)S60V+S181K+A215P+Q294H, (5)S6 0V+K119L+S181K+A215P, (6)S60V+S181K+A215P+N286D, (7)S60V+K119L+N286D, (8)S60V+K119M , (9)S60V+K119M+A215P+Q294H, (10)S60V+S181K+N286D, (11)S60V+S181K+A215P+N286E+Q296H , (12)S60V+N82M+A215P+Q296H, (13)S60V+K119L+S181K+A215P, (14)S60V+N82M+S181K+A215P.

4. The acidic protease mutant according to claim 3, characterized in that, Compared with the parental acidic protease, the acidic protease mutant exhibits higher residual enzyme activity after treatment at 60-85°C for 1-5 minutes.

5. The acidic protease mutant according to claim 4, characterized in that, Compared with the parental acidic protease, the acidic protease mutant exhibits an enzyme activity retention rate greater than 63% after treatment at 75°C for 3 minutes.

6. A nucleic acid molecule, characterized in that, Includes the nucleotide fragments shown in (a) and / or (b): (a) A nucleotide fragment encoding the acidic protease mutant of any one of claims 1-5; (b) A nucleotide fragment that is the reverse complementary to (a).

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 6.

8. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecule of claim 6 or the recombinant expression vector of claim 7.

9. The use of the acidic protease mutant according to any one of claims 1-5 in feed, wherein the acidic protease mutant can (i) Used alone; or (ii) Used in combination with direct-feed microorganisms; or (iii) Used in conjunction with at least one other enzyme; or (iv) Used in combination with direct-feed microorganisms and at least one other enzyme.

10. The application according to claim 9, wherein the other enzyme is selected from phytase, α-amylase, galactanase, α-galactosidase, protease, lipase, phospholipase, amylase, phytase, amylopectinase, β-glucanase, cellulase or xylanase.

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