Acid protease mutant as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses an acid protease mutant as well as a coding gene and application thereof. Compared with parent acid protease with an amino acid sequence as shown in SEQ ID NO: 1, the acid protease mutant comprises mutant S60V; the acid protease mutant has at least 98% and less than 100% sequence identity with the parent acid protease. Compared with parent acid protease, the thermal stability of the acid protease mutant provided by the invention is remarkably improved, and the enzyme activity retention rate is greater than 63% after the acid protease mutant is treated at 75 DEG C for 3 minutes and can reach 90.6% to the maximum. The acid protease mutant provided by the invention is beneficial to the application of protease in industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an acidic protease mutant and a coding gene and application thereof. Background Art

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

[0003] Therefore, aspartic protease, also known as acidic protease, is mainly derived from animal organs and microbial secretions. It is an enzyme suitable for hydrolyzing proteins under acidic conditions (pH 2.0-5.0) and is widely used in the fields of food, animal husbandry, medicine, leather and aquatic product processing. At present, the properties of most acidic proteases are not satisfactory, and the enzyme activity is low, which not only leads to resource waste in industrial production and food processing, but also limits its application range to a certain extent. The main reason is the difference between the optimal action conditions of the enzyme and the actual catalytic environmental conditions (such as pH, temperature, etc.), resulting in a decrease in the catalytic efficiency of the enzyme, thereby limiting its industrial application.

[0004] The gastrointestinal tract of animals is composed of a series of sections, each presenting a different pH environment. In monogastric animals such as pigs and poultry and many types of fish, the stomach is highly acidic with a potential pH as low as 2-3, while the intestine has a more neutral pH of around 6-7.5. Therefore, in order for proteases to function in the digestive tract, they need to be stable in an acidic environment, survive in the gastric environment, and at the same time be effectively active under a wide range of physiological pH in the animal's digestive tract. On the other hand, in the feed pelleting process, it is usually necessary to proceed at a higher temperature, which makes proteases with poor thermal stability easily inactivated and denatured.

[0005] Most of the acidic proteases used in industrial production are fungal acidic proteases. The optimal pH value of this type of enzyme is about 3.0. When the pH value increases, the enzyme activity will decrease significantly. In addition, this type of enzyme has poor heat tolerance and is easily unstable when the temperature reaches above 50°C, which further limits the application of proteases. Therefore, it is urgent to provide acidic proteases with high thermal stability, which is of great significance for scientific research and industrial applications. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides an acidic protease mutant and its encoding gene and application. Compared with the parent protease, the thermal stability of the acidic protease mutant is significantly improved, which is conducive to the application of protease in industrial production.

[0007] The present invention provides an acidic protease mutant.

[0008] Specifically, the acidic protease mutant, compared to the parent acidic protease of 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 parent acidic protease, for example, at least 99% sequence identity, and the acidic protease mutant has acidic protease activity.

[0009] SEQ ID NO: 1: APAQIVGRSTFQIDQVASGKVYKNGPMAMMQTYNKYAHVGAVAPAAVVAAAAAAQTGEVSANPEQYDESYLCPVTIGDQTLNLDFFDTGSADLW VFSTLTPSSESTGHTLYNPADSGTEKQGYTWNITYGDGSGAAGVVYADKVVVGGVTATSQAVEAATSVSSEFTQDTKNDGLLGLAFSSINTVQPVQQTT FFDTVKDTLAKKLFTADLKKGAAGSYGFGYIDSSKYTGTITYVPVNNENGFWQFTAGGYSIGGGNGTSGSNATTGSIGTSIADTGTTLLYLPSNVVTAYYKQVSGASYNSAQGGYTYPCGATLPDFNVAIGGKTFVVPGTDLNYAPINSAGTTCFGGIQANTGIGFNIFGDIFLKSVYAVFDQTQSSPRLGFAEQS.

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

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

[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 has a higher residual enzyme activity after treatment at 60-85°C for 1-5 minutes than the parent acidic protease. For example, the acidic protease mutant has a higher residual enzyme activity after treatment at 75°C for 3 minutes.

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

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

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

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

[0018] The present invention also provides a recombinant expression vector, which comprises the above nucleic acid molecule.

[0019] The present invention also provides a recombinant cell, which comprises the above nucleic acid molecule or the above recombinant expression vector.

[0020] Preferably, the recombinant cell comprises a bacterial or fungal cell.

[0021] More preferably, the fungal cell is a Pichia pastoris cell.

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

[0023] (1) Cultivating recombinant cells;

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

[0025] The present invention also provides the application of the acidic protease mutant.

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

[0027] (i) used alone; or

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

[0029] (iii) together with at least one other enzyme; or

[0030] (iv) in combination with a direct-fed microorganism and at least one other enzyme.

[0031] Preferably, the other enzyme is selected from phytase, α-amylase, galactanase, α-galactosidase, protease, lipase, phospholipase, amyloglucosidase, phytase, pullulanase, β-glucanase, cellulase or xylanase.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The acidic protease mutant provided by the present invention, compared with the parent acidic protease of the amino acid sequence shown in SEQ ID NO: 1, at least includes the mutation S60V, and has a sequence identity of at least 98% and less than 100% with the parent acidic protease. Compared with the parent protease, the thermal stability of the acidic protease mutant provided by the present invention is significantly improved, and the enzyme activity retention rate is greater than 63% and can reach up to 90.6% when treated at 75° C. for 3 minutes. The acidic protease mutant provided by the present invention is conducive to the application of protease in industrial production. DETAILED DESCRIPTION

[0034] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be noted that the following embodiments do not limit the protection scope of the present invention.

[0035] Unless otherwise specified, the biological materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or by existing known methods. The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Laboratory Manual (3rd Edition) by J. Sambrook, or according to the kits and product instructions.

[0036] Variant, mutant: The term "variant", "mutant" means a polypeptide having protease activity comprising a mutation (i.e., substitution, insertion and / or deletion) at one or more (e.g., several) positions relative to the position of the parent protease shown in SEQ ID NO: 1. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. The acidic protease mutant of the present invention has at least 20%, such as 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 - e.g., to detect increased expression - by techniques known in the art, such as measuring the levels of mRNA and / or translated polypeptide.

[0038] Expression vector: The present invention also relates to a recombinant expression vector comprising a polynucleotide, a promoter, and transcription and translation termination signals of a protease mutant of the present invention. A plurality of nucleotides and control sequences can be linked together to produce a recombinant expression vector, which can include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by inserting a polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. When producing an expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked to an appropriate control sequence for expression.

[0039] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0040] The term "direct-fed microorganism" refers to a source of live (viable) naturally occurring microorganisms. Direct-fed microorganisms may comprise one or more such naturally occurring microorganisms, such as bacterial strains. Classes of direct-fed microorganisms include lactic acid bacteria, lactococci, streptococci, bacillus, bezoars, enterococci, candida albicans, bacillus, propionibacterium, bifidobacterium, clostridium and macrosporidia and combinations thereof. Thus, the term direct-fed microorganism encompasses one or more of: direct-fed bacteria, direct-fed yeast, direct-fed yeast and combinations thereof.

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

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

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

[0044] In some embodiments of the present invention, the host cell can also be a prokaryotic cell, including any Gram-positive or Gram-negative bacteria. The bacterial host cell can be any Bacillus cell, including but not limited to alkaliphilic Bacillus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus radiata, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

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

[0046] The cells expressing the protease mutants used in the present invention are produced by any method known to those skilled in the art. In some embodiments of the present invention, the protease mutants can be expressed in a bacterial host, or the protein can be secreted into the periplasm or extracellular space. When the protease mutants are secreted into the nutrient medium, they can be directly recovered from the culture medium. If the protease mutants are not secreted, they can be recovered from the cell lysate.

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

[0048] The cultivation of expression organisms is prepared with appropriate volumes according to standard fermentation methods. In a preferred embodiment, cells are grown in fermentor tanks, and optionally controlled growth conditions such as pH, temperature, oxygen and / or nutrient supply. The first step of purification comprises using several technologies such as sedimentation, microfiltration, centrifugation or flocculation to separate cells from the supernatant. In a preferred embodiment, the method applicable is microfiltration. If expressed in cells, cells are processed to release protein from the intracellular gap. These processes may include, for example, pressurization, enzymatic, osmotic shock, freezing, ultrasound or other processing, thereby producing cell extracts, which may be further purified, or may not be carried out.

[0049] In some embodiments of the present invention, the protease mutant is secreted into the supernatant after induction culture, and further protein purification of the supernatant or concentrated supernatant can be performed by one or more of the following methods: extraction or classification methods such as ammonium sulfate or ethanol or acid precipitation, or chromatography, including but not limited to ion exchange, hydrophobic interaction, hydroxyapatite, particle size classification by gel filtration, phosphocellulose or lectin chromatography and affinity chromatography, or any combination thereof. In some preferred methods, the affinity-tagged protein is purified by metal chelator affinity chromatography to obtain a high-purity target protein. In other preferred embodiments, the high-purity target protein is obtained by HPLC purification.

[0050] In other embodiments of the present invention, the supernatant, or the supernatant partially purified by ultrafiltration, or the concentrated and / or diafiltrated supernatant, is further dried by any of the following technical methods: the technical methods are, for example, 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 fermentation cell suspension including the expressed protease is dried as a whole using methods such as, but not limited to, fluid bed drying, conveyor drying, spray drying or drum drying or any combination thereof.

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

[0053] (i) used alone; or

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

[0055] (iii) together with at least one other enzyme; or

[0056] (iv) in combination with a direct-fed microorganism and at least one other enzyme.

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

[0058] The at least one other enzyme may also comprise at least one selected from the group consisting of acetyl xylan esterase (EC 3.1.1.23), acylglycerol lipase (EC 3.1.1.72), α-amylase (EC 3.2.1.1), β-amylase (EC3.2.1.2), arabinofuranosidase (EC 3.2.1.55), cellobiohydrolase (EC3.2.1.91), cellulase (EC3.2.1.4), feruloyl esterase (EC 3.1.1.73), galactanase (EC3.2.1.89), α-galactosidase (EC3.2.1.22), β-galactosidase (EC 3.2.1.23), β-glucanase (EC3.2.1.6), β-glucosidase (EC3.2.1.21), triacylglycerol lipase (EC3.2.1.9), hydroxyethyl esterase (EC3.2.1.10), hydroxyethyl esterase (EC3.2.1.23), hydroxyethyl esterase (EC3.2.1.24), hydroxyethyl esterase (EC3.2.1.25), hydroxyethyl esterase (EC3.2.1.26), hydroxyethyl esterase (EC3.2.1.27), hydroxyethyl esterase (EC3.2.1.28), hydroxyethyl esterase (EC3.2.1.29), hydroxyethyl esterase (EC3.2.1.30), hydroxyethyl esterase (EC3.2 3.1.1.3), lysophospholipase (EC3.1.1.5), lysozyme (EC3.2.1.17), 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 (EC3.1.1.32), phospholipase A2 (EC3.1.1.4), phospholipase D (EC 3.1.4.4), protease (EC 3.4), pullulanase (EC 3.2.1.41), pectinesterase (EC 3.1.1.11), xylanase (EC 3.2.1.8, EC 3.2.1.136), β-xylosidase (EC 3.2.1.37) or any combination thereof.

[0059] The term "activity" or "catalytic activity" quantitatively describes the conversion of a given substrate under defined reaction conditions. The term "specific activity" quantitatively describes the catalytic activity relative to the amount of enzyme under defined 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 enzyme, plasmid extraction kit, and gel purification kit were purchased from Shanghai Bioengineering Company, and restriction endonucleases were purchased from New England Biolabs.

[0066] 3. Culture medium

[0067] The E. coli culture medium is LB (1% peptone, 0.5% yeast extract, 1% NaCL, pH 7.0). LB-Amp is LB medium plus 100 μg / ml ampicillin. LB-Zeocin is LB medium plus 25 μg / ml Zeocin. Yeast culture medium is YPD (1% yeast extract, 2% peptone, 2% glucose). Yeast induction medium BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (v / v)) and BMMY (minus 0.5% methanol instead of glycerol, the rest of the ingredients are the same as BMGY). Recombinant yeast fermentation basic salt medium: 5% diammonium hydrogen phosphate, 0.5% potassium dihydrogen phosphate, 1.5% magnesium sulfate heptahydrate, 1.95% potassium sulfate, 0.1% calcium sulfate, 0.03% defoamer. After high pressure, add 4.35 ml PTM1 per liter. PTM1 (trace salt solution): 0.6% copper sulfate, 0.018% potassium iodide, 0.3% manganese sulfate monohydrate, 0.02% sodium molybdate dihydrate, 0.002% boric acid, 0.05% cobalt chloride in running water, 2% zinc chloride, 6.5% ferrous sulfate heptahydrate, 0.5% concentrated sulfuric acid, and 0.02% biotin.

[0068] 4. Chemical reagents:

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

[0070] 5. Acid protease assay method

[0071] The activity of acidic protease was determined by the Folin phenol reagent method according to GBT23527-2009. The reaction was carried out at 40°C and pH 3.0, the absorbance of the reaction solution was measured at 680 nm, and the activity of the acidic protease was calculated based on the standard curve.

[0072] 6. Single point mutation of acid protease

[0073] The amino acid sequence of the parent acid protease PEP1 (derived from the thermophilic fungus Bispora sp.) of the acid protease mutant of the present invention is shown in SEQ ID NO: 1. The parent recombinant vector pPIC9K-PEP1 was used as a template to mutate 9 sites. The amplification primers are shown in Table 1.

[0074] Table 1

[0075]

[0076]

[0077] Agarose electrophoresis was used to detect the results of PCR amplification, and the target product of PCR amplification was purified and recovered. The template was digested with restriction endonuclease DpnI, and the decomposed product was transferred into Escherichia coli Top10 competent cells by chemical conversion heat shock method. The recombinant transformant was verified by bacterial liquid PCR, and the plasmid of the correct transformant was extracted for sequencing to determine the corresponding mutant. The mutant plasmid with correct sequencing was linearized with PmeI, the linear plasmid fragment was purified, and it was transferred into Pichia pastoris GS115 competent cells by electrotransformation method, and screened by YPD+G418 medium. The obtained yeast recombinant transformants were picked one by one with a toothpick to a 24-well plate, 1 mL of BMGY medium was added to each well, and cultured at 30°C and 220 rpm for about 24 hours, and the supernatant was centrifuged to remove the supernatant. Then 1.6 mL of BMMY medium was added for induction culture. After 24 hours of culture, the supernatant was centrifuged and 200 μL of the above supernatant was taken out to a 24-well plate for analysis of the heat resistance characteristics of acidic protease.

[0078] Example 2: Thermal stability determination of single point mutants

[0079] (1) The fermentation activity of the parent acidic protease and each mutant that was not treated with high temperature was tested respectively. The activity of the parent acidic protease was taken as 100%, and the relative fermentation activity was calculated. Relative fermentation activity = fermentation activity of mutant / fermentation activity of parent × 100%.

[0080] (2) Testing the thermal stability of the parent acidic protease and each mutant

[0081] The fermentation supernatants of the 24-well plates were treated at 75°C for 3 minutes, and then the residual enzyme activity was measured to calculate the enzyme activity retention rate (%, enzyme activity retention rate = residual enzyme activity after high temperature treatment / enzyme activity before high temperature treatment × 100%).

[0082] The test results are shown in Table 2, which show that the 9 mutants have significantly improved the thermal stability of the protease. After heat treatment, the residual enzyme activity of the parent acidic protease is only 42.96%, while the thermal stability of each mutant is improved to varying degrees, with residual enzyme activity of 48.84%-59.54% after heat treatment. Among them, S60V, N82M, S181K, and A215P have significantly improved, with retention rates of 55.18%, 56.37%, 59.54%, and 58.85%, respectively. S60V, N82M, A215P, Q294H, N286D, and N286E have higher relative fermentation enzyme activity.

[0083] Table 2

[0084]

[0085] Example 3, acid protease combination mutation

[0086] In order to further improve the heat resistance of acidic protease, the present invention uses heat resistance-enhanced sites for combined mutation screening of more heat-resistant mutants. Using pPIC9K-PEP1 as a template, multi-site combined mutation is performed. The combined sites are S60V, N82M, K119L, K119M, S181K, A215P, N286D, N286E, and Q294H for combined mutation, and these advantageous sites are combined using a multi-point mutation kit. Finally, a mutant with improved properties is obtained by a high-throughput screening method, as shown in Table 3.

[0087] Table 3 Advantageous combination mutants and mutation sites of acidic protease of the present invention

[0088]

[0089]

[0090] According to the acid protease activity determination and heat resistance determination methods of Example 2, the enzyme activity and heat resistance of each protease mutant in the present invention were determined, and the enzyme activity retention rate at 75°C-3min and the relative fermentation enzyme activity were calculated. The test results are shown in Table 4.

[0091] Table 4 Fermentation heat resistance level and relative fermentation enzyme activity of combined mutants

[0092]

[0093]

[0094] As can be seen from Table 4, the heat resistance levels of the combined mutants PEP2-PEP14 based on the PEP1 parent acidic protease have been significantly improved, among which the heat resistance levels of the mutants with mutations (2), (3), (5), (6), (7), (8), (9), (11), (12) and (13) reached more than 70%, and the highest was 90.6%. Among them, the enzyme activity retention rates of PEP2, PEP3 and PEP12 can still be maintained at more than 80%. The above results show that the present invention obtains mutants with higher thermal stability through site mutation.

[0095] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Acidic protease mutant, characterized in that Compared with the parent acidic protease of the amino acid sequence shown in SEQ ID NO: 1, it includes the following mutation: S60V; the acidic protease mutant has a sequence identity of at least 98% and less than 100% with the parent 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 a sequence identity of at least 99% and less than 100% with the parent acidic protease.

3. The acidic protease mutant according to claim 1 or 2, characterized in that: The acidic protease mutant, compared to the parent acidic protease having the amino acid sequence shown in SEQ ID NO: 1, comprises at least one of mutations (1) to (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 parent acidic protease, the acidic protease mutant has higher residual enzyme activity after being treated at 60-85° C. for 1-5 minutes.

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

6. A nucleic acid molecule, characterized in that Containing the nucleotide fragments shown in (a) and / or (b): (a) a nucleotide fragment encoding the acidic protease mutant according to any one of claims 1 to 5; (b) A nucleotide fragment that is 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 cell comprises the nucleic acid molecule of claim 6 or the recombinant expression vector of claim 7.

9. Use of the acidic protease mutant according to any one of claims 1 to 5 in feed, wherein the acidic protease mutant can (i) used alone; or (ii) in combination with direct-fed microorganisms; or (iii) together with at least one other enzyme; or (iv) in combination with a direct-fed microorganism and at least one other enzyme.

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

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