Aspartic protease heat-resistant mutant and application thereof

Abstract

The invention provides an aspartic protease heat-resistant mutant and application thereof. Wherein the aspartic protease heat-resistant mutant comprises (a) a protein which is based on wild type aspartic protease pap1 as shown in SEQ ID NO: 1, is subjected to amino acid mutation on at least one of the following sites: G133, D159, S376, T198, T103, T144, L303 or A387, has aspartic protease activity and has higher heat resistance than that of the wild type aspartic protease pap1; or (b) a protein which has more than 80% of homology with the amino acid sequence limited in (a), has aspartic protease activity and has heat resistance higher than that of wild type aspartic protease pap1. The aspartic protease can solve the problem of poor heat resistance of aspartic protease in the prior art, and is suitable for the field of enzyme catalysis.

Description

Technical Field

[0001] The present invention relates to the field of enzyme catalysis, and in particular to a heat-resistant mutant of aspartic protease and its application. Background Art

[0002] Aspartic protease, also known as acid protease, is an enzyme that degrades proteins under acidic conditions. Its optimal pH is 2-4, its relative molecular mass is 30,000-40,000, and its isoelectric point is 3.0-5.0. Acid protease is widely used in feed, brewing, leather, and other fields, and has broad application prospects. Adding a certain amount of acid protease to the fermentation process of liquor brewing helps degrade protein in the corn raw material, accelerates the utilization of saccharifying enzymes and the growth and reproduction of yeast, shortens fermentation time, and reduces costs. Furthermore, the addition of protease to feed can effectively convert large, difficult-to-use proteins into amino acids or oligopeptides that are easier for animals to absorb, thereby improving feed conversion efficiency.

[0003] Acid proteases come from a wide range of sources, including plant, animal, and microbial sources. The most widely used acid proteases in the market are primarily derived from Aspergillus niger and Trichoderma reesei. Compared to animal and plant sources, a notable feature of microbial acid proteases is their diversity and complexity; a single strain can often secrete one or more acid proteases.

[0004] Acidic proteases derived from Trichoderma reesei have achieved large-scale production and application, but most have poor thermal stability. Generally, when the temperature rises above 55°C, the enzyme activity is severely lost, which limits the enzyme's wider application. Therefore, there is an urgent need to obtain mutants of acidic proteases derived from Trichoderma reesei with improved heat resistance to further promote the application of this enzyme in the market. Summary of the Invention

[0005] The main purpose of the present invention is to provide a heat-resistant mutant of aspartic protease and its application, so as to solve the problem of poor heat resistance of aspartic protease in the prior art.

[0006] To achieve the above-mentioned object, according to a first aspect of the present invention, a thermostable mutant of aspartic protease is provided, which comprises: (a) a protein based on the wild-type aspartic protease pap1 shown in SEQ ID NO: 1, which has undergone amino acid mutation at at least one of the following sites: G133, D159, S376, T198, T103, T144, L303 or A387, and has aspartic protease activity and higher heat resistance than the wild-type aspartic protease pap1; or (b) a protein having more than 80% homology with the amino acid sequence defined in (a), has aspartic protease activity, and higher heat resistance than the wild-type aspartic protease pap1.

[0007] Furthermore, the amino acid mutations in (a) are each independently selected from the following: G133 mutates to G133C; D159 mutates to D159K; S376 mutates to S376K; T103 mutates to T103C; T144 mutates to T144C; T198 mutates to T198C; L303 mutates to L303C; A387 mutates to A387C; wherein, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0008] Furthermore, (b) is a protein having more than 85%, preferably more than 90, more preferably more than 95%, 96%, 97% or 98%, and further preferably more than 99%, 99.9% homology with the amino acid sequence defined in (a), having aspartic protease activity, and having heat resistance higher than that of wild-type aspartic protease pap1.

[0009] Furthermore, the mutation of the thermostable mutant of aspartic protease includes any one of the following amino acid mutations: G133C, D159K, S376K, T103C+T144C, G133C+T198C, L303C+A387C, G133C+S376K or G133C+T198C+S376K.

[0010] In order to achieve the above object, according to the second aspect of the present invention, a DNA molecule is provided, which encodes the above-mentioned thermostable mutant of aspartic protease.

[0011] In order to achieve the above object, according to the third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

[0012] In order to achieve the above object, according to a fourth aspect of the present invention, a host cell is provided, wherein the host cell is transformed with the above recombinant plasmid or contains the above DNA molecule.

[0013] Furthermore, the host cell includes a eukaryotic cell; preferably, the eukaryotic cell includes Pichia pastoris; preferably, Pichia pastoris is a Pichia pastoris having integrated into its genome a DNA capable of expressing a thermostable mutant of aspartic protease containing the G133C+T198C+S376K mutation; the above-mentioned Pichia pastoris is classified and named Pichia pastoris X33 Pichiapastoris X33, and was deposited in the China Center for Type Culture Collection on January 12, 2024, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC NO: M 2024092.

[0014] In order to achieve the above-mentioned purpose, according to the fifth aspect of the present invention, there is provided a use of the above-mentioned heat-resistant mutant of aspartic protease, the above-mentioned DNA molecule, the above-mentioned recombinant plasmid or the above-mentioned host cell in the preparation of feed, food or leather.

[0015] Furthermore, the application includes adding the heat-resistant mutant of aspartic protease to feed that needs to be heated and pelleted.

[0016] By applying the technical solution of the present invention, based on the spatial structure of the wild-type aspartic protease pap1 protein (PDB ID: 3C9X), the spatial conformational forces, key catalytic residues, regional distribution characteristics of amino acid residues and other properties of the wild-type aspartic protease were analyzed and simulated in detail using Discovery Studio software. Ultimately, a strategy of introducing disulfide bonds and increasing the rigidity of flexible regions was selected for design and modification. The structural stability of the mutant was predicted by the software's site-directed simulated mutagenesis and site-directed saturation simulated mutagenesis functions. The actual application effect of the mutant was then verified through experiments, and the above-mentioned active sites that have a greater impact on the heat resistance of the protein were discovered. Ultimately, an aspartic protease mutant with improved heat resistance was obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0018] Figure 1 The figure shows the enzyme activity test result according to Example 5 of the present invention. DETAILED DESCRIPTION

[0019] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0020] As mentioned in the background, aspartic proteases have been produced and used on a large scale in the prior art. However, most have poor thermal stability. Generally, when the temperature rises above 55°C, the enzyme activity is severely lost, limiting the enzyme's application in a wider range of fields. In this application, the inventors attempted to mutate wild-type aspartic proteases and discovered multiple key active amino acid sites, thereby obtaining the aforementioned various thermostable mutants of aspartic proteases, which can improve the heat resistance of aspartic proteases. Consequently, a series of protection schemes are proposed in this application.

[0021] In a first typical embodiment of the present application, a thermostable mutant of aspartic protease is provided, which thermostable mutant of aspartic protease comprises: (a) a protein based on the wild-type aspartic protease pap1 shown in SEQ ID NO: 1, which has undergone amino acid mutation at at least one of the following sites: G133, D159, S376, T198, T103, T144, L303 or A387, and has aspartic protease activity and higher heat resistance than the wild-type aspartic protease pap1; or (b) a protein having more than 80% homology with the amino acid sequence defined in (a), has aspartic protease activity, and higher heat resistance than the wild-type aspartic protease pap1.

[0022] The amino acid sequence shown in SEQ ID NO: 1 is the wild-type aspartic protease pap1 protein derived from Trichoderma reesei QM6a.

[0023] Based on the spatial structure of the aspartic protease (PDB ID: 3C9X), this application used DiscoveryStudio software to conduct a detailed and comprehensive analysis and simulation of its spatial conformational forces, key catalytic residues, and regional distribution characteristics of amino acid residues. Ultimately, a design modification strategy was selected, using the introduction of disulfide bonds and the improvement of the rigidity of flexible regions. The software's site-directed and saturation mutagenesis functions were used to predict the structural stability of the mutants. The actual application effects of the mutants were then verified experimentally, ultimately resulting in aspartic protease mutants with improved heat resistance. It was found that active sites such as G133, D159, S376, T198, T103, T144, L303, and A387 also significantly affect the protein's heat resistance. By mutating these amino acid sites, proteins with aspartic protease function and enhanced heat resistance can be obtained. For the proteins obtained above, changes can be made at non-critical mutation sites and active sites to obtain proteins with at least 80% homology to the above amino acid sequence and possessing aspartic protease function and heat resistance.

[0024] SEQ ID NO: 1:

[0025] MQTFGAFLVSFLAASGLAAALPTEGQKTASVEVQYNKNYVPHGPTALFKAKRKYGAPISDNLKSLVAARQAKQALAKRQTGSAPNHPSDSADSEYITSVSIGTPAQVLPLDFDTGSSDLWVFSSETPKSSATGHAIYTPSKSSTSKKVSGASWSISYGDGSSSSSGDVYTDKVTIGGFSVNTQGVESATRVSTEFVQDTVISGLV GLAFDSGNQVRPHPQKTWFSNAASSLAEPLFTADLRHGQNGSYNFGYIDTSVAKGPVAYTPVDNSQGFWEFTASGYSVGGGKLNRNSIDGIADTGTTLLLLD DNVVDAYYANVQSAQYDNQQEGVVFDCDEDLPSFSFGVGSSTITIPGDLLNLTPLEEGSSTCFGGLQSSSGIGINIFGDVALKAALVVFDLGNERLGWAQK.

[0026] In a preferred embodiment, the amino acid mutations in (a) are independently selected from the following: G133 mutates to G133C; D159 mutates to D159K; S376 mutates to S376K; T103 mutates to T103C; T144 mutates to T144C; L303 mutates to L303C; A387 mutates to A387C; T198 mutates to T198C; wherein the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0027] In a preferred embodiment, (b) is a protein that has more than 85%, preferably more than 90%, more preferably more than 95%, 96%, 97% or 98%, and further preferably more than 99% or 99.9% homology with the amino acid sequence defined in (a), has aspartic protease activity, and has higher heat resistance than wild-type aspartic protease pap1.

[0028] In this application, the applicant has continued to explore the above-mentioned active site and found that mutations in the active site to different amino acids can result in different protein activities, and that specific mutations can enhance the heat-resistant activity of aspartic proteases. Through experimental exploration, it was found that performing the above-mentioned specific mutations in the active site can produce aspartic proteases with enhanced heat resistance. For the amino acid mutation sites of aspartic proteases, flexible selection and combination can be made among the above-mentioned mutations.

[0029] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0030] Substitution and replacement rules generally state that amino acids with similar properties will have similar effects after substitution. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0031] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0032] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0033] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0034] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0035] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0036] In a preferred embodiment, the mutation of the thermostable mutant of aspartic protease comprises any one of the following amino acid mutations: G133C, D159K, S376K, T103C+T144C, G133C+T198C, L303C+A387C, G133C+S376K, or G133C+T198C+S376K. The above "+" indicates that the mutant simultaneously carries multiple amino acid mutations.

[0037] The above amino acid mutations were all tested and explored in the examples of this application, and all had good heat resistance. Compared with the parent having the amino acid sequence shown in SEQ ID NO: 1, a heat-resistant mutant of aspartic protease with improved heat resistance can be obtained.

[0038] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned thermostable mutant of aspartic protease.

[0039] In a third typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0040] The DNA can encode the thermostable mutant of aspartic protease and can be linked to a recombinant plasmid to form a circular DNA. Both the DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc. to obtain the thermostable mutant of aspartic protease.

[0041] In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell is transformed with the above-mentioned recombinant plasmid or contains the above-mentioned DNA molecule.

[0042] In a preferred embodiment, the host cell includes a eukaryotic cell; preferably, the eukaryotic cell includes Pichia pastoris; preferably, the Pichia pastoris is a Pichia pastoris having integrated into its genome a DNA capable of expressing a thermostable mutant of aspartic protease containing G133C+T198C+S376K mutations; the Pichia pastoris is classified and named Pichia pastoris X33 Pichia pastoris X33, and was deposited in the China Center for Type Culture Collection on January 12, 2024, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC NO: M 2024092.

[0043] Utilizing the above-mentioned host cells, recombinant plasmids can be replicated within the host cells, and the DNA molecules carried on the recombinant plasmids can also be transcribed and translated to obtain a large number of thermostable mutants of aspartic proteases. Utilizing existing technologies, the host cells can be fragmented for protein purification, followed by crude enzyme catalysis after fragmentation, or other methods to obtain thermostable mutants of aspartic proteases, which can then be used for subsequent catalytic utilization. The host cells are not of plant origin. To ensure that the thermostable mutants of aspartic proteases can fold correctly, preferred host cells include eukaryotic cells.

[0044] In a fifth typical embodiment of the present application, there is provided a use of the above-mentioned heat-resistant mutant of aspartic protease, the above-mentioned DNA molecule, the above-mentioned recombinant plasmid or the above-mentioned host cell in the preparation of feed, food or leather.

[0045] In a preferred embodiment, the application comprises adding the thermostable mutant of aspartic protease to feed that needs to be heat-pelleted.

[0046] The above-mentioned heat-resistant mutant of aspartic protease also has good aspartic protease activity at higher temperatures, which is significantly improved compared to the wild-type protein. Therefore, it is applied to the preparation of industries such as feed, food, and leather, and can withstand higher temperature environments during use, normally exerting the activity of the enzyme, without considering the impact of higher temperatures on the enzyme activity during production, which is beneficial to practical applications in industrial production. Especially in the preparation of feed, for feed that needs to be heated and granulated after mixing, aspartic proteases in the prior art are affected by high temperatures during heating and granulation, and the protein structure and activity have irreversible effects, which reduces the performance of aspartic protease activity in the feed. If the influence of temperature on protease activity is to be prevented, it is necessary to change the production industry, increase production steps and costs. However, the above-mentioned heat-resistant mutant of aspartic protease can directly undergo steps such as heating and granulation after feed mixing, can withstand high temperature processing environments, ensure the activity of protease in feed, and reduce production steps and production costs.

[0047] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0048] Example 1 Construction of wild-type aspartic protease expression plasmid

[0049] According to the Pichia pastoris codon optimization, the wild-type Trichoderma reesei aspartic protease gene shown in SEQ ID NO: 2 (encoding amino acid sequence is SEQ ID NO: 1) was obtained by whole gene synthesis technology (Wuhan Aoke Dingsheng Biotechnology Co., Ltd.), and the recombinant plasmid pUC-pap1 was obtained.

[0050] The pap1 gene fragment was amplified by PCR using the recombinant plasmid pUC-pap1 as a template and primers P1 / P2 (primer sequences are shown in Table 1). The pPICZαA vector backbone was amplified by PCR using the plasmid pPICZαA as a template and primers P3 / P4 (primer sequences are shown in Table 1). The recombinant vector was constructed using the Gibson Double Fragment Assembly Kit. The ligated product was transformed into competent Escherichia coli Top10 cells, and the transformed bacterial suspension was plated on LB solid medium (containing 25 μg / mL Zeocin). After overnight culture, transformants were selected for colony PCR verification. Plasmids from positive transformants were isolated and sent for sequencing to obtain the recombinant expression plasmid pPICZαA-pap1.

[0051] SEQ ID NO: 2:

[0052]

[0053] Experimental Materials:

[0054] Strains and vectors: Escherichia coli Top10, Pichia pastoris X33 and Pichia pastoris expression vector pPICZαA were purchased from Invitrogen.

[0055] Enzymes and kits: SacI endonuclease was purchased from Thermo; PrimeSTAR Max DNA Polymerase was purchased from TaKaRa; Gibson double fragment assembly kit was purchased from Novozymes Biotech Co., Ltd.; plasmid extraction kit and gel recovery kit were purchased from Omega.

[0056] Reagents: Zeocin was purchased from Shanghai Maokang Biotechnology Co., Ltd.; yeast powder and peptone were purchased from OXOID; Folin reagent, sodium carbonate, trichloroacetic acid, lactic acid, and sodium lactate were purchased from Xilong Chemical Co., Ltd.; NaCl and glucose were purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; glycerol was purchased from Tianjin Benchmark Chemical Reagent Co., Ltd.; biotin was purchased from Beijing Solebold Technology Co., Ltd.; other reagents can be purchased from general biochemical reagent companies.

[0057] Culture medium:

[0058] The culture medium for E. coli was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0);

[0059] Yeast medium YPD (1% yeast extract, 2% peptone, 2% glucose);

[0060] Yeast medium BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 4×10-5% Biotin, 1% glycerol (V / V), 1% potassium phosphate buffer (pH 6.0));

[0061] Yeast methanol induction medium BMMY (1% yeast extract, 2% peptone, 1.34% YNB, 4×10-5% Biotin, 1% methanol (V / V), 1% potassium phosphate buffer (pH 6.0)).

[0062] Example 2 Site-directed mutagenesis of genes

[0063] The present invention uses multiple strategies to screen mutation sites with higher stability. Disulfide bond modification and B-factor value analysis were performed on the wild-type Trichoderma reesei aspartic protease (PDB: 3C9X), and the flexible region was rationally designed and modified. Virtual saturation mutation analysis of the protein was performed using Discovery Studio software. According to the calculated energy values of the simulated mutants, the following mutations were selected at positions 133, 198, and 376 of the Trichoderma reesei aspartic protease: G133C at position 133; A135C at position 135; G158K or G158R at position 158; D159K or D159R at position 159; S376K at position 376; and G377K at position 377. or G377R; T103C+T144C at position 103 and 144; A135C+S201C at position 135 and 201; G133C+T198C at position 133 and 198; L303C+A387C at position 303 and 387; G133C+S376K at position 133 and 376; G133C+T198C+S376K at position 133, 198 and 376.

[0064] Based on the mutation sites identified above, each aspartic protease mutant was generated by introducing a point mutation via PCR using the recombinant plasmid pPICZαA-pap1 as a PCR amplification template (primers are shown in Table 1). For the construction of the pPICZαA-pap1(G133C) plasmid, the PCR system is shown in Table 2.

[0065] Table 1 Primer sequences used

[0066]

[0067]

[0068] Table 2 PCR reaction system

[0069] Components Volume (μL) Prime STAR Mix 20 Template DNA (30 ng / μL) 2 10 μM forward primer 2 10 μM reverse primer 2 <![CDATA[ddH2O]]> 14 Total volume 40

[0070] PCR amplification conditions: 98°C for 30 seconds; 98°C for 10 seconds, 55°C for 15 seconds, 72°C for 1 minute / 2 kb, 30 cycles; 72°C for 4 minutes. The PCR template was pPICZαA-pap1, and the primers were G133C-F and G133C-R. After PCR product recovery, it was digested with Dpn I at 37°C for 2 hours to remove the template DNA. The product was then dissolved in 30 μL of sterile ddH2O and transformed into Top10. Single colonies were selected for sequencing, and the plasmid that had been sequenced correctly was isolated and used for future use.

[0071] The remaining recombinant mutant plasmids were constructed in the same way, and the obtained plasmids were named as follows: pPICZαA-pap1(G133C), pPICZαA-pap1(A135C), pPICZαA-pap1(G158K), pPICZαA-pap1(G158R), pPICZαA-pap1(D159K), pPICZαA-pap1(D159R), pPICZαA-pap1(S376K), pPICZαA-pap1(G377 K), pPICZαA-pap1(G377R), pPICZαA-pap1(T103C+T144C), pPICZαA-pap1(A135C+S201C), pPICZαA-pap1(G133 C+T198C), pPICZαA-pap1(L303C+A387C), pPICZαA-pap1(G133C+S376K), pPICZαA-pap1(G133C+T198C+S376K).

[0072] Example 3 Construction of recombinant Pichia pastoris strains containing wild-type and mutant aspartic protease

[0073] (1) Preparation of Pichia pastoris X33 competent cells

[0074] Streak Pichia pastoris X33 onto a YPD plate and incubate at 30°C for 48 hours. After inoculating a single colony, inoculate it into 5 mL of liquid YPD medium and incubate overnight at 30°C with a shaker at 220 rpm. When the initial OD600 of the inoculated shake flask is 0.2, inoculate the test tube culture into 30 mL of liquid YPD medium in a 250 mL shake flask and incubate at 30°C until the OD600 reaches 0.8-1.2. Centrifuge the culture at 8500 g for 30 seconds at 4°C. Remove the supernatant and resuspend the cells once in 20 mL of pre-chilled sterile water and three times in 20 mL of pre-chilled 1 M sorbitol. Collect the cells by centrifugation at 8500 g for 30 seconds at 4°C and resuspend in approximately 200 μL of 1 M sorbitol. Prepare competent cells and keep on ice until ready to use.

[0075] (2) Preparation of transformation fragments

[0076] Before transformation, the recombinant plasmids constructed in Example 2 were digested with Sac I endonuclease. The digestion system was as follows: 5 μg of plasmid, 5 μL of Sac I, 5 μL of 10× Fast Digest buffer, and dd H₂O to 50 μL. Digestion was performed at 37°C for 2 h. The resulting fragments were analyzed by 1% agarose gel electrophoresis. The bands were recovered and dissolved in 20 μL of sterile dd H₂O. The fragments were stored at -20°C until use.

[0077] (3) Conversion

[0078] Transfer 1 μg of the recovered fragment to be transformed to competent cells, mix thoroughly, and transfer to a pre-chilled 0.1 cm electroporation cuvette. Cover and incubate on ice for 5 minutes before electroporation. Electroporation voltage: 1.5 kV. Immediately after electroporation, add 1 mL of pre-chilled 1 M sorbitol to resuspend the cell, transfer to a 1.5 mL EP tube, and incubate at room temperature for 2 hours. Centrifuge at 7000 rpm for 3 minutes, remove the supernatant, and spread approximately 200 μL of the bacterial suspension on a YPD plate (containing 100 μg / mL Zeocin) and incubate at 30°C for 72 hours. Single colonies from the plate were selected for PCR verification to confirm positive transformants. This method yielded the wild-type aspartic protease Pichia pastoris expression strain X33 / pPICZαA-pap1 and other aspartic protease mutant Pichia pastoris recombinant strains.

[0079] Among them, the Pichia pastoris whose genome is integrated with DNA capable of expressing a thermostable mutant of aspartic protease (G133C+T198C+S376K) is classified and named Pichia pastoris X33, and was deposited in the China Center for Type Culture Collection on January 12, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: M 2024092.

[0080] Example 4 Induced fermentation of recombinant strains

[0081] The recombinant strains of the wild-type and mutant aspartic protease from Example 3, frozen at -80°C, were streaked onto YPD plates containing 100 μg / mL Zeocin. Single colonies with good growth were selected and inoculated into YPD test tube culture. After overnight culture, the cells were inoculated into BMGY medium at a 1% ratio. After incubation at 30°C for 24 hours, the cells were inoculated into 30 mL of BMMY medium. Fermentation was induced at 28°C, with pure methanol added at 0.5% v / v every 24 hours. Fermentation was terminated after 4 days of induction. The cells were harvested by centrifugation, and the supernatant was obtained.

[0082] Example 5 Enzyme activity detection

[0083] The enzyme activity assay method is based on the National Food Safety Standard GB1886.174-2016, "Food Additives, Enzyme Preparations for the Food Industry." Enzyme activity is defined as the ability of 1 gram or 1 mL of enzyme to hydrolyze casein for 1 minute under specific temperature and pH conditions to produce 1 μg of tyrosine. This is expressed as 1 unit of enzyme activity, expressed as U / g (U / mL).

[0084] The main reagents used in the test are:

[0085] (1) Folin solution: Mix one part of Folin-phenol reagent with two parts of water and shake well;

[0086] (2) Sodium carbonate solution (42.4 g / L): Weigh 42.4 g of anhydrous sodium carbonate (Na2CO3), dissolve it in water and dilute to 1000 mL;

[0087] (3) Trichloroacetic acid (65.4 g / L): Weigh 65.4 g of trichloroacetic acid, dissolve it in water and dilute to 1000 mL.

[0088] (4) Sodium lactate buffer (pH = 3.0): Take 4.71 g of lactic acid (80%-90%) and 0.89 g of sodium lactate (70%), add water to 900 mL, and stir until homogeneous. Adjust the pH to 3.0 ± 0.05 with lactic acid or sodium lactate, and make up to 1000 mL.

[0089] (5) Casein solution (10.0 g / L): Weigh 1.000 g of standard casein (NICPBP National Pharmaceutical Standard Material) to the nearest 0.001 g, moisten it with a small amount of concentrated lactic acid, add approximately 80 mL of the corresponding buffer solution, and heat in a boiling water bath for 30 min, stirring occasionally until the casein is completely dissolved. After cooling to room temperature, transfer the solution to a 100 mL volumetric flask and dilute to the mark with an appropriate pH buffer solution. Before diluting to volume, check and adjust the pH to the specified value of the corresponding buffer solution. This solution should be stored in a refrigerator and is valid for 3 days.

[0090] The steps of the enzyme activity detection method are as follows: dilute the sample to be tested with sodium lactate buffer solution, take 1 mL of the diluted enzyme solution and preheat it in a 40°C water bath for 2 minutes, add the casein solution that has been preheated in a 40°C water bath for 5 minutes, mix well and react in a 40°C water bath for 10 minutes. After the reaction is completed, take out and add 2 mL of trichloroacetic acid solution, mix well and let it stand for 10 minutes, and filter with slow qualitative filter paper. Take 1 mL of the filtrate and add 5 mL of sodium carbonate solution, mix well, add 1 mL of folin phenol solution, color it in a 40°C water bath for 20 minutes, and then measure the absorbance at a wavelength of 680 nm. Calculate the enzyme activity of aspartic protease according to the standard curve. The enzyme activity test results are as follows. Figure 1 As shown, WT is the enzyme activity test result of the host cells without exogenous genes, the control group (control) is the enzyme activity test result of the aspartic protease wild-type pap1, and the relative enzyme activity is the enzyme activity expressed based on the enzyme activity test result of the wild-type pap1.

[0091] Example 6 Heat resistance performance measurement

[0092] Preferably, the supernatants of the wild-type and mutant enzyme solutions were diluted to the same enzyme activity units and then placed in a water bath at 40°C, 50°C, 55°C, and 60°C for 5 minutes, respectively. The residual enzyme activity was measured according to the method described in Example 5, and the residual enzyme activity data are shown in Table 3. The residual enzyme activity in Table 3 is relative to the enzyme activity of each mutant at 40°C and is used to evaluate the residual enzyme activity at temperatures above 40°C.

[0093] The results are shown in Table 3. After treatment at 55°C for 5 minutes, the residual enzyme activity of the aspartic protease mutant pap1(G133C) increased by 36%, the residual enzyme activity of the mutant (D159K) increased by 78%, the residual enzyme activity of the mutant (T103C+T144C) increased by 70%, the residual enzyme activity of the mutant (L303C+A387C) increased by 83%, and the residual enzyme activity of the mutant pap1(G133C+T198C+S376K) increased by approximately 89%.

[0094] After treatment at 60°C for 5 minutes, the residual enzyme activity of the mutant pap1(G133C) increased by about 17%, the residual enzyme activity of the mutant (D159K) increased by 59%, the residual enzyme activity of the mutant (T103C+T144C) increased by 57%, the residual enzyme activity of the mutant (L303C+A387C) increased by 61%, and the residual enzyme activity of the mutant pap1(G133C+T198C+S376K) increased by about 63%.

[0095] Table 3 Determination results of heat resistance of mutants

[0096]

[0097]

[0098] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: This application is based on the spatial structure of the aspartic protease derived from Trichoderma reesei (PDB ID: 3C9X), and uses DiscoveryStudio software to conduct a detailed and comprehensive analysis and simulation of its spatial conformational forces, key catalytic residues, regional distribution characteristics of amino acid residues and other properties. Finally, it is selected to use the strategy of introducing disulfide bonds and increasing the rigidity of flexible regions for design and modification. The software's site-directed simulation mutagenesis and site-directed saturation simulation mutagenesis functions are used to predict the structural stability of the mutant. The actual application effect of the mutant is then verified through experiments, and finally the above-mentioned aspartic protease mutant with improved heat resistance is obtained.

[0099] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A thermostable mutant of aspartic protease, characterized in that: The heat-resistant mutants of aspartic protease include: (a) a protein based on the wild-type aspartic protease pap1 as shown in SEQ ID NO: 1, which has undergone amino acid mutation at at least one of the following sites: G133, D159, S376, T198, T103, T144, L303 or A387, thereby having aspartic protease activity and having higher heat resistance than the wild-type aspartic protease pap1; or (b) A protein having a homology of more than 80% with the amino acid sequence defined in (a), having aspartic protease activity, and having a heat resistance higher than that of the wild-type aspartic protease pap1.

2. The thermostable mutant of aspartic protease according to claim 1, characterized in that The amino acid mutations in (a) are each independently selected from the following: G133 mutated to G133C; D159 mutated to D159K; S376 mutated to S376K; T103 mutated to T103C; T144 mutated to T144C; T198 mutated to T198C; L303 mutated to L303C; A387 mutated to A387C; The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

3. The thermostable mutant of aspartic protease according to claim 2, characterized in that The protein in (b) has more than 85%, preferably more than 90%, more preferably more than 95%, 96%, 97% or 98%, and further preferably more than 99% or 99.9% homology with the amino acid sequence defined in (a), has aspartic acid protease activity, and has higher heat resistance than the wild-type aspartic acid protease pap1.

4. The thermostable mutant of aspartic protease according to claim 2 or 3, characterized in that The mutation of the thermostable mutant of aspartic protease includes any one of the following amino acid mutations: G133C, D159K, S376K, T103C+T144C, G133C+T198C, L303C+A387C, G133C+S376K, or G133C+T198C+S376K.

5. A DNA molecule, characterized in that The DNA molecule encodes the thermostable mutant of the aspartic protease according to any one of claims 1 to 4.

6. A recombinant plasmid, characterized in that The recombinant plasmid is connected to the DNA molecule according to claim 5.

7. A host cell, characterized in that The host cell is transformed with the recombinant plasmid according to claim 6 or contains the DNA molecule according to claim 5.

8. The host cell according to claim 7, characterized in that The host cells include eukaryotic cells; Preferably, the eukaryotic cell comprises Pichia pastoris; Preferably, the Pichia pastoris is a Pichia pastoris having integrated into its genome a DNA capable of expressing the thermostable mutant of the aspartic protease containing the G133C+T198C+S376K mutation; The Pichia pastoris is classified and named Pichia pastoris X33, and was deposited in the China Center for Type Culture Collection on January 12, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 2024092.

9. Use of the thermostable mutant of aspartic protease according to any one of claims 1 to 4, the DNA molecule according to claim 5, the recombinant plasmid according to claim 6, or the host cell according to claim 7 or 8 in the preparation of feed, food, or leather.

10. The use according to claim 9, characterized in that The application comprises adding the heat-resistant mutant of aspartic protease to feed requiring heating and pelleting.

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