Aspartic protease mutants and their applications

Abstract

The present invention provides an aspartic protease mutant and its applications. The aspartic protease mutant includes: 1) a protein having an amino acid sequence with mutations at at least one of the following sites in SEQ ID NO: 4: R110, S38, S51, Q134, N169, V182, A183, V250, R322, N101, T113 or Q239, and having aspartic protease activity; or 2) a protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more homology with the protein in 1), and having aspartic protease activity. Compared with the wild-type aspartic protease, the aspartic protease mutant of the present invention has the advantages of high specific activity and strong thermal stability, which is helpful for its wide application in the fields of food, medicine, feed and leather, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of aspartic proteases, and in particular, to an aspartic protease mutant and its application. Background Art

[0002] Aspartic protease is an enzyme that can degrade proteins under acidic conditions, also known as acid protease. Its optimal pH for action is 2-4, the relative molecular mass is 30,000-40,000, and the isoelectric point is 3.0-5.0. Acid proteases are widely used in the fields of feed, brewing, leather, etc., and have broad application prospects. For example, adding a certain amount of acid protease during the fermentation process of liquor brewing helps to degrade the proteins in corn raw materials, accelerate the utilization of saccharifying enzymes and the growth and reproduction of yeast, shorten the fermentation time, and reduce costs. In addition, adding protease to feed can effectively convert macromolecular proteins that are difficult to utilize into amino acids or oligopeptides that are beneficial for animal absorption, improving the feed conversion efficiency.

[0003] The sources of aspartic proteases are very extensive, including plant sources, animal sources, and microbial sources. The aspartic proteases that have been maturely applied in the market mainly come from Aspergillus niger and Trichoderma reesei. Compared with aspartic proteases from animal and plant sources, the proteases from microbial sources have the characteristics of diversity and complexity. Usually, one strain can secrete one or more aspartic proteases.

[0004] Directed evolution of enzyme molecules is a technology that mimics the natural evolution process (random mutation and natural selection). First, artificial random mutations of enzyme genes are carried out in vitro to establish a mutant gene library, and then mutants of enzymes with excellent catalytic characteristics are obtained by directed selection under special environments with artificially controlled conditions.

[0005] The large-scale production and application of aspartic protease have been realized, but its specific enzyme activity and thermal stability still need to be improved. Therefore, there is an urgent need to obtain aspartic protease mutants with improved specific enzyme activity and heat resistance to realize the application of this enzyme in a wider range of fields. Summary of the Invention

[0006] The main object of the present invention is to provide an aspartic protease mutant and its application to solve the problem of poor specific activity and thermal stability of aspartic protease in the prior art.

[0007] To achieve the above object, according to one aspect of the present invention, there is provided an aspartic protease mutant, which comprises: 1) a protein having an amino acid sequence mutated at at least one of the following sites in SEQ ID NO: 4: R110, S38, S51, Q134, N169, V182, A183, V250, R322, N101, T102, T113 or Q239, and having aspartic protease activity; or 2) a protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more homology with the protein in 1), and having aspartic protease activity.

[0008] Further, in the above 1), the mutated sites include R110S, S38A, S51T, Q134T, N169G, V182I, A183N, V250Y, R322S, N101T+T102S, T113S or Q239S; wherein, the letter before the number represents the wild-type amino acid, and the letter after the number represents the mutated amino acid.

[0009] To achieve the above object, according to the second aspect of the present invention, there is provided a gene encoding the above aspartic protease mutant.

[0010] Further, the above gene has the nucleotide sequence shown in SEQ ID NO: 2.

[0011] To achieve the above object, according to the third aspect of the present invention, there is provided a plasmid containing the above gene.

[0012] Further, the above plasmid includes pPICZαA, pPIC9K or pPIC3.5K.

[0013] To achieve the above object, according to the fourth aspect of the present invention, there is provided a non-animal and non-plant cell, which includes the above gene or the above plasmid.

[0014] Further, the above non-animal and non-plant cell is selected from any one of the following: Escherichia coli, Pichia pastoris or Trichoderma reesei.

[0015] Further, the above Pichia pastoris includes X33; the above Trichoderma reesei includes QM6a; the above Escherichia coli includes DH5α.

[0016] To achieve the above object, according to the fifth aspect of the present invention, there is provided an application of the above aspartic protease mutant, the above gene, the above plasmid or the above non-animal and non-plant cell in the preparation of feed additives, the preparation of food additives, brewing, medicine and / or the leather industry.

[0017] Applying the technical solution of the present invention, that is, the aspartic protease mutant of the present invention includes: 1) a protein having an amino acid sequence with at least one of the following sites in SEQ ID NO: 4: R110, S38, S51, Q134, N169, V182, A183, V250, R322, N101, T102, T113 or Q239 sites mutated, and having aspartic protease activity; or 2) a protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the protein in 1), and having aspartic protease activity.

[0018] Compared with the wild-type aspartic protease, the aspartic protease mutant of the present invention has the advantages of high specific activity and strong thermal stability, and can be widely used in fields such as food additives and feed additives that require high specific activity and thermal stability of aspartic protease, and has high economic value. In addition, with the improvement of the thermal stability of aspartic protease, its heat resistance is also improved. This enables aspartic protease to maintain its activity in extreme environments such as high temperature, broadening the application fields of aspartic protease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 Shows the preliminary screening of aspartic protease pap1 error-prone PCR transformants by the plate method according to an embodiment of the present invention.

[0021] Figure 2 Shows the SDS-PAGE diagram of detecting the purified aspartic protease mutant according to an embodiment of the present invention.

[0022] Figure 3 Shows the heat resistance of the wild-type aspartic protease pap1 in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0024] TERMINOLOGY EXPLANATION:

[0025] Thermal stability of protease: The ability of protease to maintain its structural and functional stability at high temperature.

[0026] Thermotolerance of protease: The ability of protease to maintain its activity in a high-temperature environment. The thermal stability of protease directly affects its thermotolerance. If protease has good thermal stability, it can maintain its activity in a high-temperature environment, that is, it has high thermotolerance.

[0027] Specific activity: The number of enzyme activity units in a unit weight of protein, generally expressed as U / mg protein. Specific activity calculation formula: Specific activity (U / mg) = Enzyme activity (U / mL) / Protein content (mg / mL).

[0028] As mentioned in the background art, there are problems of low specific activity and poor thermal stability of aspartic protease in the prior art. In the present invention, the inventors attempted to randomly mutate the acidic protease PAP1 gene (the amino acid sequence of the encoded protein is shown in SEQ ID NO: 4) derived from Trichoderma reesei QM6a (deposit number: ATCC13631) by error-prone PCR to obtain a variety of aspartic protease mutants with improved specific activity and / or improved thermal stability, and thus proposed a series of protection schemes of the present invention.

[0029] In a first typical embodiment of the present invention, an aspartic protease mutant is provided. The aspartic protease mutant includes: 1) a protein having an amino acid sequence mutated at at least one of the following sites in SEQ ID NO: 4: R110, S38, S51, Q134, N169, V182, A183, V250, R322, N101, T102, T113 or Q239, and having aspartic protease activity; or 2) an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more homology with the protein in 1), and having aspartic protease activity.

[0030] The aspartic protease mutant of the present invention has higher specific activity and stronger thermal stability than the wild-type aspartic protease, can adapt to the extreme environment (high temperature) of industrial production, and is widely used in fields such as feed additives or food additives. In addition, improving the thermal stability of aspartic protease can help extend its shelf life and result in less loss of specific activity during post-treatment processes such as enzyme protein granulation, which is beneficial to the wide application of aspartic protease in multiple fields.

[0031] Based on this application, on the basis of retaining the above site mutations, mutating the remaining sites can obtain a protein having a certain homology with the protein in 1) and having the same aspartic protease activity.

[0032] It should be noted that the homology in the present invention refers to the "sequence identity" between two amino acid sequences, that is, the percentage of identical amino acids between the sequences. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, the amino acid sequence identity is usually measured by sequence analysis software. For example, it can be determined by the BLAST program of the NCBI database. For the determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Primer of Sequence Analysis, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0033] The above proteins having a homology of 70%, 75%, 80%, 85%, 90%, 95%, more than 99% (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, more than 99.9%) with the mutant shown in 1) and having aspartic protease activity, their active sites, active pockets, active mechanisms, protein structures, etc. are probably the same as the proteins provided in 1) of the corresponding proteins.

[0034] Amino acid residues can be represented according to the well-known and agreed-upon standard three-letter or one-letter amino acid codes in the art. In this article, the abbreviations of amino acid residues are 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).

[0035] Conservative amino acid substitutions or replacements are well-known in the art. For example, conservative amino acid substitutions preferably involve one amino acid residue in the following groups (1)-(5) being replaced by another amino acid in the same group: (1) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (2) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (3) polar positively charged residues: His, Arg, and Lys; (4) larger aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys; and (5) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln, or Glu; Met is replaced by Leu, Tyr, or Ile; Phe is replaced by Met, Leu, or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; and Val is replaced by Ile or Leu.

[0036] Those skilled in the art can 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.

[0037] In a preferred embodiment of the present invention, in the above (1), the sites where mutations occur include R110S, S38A, S51T, Q134T, N169G, V182I, A183N, V250Y, R322S, N101T+T102S, T113S, or Q239S; wherein, the letter before the number represents the wild-type amino acid, and the letter after the number represents the mutant amino acid. The aspartic protease mutant with this mutation has the amino acid sequence shown in SEQ ID NO: 1. According to the amino acid sequence of the aspartic protease mutant above, and based on common general knowledge in the art, the nucleotide sequence of its encoding gene can be obtained. In a preferred embodiment of the present invention, this gene has the nucleotide sequence shown in SEQ ID NO: 2.

[0038] Among them, the sequence of SEQ ID NO: 1 is:

[0039] TGSAPNHPSDSADSEYITSVSIGTPAQVLPLDFDTGSSDLWVFSSETPKSSATGHAIYTPSKSSTSKKVSGASWSISYGDGSSSSGDVYTDKVTIGGFSVNTQGVESATSVSTEFVQDTVISGLVGLAFDSGNQVRPHPQKTWFSNAASSLAEPLFTADLRHGQNGSYNFGYIDTSVAKGPVAYTPVDNSQGFWEFTASGYSVGGGKLNRNSIDGIADTGTTLLLLDDNVVDAYYANVQSAQYDNQQEGVVFDCDEDLPSFSFGVGSSTITIPGDLLNLTPLEEGSSTCFGGLQSSSGIGINIFGDVALKAALVVFDLGNERLGWAQK。

[0040] The sequence of SEQ ID NO: 2 is as follows:

[0041] ACCGGCTCGGCGCCCAACCACCCCAGTGACAGCGCCGATTCGGAGTACATCACCTCCGTCTCCATCGGCACTCCGGCTCAGGTCCTCCCCCTGGACTTTGACACCGGCTCCTCCGACCTGTGGGTCTTTAGCTCCGAGACGCCCAAGTCTTCGGCCACCGGCCACGCCATCTACACGCCCTCCAAGTCGTCCACCTCCAAGAAGGTGTCTGGCGCCAGCTGGTCCATCAGCTACGGCGACGGCAGCAGCTCCAGCGGCGATGTCTACACCGACAAGGTCACCATCGGAGGCTTCAGCGTCAACACCCAGGGCGTCGAGTCTGCCACCAGCGTGTCCACCGAGTTCGTCCAGGACACGGTCATCTCTGGCCTCGTCGGCCTTGCCTTTGACAGCGGCAACCAGGTCAGGCCGCACCCGCAGAAGACGTGGTTCTCCAACGCCGCCAGCAGCCTGGCTGAGCCCCTTTTCACTGCCGACCTGAGGCACGGACAGAACGGCAGCTACAACTTTGGCTACATCGACACCAGCGTCGCCAAGGGCCCCGTTGCCTACACCCCCGTTGACAACAGCCAGGGCTTCTGGGAGTTCACTGCCTCGGGCTACTCTGTCGGCGGCGGCAAGCTCAACCGCAACTCCATCGACGGCATTGCCGACACCGGCACCACCCTGCTCCTCCTCGACGACAACGTCGTCGATGCCTACTACGCCAACGTCCAGTCGGCCCAGTACGACAACCAGCAGGAGGGTGTCGTCTTCGACTGCGACGAGGACCTCCCTTCGTTCAGCTTCGGTGTTGGAAGCTCCACCATCACCATCCCTGGCGATCTGCTGAACCTGACTCCCCTCGAGGAGGGCAGCTCCACCTGCTTCGGTGGCCTCCAGAGCAGCTCCGGCATTGGCATCAACATCTTTGGTGACGTTGCCCTCAAGGCTGCCCTGGTTGTCTTTGACCTCGGCAACGAGCGCCTGGGCTGGGCTCAGAAATAA。

[0042] In the second typical embodiment of the present invention, a gene is provided, and the gene encodes the above-mentioned aspartic protease mutant. The gene encoding the aspartic protease mutant expresses an aspartic protease that not only has a higher specific activity but also has improved thermal stability compared to the wild-type aspartic protease, and can be applied to fields such as food additives and feed additives.

[0043] In the third typical embodiment of the present invention, a plasmid is provided, and the plasmid contains the above-mentioned gene. When the plasmid containing the above-mentioned gene is transferred into a specific host cell, it can express an aspartic protease in the host cell with both specific activity and thermal stability improved compared to the wild-type aspartic protease. In a preferred embodiment of the present invention, the above-mentioned plasmid includes: pPICZαA, pPIC9K or pPIC3.5K. The above three plasmids are all common plasmids in the art for expressing foreign proteins.

[0044] In the fourth typical embodiment of the present invention, a non-animal and non-plant cell is provided, and the non-animal and non-plant cell includes the above-mentioned plasmid or the above-mentioned gene. After induced expression, the non-animal and non-plant cell can produce aspartic protease, and the specific enzyme activity and thermal stability of the aspartic protease are higher than those of the wild-type aspartic protease.

[0045] In a preferred embodiment of the present invention, the above-mentioned non-animal and non-plant cell is selected from any one of the following: Escherichia coli, Pichia pastoris or Trichoderma reesei. In a preferred embodiment of the present invention, the above-mentioned Pichia pastoris includes X33; the above-mentioned Trichoderma reesei includes QM6a; the above-mentioned Escherichia coli includes DH5α. The present invention takes Escherichia coli as an example to construct a variety of genetically engineered bacteria that can produce aspartic protease.

[0046] In the fifth typical embodiment of the present invention, an application is provided. The application includes the application of the above-mentioned aspartic protease mutant, the above-mentioned gene, the above-mentioned plasmid or the above-mentioned non-animal and non-plant cell in the preparation of feed additives, the preparation of food additives, brewing, medicine and / or the leather industry.

[0047] The following further describes the present invention in detail with specific embodiments, and these embodiments should not be construed as limiting the scope claimed by the present invention.

[0048] 1. Experimental materials

[0049] (1) Strains and vectors: Escherichia coli Top10, Pichia pastoris X33 and Pichia pastoris expression vector pPICZαA were purchased from Invitrogen.

[0050] (2) Enzymes and kits: Sal I, Not I, and Cpo I restriction endonucleases, and T4 DNA ligase were purchased from TaKaRa; DNA polymerase, plasmid extraction kit, and gel extraction kit were purchased from Novoprotein Scientific Inc.

[0051] (3) Reagents: Yeast extract and peptone were purchased from OXOID; Folin-Ciocalteu reagent, sodium carbonate, trichloroacetic acid, lactic acid, sodium lactate, NaCl, glucose, glycerol, etc. were all purchased from Sinopharm Group.

[0052] The Escherichia coli medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).

[0053] The yeast medium YPD (1% yeast extract, 2% peptone, 2% glucose).

[0054] The yeast medium BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 1% glycerol (V / V), 1% 1M potassium phosphate buffer (pH 6.0).

[0055] The yeast methanol induction medium BMMY (1% yeast extract, 2% peptone, 1.34% YNB, 1% 1M potassium phosphate buffer (pH 6.0).

[0056] Skim milk screening medium:

[0057] Solution A: Dissolve 6 g of skim milk powder (BD) in 100 ml of citric acid buffer solution with pH 3.0, and adjust the pH to 3.0. Sterilize at 108 °C under high temperature and high pressure for 30 min;

[0058] Solution B: Dissolve 4 g of agar in 100 ml of aqueous solution, and sterilize at 121 °C under high temperature and high pressure for 20 min;

[0059] Solution C: Prepare a solid BMMY medium containing 2% agar

[0060] Mix Solution A and Solution B in a 1:1 ratio to prepare a 3% solid milk medium (pH 3.0). Take an appropriate amount and pour it into a sterile petri dish. Wait for the milk medium to solidify; pour an appropriate amount of solid BMMY induction medium containing 0.5% methanol on the upper layer of the milk medium. Wait for the induction medium to solidify, and the preparation of the milk screening medium is completed.

[0061] Example 1 Construction of wild-type aspartic protease expression plasmid

[0062] Optimized according to the codons of Pichia pastoris, the mature peptide coding gene pap1 of Trichoderma reesei aspartic protease of wild type shown in SEQ ID NO: 3 (the amino acid sequence of the encoded protein is shown in SEQ ID NO: 4) was obtained through total gene synthesis technology (Wuhan Aoke Dingsheng Biotechnology Co., Ltd.), and the recombinant plasmid pUC-pap1 was obtained. Figure 3 The thermotolerance of the above-mentioned wild-type aspartic protease pap1 is shown. The experimental results show that the wild-type aspartic protease pap1 loses its enzyme activity at 60 °C.

[0063] Among them, the sequence of SEQ ID NO: 3 is:

[0064] ACCGGCTCGGCGCCCAACCACCCCAGTGACAGCGCCGATTCGGAGTACATCACCTCCGTCTCCATCGGCACTCCGGCTCAGGTCCTCCCCCTGGACTTTGACACCGGCTCCTCCGACCTGTGGGTCTTTAGCTCCGAGACGCCCAAGTCTTCGGCCACCGGCCACGCCATCTACACGCCCTCCAAGTCGTCCACCTCCAAGAAGGTGTCTGGCGCCAGCTGGTCCATCAGCTACGGCGACGGCAGCAGCTCCAGCGGCGATGTCTACACCGACAAGGTCACCATCGGAGGCTTCAGCGTCAACACCCAGGGCGTCGAGTCTGCCACCCGCGTGTCCACCGAGTTCGTCCAGGACACGGTCATCTCTGGCCTCGTCGGCCTTGCCTTTGACAGCGGCAACCAGGTCAGGCCGCACCCGCAGAAGACGTGGTTCTCCAACGCCGCCAGCAGCCTGGCTGAGCCCCTTTTCACTGCCGACCTGAGGCACGGACAGAACGGCAGCTACAACTTTGGCTACATCGACACCAGCGTCGCCAAGGGCCCCGTTGCCTACACCCCCGTTGACAACAGCCAGGGCTTCTGGGAGTTCACTGCCTCGGGCTACTCTGTCGGCGGCGGCAAGCTCAACCGCAACTCCATCGACGGCATTGCCGACACCGGCACCACCCTGCTCCTCCTCGACGACAACGTCGTCGATGCCTACTACGCCAACGTCCAGTCGGCCCAGTACGACAACCAGCAGGAGGGTGTCGTCTTCGACTGCGACGAGGACCTCCCTTCGTTCAGCTTCGGTGTTGGAAGCTCCACCATCACCATCCCTGGCGATCTGCTGAACCTGACTCCCCTCGAGGAGGGCAGCTCCACCTGCTTCGGTGGCCTCCAGAGCAGCTCCGGCATTGGCATCAACATCTTTGGTGACGTTGCCCTCAAGGCTGCCCTGGTTGTCTTTGACCTCGGCAACGAGCGCCTGGGCTGGGCTCAGAAATAA。

[0065] The sequence of SEQ ID NO: 4 is as follows:

[0066] TGSAPNHPSDSADSEYITSVSIGTPAQVLPLDFDTGSSDLWVFSSETPKSSATGHAIYTPSKSSTSKKVSGASWSISYGDGSSSSGDVYTDKVTIGGFSVNTQGVESATRVSTEFVQDTVISGLVGLAFDSGNQVRPHPQKTWFSNAASSLAEPLFTADLRHGQNGSYNFGYIDTSVAKGPVAYTPVDNSQGFWEFTASGYSVGGGKLNRNSIDGIADTGTTLLLLDDNVVDAYYANVQSAQYDNQQEGVVFDCDEDLPSFSFGVGSSTITIPGDLLNLTPLEEGSSTCFGGLQSSSGIGINIFGDVALKAALVVFDLGNERLGWAQK.

[0067] Using the recombinant plasmid pUC-pap1 as a template and P1 / P2 as primers (the primer sequences are shown in Table 1), the pap1 gene fragment was obtained by PCR amplification. The 6xhis tag coding sequence was added to the 3' end of the gene, and it was cloned into the plasmid pPICZαA (carrying the start codon ATG, product number: V19020, manufacturer: Invitrogen), and then transformed into Escherichia coli Top10 competent cells. The transformed bacterial solution was spread on SLB (containing 25 μg / mL Zeocin). After overnight culture, transformants were picked for colony PCR verification, and the plasmids of positive transformants were extracted and sent for sequencing, obtaining the recombinant expression plasmid pPICZαA-pap1.

[0068] Table 1 Primer sequences used

[0069]

[0070] Example 2 Error-prone PCR mutagenesis

[0071] In the present invention, the nucleotide sequence shown in SEQ ID NO: 3 of the wild-type Trichoderma reesei aspartic protease pap1 was used as a template, and error-prone PCR mutagenesis was performed on pap1. The PCR system is shown in Table 2.

[0072] Table 2 Error-prone PCR reaction system

[0073]

[0074] The amplification procedure was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, with 30 cycles; extension at 72°C for 10 min. The PCR amplification products were electrophoresed on a 0.8% agarose gel, and the target PCR products were recovered using a DNA gel recovery kit to obtain the aspartic protease gene fragment with random mutations.

[0075] The aspartic protease random mutant gene fragment was ligated to the expression vector pPICZαA and then transformed into Escherichia coli TOP10. Twenty transformants were randomly selected to sequence the coding region of aspartic protease to verify the quality of error-prone PCR (Table 3). The mixed plasmids of the verified library transformants were extracted to obtain the recombinant plasmid pPICZαA-pap1m. Then, the recombinant plasmid pPICZαA-pap1m was transformed into Pichia pastoris X33 (a commonly used eukaryotic expression system strain, widely used in the industrial production of recombinant proteins) to obtain the recombinant strain Pichia pastoris X33 / pPICZαA-pap1m.

[0076] Table 3 Sequencing verification of error-prone PCR transformants

[0077]

[0078] Example 3 Screening of aspartic protease mutants

[0079] The transformants were streaked onto skim milk powder medium and cultured at 30°C. Every 24 h, 200 μL of methanol was added dropwise onto the lid of the culture dish. After culturing for 4 d, the hydrolysis zone and colony diameter were measured, and the transformants were initially screened based on the ratio of the hydrolysis zone diameter to the colony diameter (see Figure 1 )

[0080] Example 4 Expression and purification of aspartic protease mutants

[0081] The transformants obtained from the initial screening in Example 3 and the recombinant strain of wild-type aspartic protease Pichia pastoris X22 were streaked onto YPD plates containing 100 μg / mL Zeocin. Single colonies with good colony growth status were picked and inoculated into YPD test tube medium. After overnight culture, they were inoculated into BMGY medium at a ratio of 1%, cultured at 30°C for 24 h, then inoculated into 30 mL of BMMY medium, and induced for fermentation at 28°C. Pure methanol was added at a ratio of 0.5% v / v every 24 h. Fermentation was terminated after 4 d of induction. The cells were collected by centrifugation to obtain the supernatant.

[0082] The aspartic protease mutants in the supernatant were purified by Ni column affinity chromatography. The main process was as follows:

[0083] 1) Equilibrate the Ni column with Binding buffer (sodium lactate buffer, pH 3.0, 10 mM imidazole, 0.5 M NaCl), about 2 column volumes;

[0084] 2) Add the fermentation supernatant to the purification column. After incubating for 10 min, remove the flow-through by gravity column;

[0085] 3) Wash away the non-Ni 2+ -bound impurity proteins with Washing buffer (sodium lactate buffer, pH 3.0, 25 mM imidazole, 0.5 M NaCl), about 5 column volumes;

[0086] 4) Perform gradient elution with Elution buffer containing 50, 100, and 300 mM imidazole respectively (sodium lactate buffer, pH 3.0, different concentrations of imidazole, 0.5 M NaCl), collect the elution peaks at each stage, and let each concentration of imidazole buffer flow through 1 column volume.

[0087] 5) Detect the molecular weight and purity of the fusion protein in the collected protein sample by SDS-PAGE.

[0088] The SDS-PAGE detection results of the partially purified aspartic protease mutant are as Figure 2 shown.

[0089] Example 5 Detection of specific activity and thermal stability of aspartic protease mutant

[0090] The method for detecting the activity of the aspartic protease used in the present invention is "GB 1886.174-2016 National Food Safety Standard Food Additive Enzyme Preparations for Food Industry". The enzyme activity is defined as 1 mL of enzyme, under certain temperature (40 °C) and pH (pH 3) conditions, hydrolyzing casein to produce 1 μg of tyrosine in 1 min, which is 1 enzyme activity unit, expressed as U / mL.

[0091] The protein concentration was determined using a BCA protein concentration assay kit (Model: P0011, Manufacturer: Beyotime).

[0092] The main reagents used for detection are:

[0093] (1) Folin working solution: Mix one part of Folin-Ciocalteu reagent with two parts of water and shake well;

[0094] (2) Sodium carbonate solution (42.4 g / L): Weigh 42.4 g of anhydrous sodium carbonate (Na2CO3), dissolve it in water and make up the volume to 1000 mL;

[0095] (3) Trichloroacetic acid (65.4 g / L): Weigh 65.4 g of trichloroacetic acid, dissolve it in water and make up the volume to 1000 mL.

[0096] (4) Sodium lactate buffer solution (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 the volume to 1000 mL.

[0097] (5) Casein solution (10.0 g / L): Weigh 1.000 g of standard casein (NICPBP national drug standard substance), accurate to 0.001 g. After moistening with a small amount of concentrated lactic acid, add about 80 mL of the corresponding buffer solution, heat and boil in a boiling water bath for 30 min, and stir constantly until all the casein is dissolved. After cooling to room temperature, transfer it to a 100 mL volumetric flask and dilute it to the mark with an appropriate pH buffer solution. Check and adjust the pH to the specified value of the corresponding buffer solution before making up the volume. This solution is stored in the refrigerator and the validity period is 3 days.

[0098] The steps of the above enzyme activity detection method are as follows: Dilute the purified aspartic protease mutant sample with the sodium lactate buffer solution. Take 1 mL of the diluted enzyme solution and preheat it in a 40 °C water bath for 2 min, add the casein solution preheated in a 40 °C water bath for 5 min, mix well and react in a 40 °C water bath for 10 min. After the reaction is completed, take it out and add 2 mL of trichloroacetic acid solution, mix well and let it stand for 10 min, and filter it with a slow qualitative filter paper. Take 1 mL of the filtrate, add 5 mL of sodium carbonate solution and mix well, add 1 mL of Folin-Ciocalteu reagent, develop color in a 40 °C water bath for 20 min, and then measure the absorbance at a wavelength of 680 nm. Calculate the enzyme activity of aspartic protease according to the standard curve.

[0099] Sequence the mutants with an enzyme specific activity higher than that of the wild-type aspartic protease PAP1, and finally obtain the following mutants with increased enzyme specific activity (see Table 4).

[0100] After diluting the obtained wild-type and mutant enzyme solution supernatants to the same protein content, place them in a 50 °C water bath for 10 min respectively, then detect the enzyme activity and protein concentration of the enzyme, and calculate the enzyme specific activity. The experimental results are shown in Table 4. It should be noted that since the wild-type aspartic protease pap1 has lost its enzyme activity at 60 °C. Therefore, in this invention, 50 °C is selected to explore the thermal stability test of the protease.

[0101] Among them, the calculation formula for the degree of change in the specific activity of the protease (compared with the wild-type) is: (specific activity of the protease of the mutant - specific activity of the protease of the wild-type) / specific activity of the protease of the wild-type * 100%.

[0102] The calculation formula for the remaining specific enzyme activity of protease is: the specific enzyme activity of protease after water bath heating / the specific enzyme activity of protease before water bath heating * 100%.

[0103] The calculation formula for the degree of change in the remaining specific activity of protease (compared with the wild type) is: the remaining specific enzyme activity of mutant protease - the remaining specific enzyme activity of wild type protease.

[0104] Table 4

[0105]

[0106] It should be noted that in the column of "Degree of change in specific activity of protease (compared with the wild type)" in Table 4, negative values indicate that the specific activity of this mutant is lower than that of the wild type protease, and positive values indicate that the specific activity of this mutant is higher than that of the wild type protease. Positive values in the column of "Degree of change in remaining specific enzyme activity of protease (compared with the wild type)" indicate that the thermal stability of this mutant is higher than that of the wild type protease, and 0 indicates that the thermal stability of this mutant protease is similar to that of the wild type protease.

[0107] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The present invention conducts directed evolution modification on aspartic protease derived from Trichoderma reesei through error-prone PCR, and ingeniously designs the skim milk plate method for high-throughput screening of the mutant library, obtaining a series of mutants with improved specific enzyme activity and thermal stability, promoting the wider application of aspartic protease derived from Trichoderma reesei. This characteristic has important economic significance for industrial fields such as feed and food. In addition, the directed evolution and screening methods used in the present invention can provide guidance for the modification of other types of enzyme proteins, especially aspartic protease.

[0108] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An aspartic protease mutant, characterized in that, The aspartic protease mutant is: arginine at position 110 of SEQ ID NO: 4 is mutated to serine.

2. A gene, characterized in that, The gene encodes the aspartic protease mutant according to claim 1.

3. The gene according to claim 2, wherein The gene has the nucleotide sequence shown in SEQ ID NO:

2.

4. A plasmid, characterized in that, The plasmid contains the gene according to claim 2 or 3.

5. The plasmid according to claim 4, characterized in that, The plasmid includes pPICZαA, pPIC9K or pPIC3.5K.

6. A non-animal and non-plant cell, characterized in that, The non-animal and non-plant cell contains the gene according to claim 2 or 3 or the plasmid according to claim 4 or 5.

7. The non-animal and non-plant cell according to claim 6, wherein The non-animal and non-plant cell is selected from any one of the following: Escherichia coli, Pichia pastoris or Trichoderma reesei.

8. The non-animal and non-plant cell according to claim 7, wherein The Pichia pastoris includes X33; the Trichoderma reesei includes QM6a; the Escherichia coli includes DH5α.

9. Use of the aspartic protease mutant according to claim 1, the gene according to claim 2 or 3, the plasmid according to claim 4 or 5, or the non-animal and non-plant cell according to any one of claims 6-8 in the preparation of feed additives and the brewing industry.

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