Aspartic protease mutant and application thereof

Abstract

The invention provides an aspartic protease mutant and application thereof. The aspartic protease mutant comprises: 1) a protein having an amino acid sequence mutated at at least one of the following sites of 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 having 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. Compared with wild type aspartic protease, the aspartic protease mutant disclosed by the invention has the advantages of high specific activity and high thermal stability, and can be widely applied to the fields of foods, medicines, feeds, leather and the like.

Description

Technical Field

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

[0002] Aspartic protease is an enzyme that can degrade proteins under acidic conditions, also known as acid protease. Its optimal pH is 2-4, its relative molecular mass is 30000-40000, and its isoelectric point is 3.0-5.0. Acid protease is widely used in the fields of feed, brewing, leather, etc., and has broad application prospects. For example, adding a certain amount of acid protease during the fermentation process of liquor brewing can help degrade protein in corn raw materials, accelerate the utilization of saccharifying enzymes and the growth and reproduction of yeast, shorten fermentation time, and reduce costs. In addition, adding protease to feed can effectively convert large molecular proteins that are difficult to use into amino acids or oligopeptides that are easy for animals to absorb, thereby improving feed conversion efficiency.

[0003] Aspartic proteases come from a wide range of sources, including plant, animal and microbial sources. The mature aspartic proteases on the market are mainly from Aspergillus niger and Trichoderma reesei. Compared with aspartic proteases from animal and plant sources, proteases from microbial acids are diverse and complex. Usually, a strain can secrete one or more aspartic proteases.

[0004] Directed evolution of enzyme molecules is a technology that simulates the natural evolution process (random mutation and natural selection). It first conducts artificial random mutation of enzyme genes in vitro to establish a mutant gene library, and then selectively selects mutants of enzymes with excellent catalytic properties under a special environment with artificially controlled conditions.

[0005] Aspartic acid protease has been produced and applied on a large scale, but its enzyme specific activity and thermal stability still need to be improved. Therefore, there is an urgent need to obtain aspartic acid protease mutants with improved enzyme specific activity and improved heat resistance to realize the application of this enzyme in a wider range of fields. Summary of the invention

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

[0007] To achieve the above object, according to one aspect of the present invention, an aspartic protease mutant is provided, the aspartic protease mutant comprising: 1) a protein having an amino acid sequence in which at least one of the following sites of SEQ ID NO: 4 is mutated: 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% homology with the protein in 1), and having aspartic protease activity.

[0008] Furthermore, in the above 1), the mutated sites include R110S, S38A, S51T, Q134T, N169G, V182I, A183N, V250Y, R322S, N101T+T102S, T113S or Q239S; wherein the letters before the numbers represent the wild-type amino acids, and the letters after the numbers represent the mutant amino acids.

[0009] In order to achieve the above object, according to the second aspect of the present invention, a gene is provided, wherein the gene encodes the above aspartic protease mutant.

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

[0011] In order to achieve the above object, according to the third aspect of the present invention, a plasmid is provided, wherein the plasmid comprises the above gene.

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

[0013] In order to achieve the above objectives, according to four aspects of the present invention, a non-animal or plant cell is provided, wherein the non-animal or plant cell comprises the above gene or the above plasmid.

[0014] Furthermore, the non-animal and plant cells are selected from any one of the following: Escherichia coli, Pichia pastoris or Trichoderma reesei.

[0015] Furthermore, the above-mentioned Pichia pastoris includes X33; the above-mentioned Trichoderma reesei includes QM6a; and the above-mentioned Escherichia coli includes DH5α.

[0016] In order to achieve the above objectives, according to five aspects of the present invention, there is provided an application of the above-mentioned aspartic protease mutant, the above-mentioned gene, the above-mentioned plasmid or the above-mentioned non-animal and plant cells in the preparation of feed additives, the preparation of food additives, brewing, medicine and / or leather industry.

[0017] Applying the technical scheme of the present invention, the aspartic protease mutants of the present invention include: 1) a protein having an amino acid sequence in which at least one of the following sites of SEQ ID NO: 4 is mutated: 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 that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to the protein in 1), and having aspartic protease activity.

[0018] Compared with 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, the thermal stability of aspartic protease is improved, and its temperature resistance is also improved. This allows aspartic protease to maintain activity in extreme environments such as high temperature, thereby broadening the application field of aspartic protease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary 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 accompanying drawings:

[0020] Figure 1 The plate method according to an embodiment of the present invention is shown to initially screen error-prone PCR transformants of aspartic protease pap1.

[0021] Figure 2 The SDS-PAGE diagram of the purified aspartic protease mutants is shown according to an embodiment of the present invention.

[0022] Figure 3 The temperature resistance of the wild-type aspartic protease pap1 according to the examples of the present invention is shown. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] Terminology explanation:

[0025] Thermal stability of proteases: the ability of proteases to maintain their structural and functional stability at high temperatures.

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

[0027] Specific activity: The number of enzyme activity units per 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 technology, aspartic acid proteases in the prior art have the problems of low specific activity and poor thermal stability. In the present invention, the inventors attempted to perform random mutations on the acidic protease PAP1 gene (the amino acid sequence of the encoded protein is shown in SEQ ID NO: 4) from Trichoderma reesei QM6a (Accession No.: ATCC13631) through error-prone PCR to obtain a variety of aspartic acid 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, and the aspartic protease mutant includes: 1) a protein having an amino acid sequence in which at least one of the following sites of SEQ ID NO: 4 is mutated: 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% homology with the protein in 1), and having aspartic protease activity.

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

[0031] Based on this application, while retaining the mutations at the above sites, the remaining sites are mutated to obtain a protein having a certain homology with the protein in 1) and the same aspartic acid protease activity.

[0032] It should be noted that 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, amino acid sequence identity is usually measured using 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 Sequence Analysis Primers, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0033] The above proteins that have 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (for example, 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% or more, or even 99.9% or more) homology with the mutants shown in 1) and have aspartic protease activity, their active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as those of the corresponding proteins provided in 1).

[0034] Amino acid residues can be represented according to the standard three-letter or one-letter amino acid code known and agreed in the art. Herein, the 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).

[0035] Conservative amino acid substitutions or replacements are well known in the art. For example, conservative amino acid substitutions preferably involve replacing one amino acid residue in the following groups (1)-(5) with another amino acid residue 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] A person 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.

[0037] In a preferred embodiment of the present invention, in the above 1), the mutation sites include R110S, S38A, S51T, Q134T, N169G, V182I, A183N, V250Y, R322S, N101T+T102S, T113S or Q239S; wherein the letters before the numbers represent the wild-type amino acids, and the letters after the numbers represent the mutant amino acids. The aspartic acid protease mutant having the mutation has the amino acid sequence shown in SEQ ID NO: 1. According to the amino acid sequence of the aspartic acid protease mutant, according to the common knowledge in the art, the nucleotide sequence of its encoding gene can be obtained. In a preferred embodiment of the present invention, the 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 a second typical embodiment of the present invention, a gene is provided, the gene encoding the above-mentioned aspartic protease mutant. Compared with the wild-type aspartic protease, the aspartic protease expressed by the gene encoding the above-mentioned aspartic protease mutant has not only higher specific activity but also improved thermal stability, and can be applied to the fields of food additives and feed additives.

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

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

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

[0046] In a fifth typical embodiment of the present invention, an application is provided, which 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 plant cells in the preparation of feed additives, the preparation of food additives, brewing, medicine and / or leather industry.

[0047] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection 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, Cpo I restriction endonucleases, and T4 DNA ligase were purchased from TaKaRa; DNA polymerase, plasmid extraction kit, and gel recovery kit were purchased from Novazonic Biotech Co., Ltd.

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

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

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

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

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

[0056] Skim Milk Screening Medium:

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

[0058] Solution B: Dissolve 4 g agar in 100 ml aqueous solution and sterilize at 121°C for 20 min.

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

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

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

[0062] According to the optimization of Pichia pastoris codons, the wild-type Trichoderma reesei aspartic acid protease mature peptide encoding gene pap1 shown in SEQ ID NO: 3 (the amino acid sequence of the encoded protein is shown in SEQ ID NO: 4) was obtained by whole gene synthesis technology (Wuhan Aoke Dingsheng Biotechnology Co., Ltd.), and the recombinant plasmid pUC-pap1 was obtained. Figure 3 The temperature resistance of the 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:

[0066] TGSAPNHPSDSADSEYITSVSIGTPAQVLPLDFDTGSSDLWVFSSETPKSSATGHAIYTPSKSSTSKKVSGASWSISYGDGSSSSSGDVYTDKVTIGGFSVNTQGVESATRVSTEFVQDTVISGLVGLAFDSGNQVRPHPQKTWFSNAASSLAEPLFTADLRHGQ NGSYNFGYIDTSVAKGPVAYTPVDNSQGFWEFTASGYSVGGGKLNRNSIDGIADTGTTLLLLDDNVVDAYYANVQSAQYDNQQEGVVFDCDEDLPSFSFGVGSSTITIPGDLLNLTPLEEGSSTCFGGLQSSSGIGINIFGDVALKAALVVFDLGNERLGWAQK.

[0067] Using the recombinant plasmid pUC-pap1 as a template and P1 / P2 as primers (primer sequences refer to Table 1), PCR amplification was performed to obtain the pap1 gene fragment, and a 6xhis tag coding sequence was added to the 3' end of the gene, cloned into the plasmid pPICZαA (with start codon ATG, catalog number: V19020, manufacturer: Invitrogen), transformed into E. coli Top10 competent cells, and the transformation liquid was spread on SLB (containing Zeocin 25 μg / mL). After overnight culture, the transformants were picked for colony PCR verification, and the positive transformant plasmids were extracted and sent for sequencing to obtain the recombinant expression plasmid pPICZαA-pap1.

[0068] Table 1 Primer sequences used

[0069]

[0070] Example 2 Error-prone PCR mutation

[0071] In the present invention, the nucleotide sequence shown in SEQ ID NO: 3 of wild-type Trichoderma reesei aspartic protease pap1 is used as a template to perform error-prone PCR mutation 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: 95°C pre-denaturation for 10 min; 95°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 1 min, 30 cycles; 72°C extension for 10 min. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and the target PCR product was recovered using a DNA gel recovery kit to obtain an aspartic protease gene fragment with random mutations.

[0075] The random mutant gene fragment of aspartic protease was connected to the expression vector pPICZαA and transformed into Escherichia coli TOP10. Twenty transformants were randomly selected to sequence the aspartic protease coding region to verify the quality of error-prone PCR (Table 3). The recombinant plasmid pPICZαA-pap1m was obtained by extracting the mixed plasmid of the verified library transformants, and 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] Transformants were inoculated into skim milk powder medium and cultured at 30°C. 200 μL of methanol was added to the lid of the culture dish every 24 hours. After 4 days of culture, the hydrolysis zone and colony diameter were measured. The ratio of hydrolysis zone diameter to colony diameter was used for preliminary screening of transformants (see Figure 1 ).

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

[0081] The transformants obtained from the initial screening of Example 3 and the wild-type aspartic protease Pichia pastoris X22 recombinant strain were streaked on a YPD plate containing 100 μg / mL Zeocin, and a single colony with good colony growth was picked and inoculated into a YPD test tube medium, and inoculated into a BMGY medium at a ratio of 1% after overnight culture, and then inoculated into 30 mL BMMY medium after culturing at 30°C for 24 hours, and fermented at 28°C, pure methanol was added at 0.5% v / v every 24 hours, and fermentation was terminated after 4 days of induction. The bacteria were collected by centrifugation and the supernatant was obtained.

[0082] The aspartic acid protease mutant in the supernatant was 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) for about 2 column volumes;

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

[0085] 3) Use washing buffer (sodium lactate buffer pH 3.0, 25mM imidazole, 0.5M NaCl) to wash away the unattached Ni 2+ Bound impurities, 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) Collect the protein samples and use SDS-PAGE to detect the molecular weight and purity of the fusion protein.

[0088] The results of SDS-PAGE detection of partially purified aspartic protease mutants are shown in Figure 2 shown.

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

[0090] The aspartic protease activity detection method used in the present invention is "GB1886.174-2016 National Food Safety Standard Food Additives 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, 1 minute hydrolysis of casein produces 1 μg of tyrosine, which is 1 enzyme activity unit, expressed as U / mL.

[0091] The protein concentration was determined using the BCA protein concentration assay kit (model: P0011, manufacturer: Biyuntian).

[0092] The main reagents used in the test are:

[0093] (1) Folin solution: Mix one part of Folin phenol 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 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 to 1000 mL.

[0096] (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 uniform. Adjust the pH to 3.0±0.05 with lactic acid or sodium lactate, and make up to 1000 mL.

[0097] (5) Casein solution (10.0 g / L): Weigh 1.000 g of standard casein (NICPBP national drug standard substance) to the nearest 0.001 g, moisten with a small amount of concentrated lactic acid, add about 80 mL of the corresponding buffer solution, heat in a boiling water bath and boil for 30 min, stirring from time to time until the casein is completely dissolved. After cooling to room temperature, transfer to a 100 mL volumetric flask and dilute 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 to volume. This solution is stored in a refrigerator and is valid for 3 days.

[0098] The above enzyme activity detection method comprises the following steps: dilute the purified aspartic protease mutant sample 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 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 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 in a 40°C water bath for 20 minutes, and measure the absorbance at a wavelength of 680 nm. Calculate the enzyme activity of aspartic protease according to the standard curve.

[0099] The mutants with improved enzyme specific activity compared with the wild-type aspartic protease PAP1 were sequenced, and finally the following mutants with improved enzyme specific activity were obtained (see Table 4).

[0100] After the obtained wild-type and mutant enzyme supernatants were diluted to the same protein content, they were placed in a 50°C water bath for 10 min, and then the enzyme activity and protein concentration were detected, and the enzyme specific activity was calculated. 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, the present invention selected 50°C to explore the thermal stability test of the protease.

[0101] The calculation formula for the change degree of 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 of the residual enzyme specific activity of the protease is: the enzyme specific activity of the protease after heating in a water bath / the enzyme specific activity of the protease before heating in a water bath*100%.

[0103] The degree of change in the residual specific activity of the protease (compared to the wild type) was calculated as follows: residual specific activity of the mutant protease - residual specific activity of the wild type protease.

[0104] Table 4

[0105]

[0106] It should be noted that the negative value in the column "Change in specific activity of protease (compared with wild type)" in Table 4 indicates that the specific activity of the mutant is lower than that of the wild type protease, and the positive value indicates that the specific activity of the mutant is higher than that of the wild type protease. The positive value in the column "Change in residual enzyme specific activity of protease (compared with wild type)" indicates that the thermal stability of the mutant is improved compared with that of the wild type protease, and 0 indicates that the thermal stability of the 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 uses error-prone PCR to perform directed evolution transformation on aspartic protease from Trichoderma reesei, cleverly designs skim milk plate method to perform high-throughput screening on mutant library, obtains a series of mutants with improved enzyme specific activity and thermal stability, promotes the wider application of aspartic protease from Trichoderma reesei, and 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 transformation of other types of enzyme proteins, especially aspartic proteases.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An aspartic acid protease mutant, characterized in that: The aspartic protease mutants include: The amino acid sequence is a protein having at least 99% homology to SEQ ID NO: 4 and having aspartic protease activity.

2. The aspartic protease mutant according to claim 1, characterized in that In the above 1), the mutated site is selected from any one of the following: R110S, S38A, S51T, Q134T, N169G, V182I, A183N, V250Y, R322S, N101T+T102S, T113S or Q239S; wherein the letters before the numbers represent the wild-type amino acids, and the letters after the numbers represent the mutant amino acids.

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

4. The gene according to claim 3, characterized in that The gene has a nucleotide sequence shown in SEQ ID NO:

2.

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

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

7. A non-animal or plant cell, characterized in that: The non-animal or plant cell comprises the gene according to claim 3 or 4 or the plasmid according to claim 5 or 6.

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

9. The non-animal or plant cell according to claim 8, characterized in that: The Pichia pastoris includes X33; the Trichoderma reesei includes QM6a; and the Escherichia coli includes DH5α.

10. Use of the aspartic protease mutant according to claim 1 or 2, the gene according to claim 3 or 4, the plasmid according to claim 5 or 6, or the non-animal or plant cell according to any one of claims 7 to 9 in the preparation of feed additives, food additives, brewing, medicine and / or leather industry.

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