A Rhizomucor miehei aspartic protease with coagulating activity, its preparation method and application

Abstract

The present invention discloses an aspartic protease from Rhizomucor miehei with rennet activity, its preparation method and application. The expression plasmid of this aspartic protease is constructed and transformed into Aspergillus niger for heterologous expression. The protease activity of the fermentation broth of the recombinant Aspergillus niger strain reaches 2000 U / mL, the protein content is 6.1 mg / mL, the optimal pH is 2.5, the optimal temperature is 40 °C, and it maintains a relatively high enzyme activity below 45 °C, and its substrate specificity is wide. This enzyme has a significant renneting effect, the rennet activity is 3894.1 SU / mg, and it specifically hydrolyzes the peptide bonds of Lys22-Ile23, Leu33-Ser34, Leu51-Ile52, Lys64-Pro65 and Phe105-Met106 of κ-casein. The aspartic protease of the present invention has good application value in the fields such as cheese making.

Description

Technical Field

[0001] The present invention relates to an aspartic protease of Rhizomucor miehei with coagulating activity, a preparation method thereof, and an application thereof in the field of food biotechnology. Background Art

[0002] Protease refers to a large class of enzymes that can hydrolyze peptide bonds in proteins to generate amino acids or small peptides. Proteases are rich in sources and widely exist in animals, plants and microorganisms. As one of the three major industrial enzyme preparations, proteases have a wide range of applications in the fields of food, washing, leather making, medicine, etc. Aspartic protease (EC 3.4.23.X) is a kind of protease with optimal catalytic activity in an acidic environment and belongs to acidic protease. Aspartic protease is widely used in cheese making, condiment production, active peptide preparation, meat tenderization, etc. (Abdul Razzaq, Sadia Shamsi, Arfan Ali, et al. Microbial proteases applications. Frontiers in Bioengineering and Biotechnology, 2019, 7: 110).

[0003] Some aspartic proteases have coagulating activity. Among them, chymosin from calf abomasum is the most widely used aspartic protease in cheese making currently. In the coagulation reaction involving proteases, the protease hydrolyzes the peptide bond of Phe105-Met106 in κ-casein, generating caseinomacropeptide and para-κ-casein. Among them, para-κ-casein proteins aggregate to form a three-dimensional network gel, and then, under the action of calcium ions, the casein micelles aggregate and become unstable, forming curds (Douglas G. Dalgleish, Milena Corredig. The structure of the casein micelle of milk and its changes during processing. Annual Review of Food Science and Technology, 2012, 3:449–67). Therefore, specific cleavage of κ-casein and a high ratio of coagulating activity / hydrolytic activity (CA / PA) are important parameters for measuring the coagulating properties of proteases (Louwrens W. Theron. Microbial aspartic proteases: current and potential applications in industry. Applied Microbiology and Biotechnology, 2014, 98:8853-8868). With the growth of cheese consumption and the demand for different flavored cheeses, aspartic proteases with coagulating activity have received increasing attention. The types and properties of aspartic proteases from microorganisms are the most diverse. Currently, a variety of aspartic proteases from microorganisms have been found to have coagulating activity. Among them, the natural aspartic proteases of Rhizomucor miehei, Rhizomucor pusillus, and Mucor mucedo from fungi have been applied to the production of commercial cheeses (Ronivaldo Rodrigues da Silva. Exploring microbial peptidases for cheese production: a viewpoint on the current conjecture. Journal of Agricultural and Food Chemistry, 2018, 66:1305-1306). Some individual types of coagulases from microorganisms have been industrially applied, but most are natural enzymes. The development of recombinant coagulases from microorganisms lags behind relatively.

[0004] High - efficiency expression by genetic engineering is an important way to explore new proteases and carry out large - scale industrial production. Aspergillus niger is a filamentous fungal expression system with good safety for food - grade product fermentation. Aspergillus niger has excellent post - translational processing ability and secretion ability of proteins. Its modification of protein molecules is closer to that of higher eukaryotic cells, and it has strong advantages in producing active proteins (Dominik Mojzita, Anssi Rantasalo, Jussi Gene expression engineering in fungi. Current Opinion in Biotechnology, 2019, 59:141 - 149). Currently, there is a patent reporting an Aspergillus niger strain with high - yield acid protease. A recombinant Aspergillus niger engineering strain expressing acid protease from Aspergillus niger was constructed, and its enzyme - producing ability was further improved by ultraviolet mutagenesis. The enzyme - producing level of the mutant strain in a 20L fermenter was 17428 U / mL (patent number: 201810793553.5, application date: 2018.07.18). Recombinant bovine chymosin heterologously expressed in Aspergillus niger, as a widely used commercial rennet, still occupies an important market position at present (S.V. Belenkaya, D.V. Balabova, A.N. Belov, et al. Basic biochemical properties of recombinant chymosins (review). Applied Biochemistry and Microbiology, 2020, 56:363 - 372).

[0005] Mucor miehei is an important industrial strain for the production of protease and lipase. The aspartic protease produced by it is applied in cheese coagulation and meat tenderization. There are patent reports on the method of using Mucor miehei (the same as Mucor miehei) to produce rennet. Using Mucor miehei ATCC16457 to produce rennet, the rennet activity of its fermentation broth is 410 IMCU / mL (patent number: 201310472404.6, application date: October 11, 2013). There are also patent reports on a method for the production and application of a novel aspartic protease from Mucor miehei. Heterologously express an aspartic protease from Mucor miehei in Pichia pastoris GS115, and the protease activity is 3400 U / mL. This enzyme can be used for tenderizing pork and preparing low-molecular-weight polypeptides (patent number: 201710383874.3, application time: May 26, 2017). The genome of Mucor miehei contains multiple protease genes, and only a few of them have been cloned, expressed, and studied for application. Using genetic engineering technology to mine and highly express novel aspartic proteases from Mucor miehei can expand the types of aspartic proteases. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a new aspartic protease with coagulation activity.

[0007] To solve the above technical problems, the present invention provides a protein, which is an aspartic protease, derived from Mucor miehei, named RmproB, and is any one of the following A1)-A4):

[0008] A1) A protein whose amino acid sequence is the amino acid residues at positions 20 to 439 of Sequence 1 in the sequence listing;

[0009] A2) A protein whose amino acid sequence is the amino acid residues at positions 1 to 439 of Sequence 1 in the sequence listing;

[0010] A3) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the protein of A1) or A2), having more than 80% identity with the protein shown in A1) and having aspartic protease activity;

[0011] A4) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of any one of A1)-A3).

[0012] Among them, Sequence 1 in the sequence listing consists of 439 amino acid residues, and the amino acid residues at positions 1-19 are the signal peptide sequence.

[0013] The above protein can be artificially synthesized, or its coding gene can be synthesized first and then obtained by biological expression.

[0014] Among the above-mentioned proteins, the protein tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be any one or more of the tag sequences shown in Table 1.

[0015] Table 1 Sequences of Tags

[0016] Label Residue Sequence Poly-Arg 5 - 6 (usually 5) RRRRR Poly-His 2 - 10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tagⅡ 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0017] Among the above-mentioned proteins, the substitution and / or deletion and / or addition of one or several amino acid residues are substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0018] Among the above-mentioned proteins, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined by using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search to calculate the identity of the amino acid sequences, and then the identity value (%) can be obtained.

[0019] Among the above-mentioned proteins, the identity of more than 80% can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity.

[0020] Biological materials related to RmproB also fall within the scope of protection of the present invention.

[0021] The biological materials related to the protein RmproB provided by the present invention are any one of the following B1) to B6):

[0022] B1) A nucleic acid molecule encoding RmproB;

[0023] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0024] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

[0025] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0026] Wherein, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

[0027] Among the above biological materials, the nucleic acid molecule described in B1) is a coding gene shown in any one of C1-C5 as follows:

[0028] C1) A cDNA molecule or DNA molecule whose coding sequence of the coding strand is the nucleotide positions 58-1320 of Sequence 2 in the Sequence Listing;

[0029] C2) A cDNA molecule or DNA molecule whose nucleotide of the coding strand is the nucleotide positions 58-1320 of Sequence 2 in the Sequence Listing;

[0030] C3) A cDNA molecule or DNA molecule whose coding sequence of the coding strand is the nucleotide positions 1-1320 of Sequence 2 in the Sequence Listing;

[0031] C4) A cDNA molecule or DNA molecule whose nucleotide of the coding strand is the nucleotide positions 1-1320 of Sequence 2 in the Sequence Listing;

[0032] C5) A DNA molecule having 75% or more identity with the nucleotide sequence defined in any one of C1)-C4) and encoding the said protein;

[0033] C6) A DNA molecule that hybridizes with the nucleotide sequence defined in any one of C1)-C4) under stringent conditions and encodes the said protein.

[0034] Wherein, the nucleotide positions 1-1320 of Sequence 2 in the Sequence Listing consist of 1320 nucleotides and encode the protein shown in Sequence 1 in the Sequence Listing.

[0035] The identity of more than 75% may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98% or 99% identity.

[0036] Among the above biological materials, the expression cassette containing the nucleic acid molecule encoding RmproB (RmproB gene expression cassette) described in B2) refers to DNA that can express RmproB in a host cell. This DNA may not only include a promoter that initiates the transcription of RmproB, but also include a terminator that terminates the transcription of RmproB. Further, the expression cassette may also include an enhancer sequence.

[0037] Among the above biological materials, the recombinant vector may be Blunt-Pamy / gla-HygB-RmproB(P2A). Blunt-Pamy / gla-HygB-RmproB(P2A) is a recombinant expression vector of the RmproB gene obtained by replacing a small fragment between the NotI recognition site and the XhoI recognition site of the Blunt plasmid with the nucleotide fragment of Sequence 3 in the Sequence Listing, while keeping the other nucleotide sequences of the Blunt plasmid unchanged.

[0038] Among the above biological materials, the recombinant microorganism described in B4) may be D1) or D2):

[0039] D1) The recombinant microorganism is obtained by introducing the coding gene of the protein into a recipient microorganism to obtain a recombinant microorganism that expresses the protein. The recipient microorganism may be any one of E1)-E3):

[0040] E1) Eukaryotic microorganism;

[0041] E2) Microorganism of the genus Aspergillus;

[0042] E3) Aspergillus niger, such as Aspergillus niger AT.

[0043] D2) The recombinant microorganism is a recombinant Aspergillus niger that secretes and expresses a recombinant protein (named RmproB) with the amino acid sequence from position 20 to position 439 of Sequence 1 in the Sequence Listing, obtained by introducing the Blunt-Pamy / gla-HygB-RmproB(P2A) into Aspergillus niger.

[0044] Among the above biological materials, B4) to B6) may or may not include propagation materials.

[0045] To solve the above technical problems, the present invention provides a method for preparing a protein with aspartic protease activity.

[0046] The method for preparing a protein with aspartic protease activity provided by the present invention includes: expressing a DNA molecule containing the coding gene of the protein in an organism to obtain a protein with aspartic protease activity; the organism is a microorganism.

[0047] In the above method, the microorganism can be any one of E1)-E3):

[0048] E1) Eukaryotic microorganism;

[0049] E2) Aspergillus microorganism;

[0050] E3) Aspergillus niger, such as Aspergillus niger AT.

[0051] In the above method, expressing the DNA molecule containing the coding gene of the protein in an organism includes introducing the coding gene of the protein into a recipient microorganism to obtain a recombinant microorganism expressing the protein, culturing the recombinant microorganism, and expressing to obtain the protein.

[0052] In the above method, the recipient microorganism can be any one of E1)-E3):

[0053] E1) Eukaryotic microorganism;

[0054] E2) Aspergillus

[0055] E3) Aspergillus niger, such as Aspergillus niger AT.

[0056] In the above method, the recombinant microorganism can be a recombinant Aspergillus niger that secretes and expresses a recombinant protein (named RmproB) with the amino acid sequence from position 20 to position 439 of Sequence 1 in the Sequence Listing by introducing the Blunt-Pamy / gla-HygB-RmproB(P2A) into Aspergillus niger.

[0057] In the above method, after expression, it includes collecting the protein secreted extracellularly (the secreted and expressed protein) to obtain a protein with aspartic protease activity.

[0058] The present invention also provides a coagulant.

[0059] The active ingredient of the above coagulant contains the protein and / or biological material. The active ingredient of the above coagulant can also contain other biological components or / and non-biological components. Those skilled in the art can determine other active ingredients of the above coagulant according to the antibacterial effect.

[0060] Any one of the following P1-P3 applications of the above protein or the above biological material also belongs to the protection scope of the present invention:

[0061] P1. Application of the protein or the biological material in the preparation of aspartic protease;

[0062] P2. Application of the protein or the biological material in the preparation of a coagulant;

[0063] P3. Use of the protein or the biological material in preparing cheese.

[0064] The present invention discovered a novel aspartic protease gene from Rhizomucor miehei CAU432 and heterologously expressed it in Aspergillus niger. The aspartic protease has both protease and chymosin activity. When the enzyme-encoding gene was expressed in Aspergillus niger and subjected to liquid fermentation, the protease activity reached 2000 U / mL and the protein content reached 6.1 mg / mL. The aspartic protease has an optimal pH of 2.5 and remains stable at pH 1.5-6.5. Its optimal temperature is 40°C, and it maintains high enzyme activity below 45°C. It also has broad substrate specificity, exhibiting high hydrolysis activity against casein, hemoglobin, myoglobin, bovine serum albumin, and skim milk. The enzyme has a significant milk-clotting effect, with a chymosin activity of 3894.1 SU / mg. It can specifically hydrolyze the Lys22-Ile23, Leu33-Ser34, Leu51-Ile52, Lys64-Pro65, and Phe105-Met106 peptide bonds in κ-casein. These properties give it great potential for application in the food industry and are valuable in cheesemaking. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is the plasmid map of the strongly expressing double-copy plasmid Blunt-Pamy / gla-HygB-RmproB (P2A) in Example 1 of the present invention.

[0066] Figure 2 This is the enzyme production process of the recombinant aspartic protease RmproB Aspergillus niger transformant in Example 1 of the present invention in a 5-L fermentor, wherein ● is the enzyme activity of the protease in the fermentation supernatant; ■ is the protein content in the fermentation supernatant; ▲ is the wet weight of the bacteria.

[0067] Figure 3 This is an electrophoresis diagram of the purification of the recombinant aspartic protease RmproB in Example 2 of the present invention. In the figure, lane M: low molecular weight standard protein; lane 1: Aspergillus niger empty strain control; lane 2: RmproB transformant fermentation broth; lane 3: Q column purified enzyme solution; lane 4: pure enzyme solution.

[0068] Figure 4 This is a graph showing the optimal pH of the recombinant aspartic protease RmproB in Example 2 of the present invention. In the graph, ■ represents KCl-HCl (pH 1.0-2.0); ● represents lactate (pH 2.0-4.0); ▲ represents citrate (pH 3.5-6.5); and ◆ represents phosphate (pH 6.5-8.0).

[0069] Figure 5This is the pH stability determination curve of recombinant aspartic protease RmproB in Example 2 of the present invention. In the figure, ■ represents KCl-HCl (pH 1.0 - 2.0); ● represents lactate (pH 2.0 - 4.0); ▲ represents citrate (pH 3.5 - 6.5); ◆ represents phosphate (pH 6.5 - 8.0).

[0070] Figure 6 This is the optimal temperature determination curve of recombinant aspartic protease RmproB in Example 2 of the present invention.

[0071] Figure 7 This is the temperature stability determination curve of recombinant aspartic protease RmproB in Example 2 of the present invention.

[0072] Figure 8 This is the optimal pH of the curdling activity of recombinant aspartic protease RmproB and the ratio of curdling activity to protease activity at different pH values in Example 3 of the present invention. In the figure, is the relative curdling activity; is the ratio of curdling activity to protease activity.

[0073] Figure 9 This is the optimal calcium ion concentration for the curdling activity of recombinant aspartic protease RmproB in Example 3 of the present invention.

[0074] Figure 10 This is the hydrolysis schematic diagram of recombinant aspartic protease RmproB on different components of casein in Example 3 of the present invention.

[0075] Instructions for Deposit of Biological Material

[0076] Classification and naming of biological material: Rhizomucor miehei

[0077] Latin scientific name of biological material: Rhizomucor miehei

[0078] Strain number of biological material: CAU432

[0079] Full name of the depository: General Microbiology Center of China Committee for Culture Collection of Microorganisms

[0080] Abbreviation of the depository: CGMCC

[0081] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101

[0082] Date of deposit: June 21, 2011

[0083] Deposit number: CGMCC No. 4967 Detailed implementation mode

[0084] The present invention will be further described in detail below in conjunction with specific embodiments. The embodiments given are only for clarifying the present invention and not for limiting the scope of the present invention. In the following embodiments, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5'-terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3'-terminal nucleotide of the corresponding DNA / RNA. In the quantitative experiments in the following embodiments, unless otherwise specified, 3 replicates are set. In the experimental methods in the following embodiments, unless otherwise specified, they are all conventional methods. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0085] In the following embodiments, the protease activity was determined with reference to Anson et al. (Anson, M. L. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. Journal of General Physiology, 1938, 22: 79-89): A substrate casein solution (concentration 1%) was prepared using 50 mM lactic acid-sodium lactate buffer (pH 2.5, solvent water). After incubating 100 μL of the enzyme solution and 100 μL of the casein solution at 40 °C for 10 min, 200 μL of 0.4 M trichloroacetic acid solution was added to terminate the reaction. After centrifuging at 12,000 rpm for 5 min, 100 μL of the supernatant was taken and added to 500 μL of 0.4 M Na2CO3 solution and 100 μL of Folin reagent, and incubated at 40 °C for 20 min. The absorbance was measured at OD 660nm . The enzyme solution with trichloroacetic acid added first to terminate the reaction was used as a control. The amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute was defined as 1 enzyme activity unit (U).

[0086] In the following examples, the rennet activity was determined with reference to Kei et al. (Kei Arima, Shinjiro Iwasaki, Gakuzo Tamura. Milkclotting enzyme from microorganisms. Agricultural and Biological Chemistry, 1967, 31:540 - 545): A skim milk powder emulsion with a concentration of 10% was prepared using a 50 mM sodium citrate buffer at pH 4.0, and calcium chloride was added to a final concentration of 10 mM to obtain a skim milk emulsion. Take 5 mL of the skim milk emulsion and incubate it at 40 °C for 5 min, add 0.5 mL of the enzyme solution, mix immediately, start timing, and the end point is reached when flocculent precipitates appear in the emulsion. The amount of enzyme required to coagulate 1 mL of a 10% skim milk emulsion in 40 min was defined as one Soxhlet unit (SU). The rennet activity was calculated according to the following formula:

[0087] MC (SU / mL) = 2400 / t × S / E

[0088] Where: MC is the rennet activity (SU / mL); S is the volume of the skim milk solution (mL); E is the volume of the added enzyme (mL); t is the clotting time (s).

[0089] The formulations and specific preparation methods of the media and solutions involved in the following examples are as follows:

[0090] 1) The composition of the DPY liquid medium is: glucose 2%, tryptone 1%, yeast extract powder 0.5%, KH2PO4 0.5%, MgSO4·7H2O 0.05%, and the rest is water. The preparation method of the DPY liquid medium can be: 2 g of glucose, 1 g of tryptone, 0.5 g of yeast extract powder, 0.5 g of KH2PO4, 0.05 g of MgSO4·7H2O, dissolved in deionized water and made up to 100 mL, and sterilized at 121 °C for 20 min.

[0091] 2) The composition of the CD hypertonic medium is: sucrose 34.2%, NaNO3 0.3%, KCl 0.2%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.01%, agar 2%, pH 5.5, and the rest is water. The preparation method of the CD hypertonic medium can be: 34.2 g of sucrose, 0.3 g of NaNO3, 0.2 g of KCl, 0.1 g of KH2PO4, 0.05 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, 2 g of agar, dissolved in deionized water and made up to 100 mL, adjust the pH value to 5.5, and sterilize at 121 °C for 20 min.

[0092] 3) The composition of the CD hypertonic soft agar is as follows: sucrose 34.2%, NaNO3 0.3%, KCl 0.2%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.01%, agar 0.05%, pH 5.5, and the rest is water. The preparation method of the CD hypertonic soft agar medium can be: 34.2 g of sucrose, 0.3 g of NaNO3, 0.2 g of KCl, 0.1 g of KH2PO4, 0.05 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, 0.05 g of agar, dissolved in deionized water and made up to 100 mL, adjust the pH value to 5.5, and sterilize at 121 °C for 20 min.

[0093] 4) The composition of the CD solid medium is as follows: glucose 2%, NaNO3 0.3%, KCl 0.2%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.01%, agar 2%, pH 5.5, and the rest is water. The preparation method of the CD solid medium can be: 2 g of glucose, 0.3 g of NaNO3, 0.2 g of KCl, 0.1 g of KH2PO4, 0.05 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, 2 g of agar, dissolved in deionized water and made up to 100 mL, adjust the pH value to 5.5, and sterilize at 120 °C under high pressure.

[0094] 5) The composition of the fermentation medium is as follows: corn starch hydrolyzate 10%, corn steep liquor 3%, soybean meal powder 3%, and the rest is water. The preparation method of the fermentation medium can be: 100 g of corn starch hydrolyzate, 30 g of corn steep liquor, 30 g of soybean meal powder, dissolved in deionized water and made up to 1 L, and sterilize at 121 °C for 20 min.

[0095] 6) The composition of the enzyme solution is as follows: lysing enzyme 1.5%, lysozyme 0.5%, snail enzyme 1.5%, cellulase 2%, NaCl 4.8%, and the rest is 50 mM pH 6.0 phosphate buffer. The preparation method of the enzyme solution can be: 1.5 g of lysing enzyme, 0.5 g of lysozyme, 1.5 g of snail enzyme, 2 g of cellulase, 4.8 g of NaCl, dissolved in 50 mM pH 6.0 phosphate buffer and made up to 100 mL, and sterilize at 121 °C for 20 min.

[0096] The biological materials involved in the following examples:

[0097] Rhizomucor miehei CAU432 is the Rhizomucor miehei strain provided in the patent "Rhizomucor miehei Strain and Its Application in the Preparation of β-Glucanase and Rennin" (authorized announcement number: CN103045485B). It was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on June 21, 2011 (the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode 100101). The taxonomic name is Rhizomucor miehei, and the deposit number is: CGMCC No. 4967.

[0098] The vector Blunt plasmid (full name: Simple Cloning Vector; catalog number: CB111-01) is a product of TransGen Biotech Co., Ltd.

[0099] The Aspergillus niger host (A. niger) AT (product number AS3.4309) is a product of Allee Biotechnology (Shanghai) Co., Ltd.

[0100] Casein, hemoglobin, myoglobin, and skim milk powder are all products of Sigma.

[0101] Example 1: Cloning of the Aspartic Protease Gene and Its Expression in Aspergillus niger

[0102] I. Construction of Recombinant Bacteria

[0103] An aspartic protease RmproB from Rhizomucor miehei, the amino acid sequence is shown as positions 20 to 439 of Sequence 1 in the sequence listing, and the nucleotide sequence of its encoding gene is shown as positions 58 - 1320 of Sequence 2 in the sequence listing.

[0104] Design primers RmproB-F and RmproB-F:

[0105] RmproB-F: 5′-GCTCTCACCAGAATTCCTATCAAGAAGACA-3′;

[0106] RmproB-R: 5′-CTAGCTTGCGACAATCTGATGTAGATGAAG-3′.

[0107] Using the cDNA of Rhizomucor miehei CAU432 as a template, and RmproB-F and RmproB-F as primers for PCR amplification. The polymerase chain reaction (PCR) amplification conditions are: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, for 35 cycles; finally, extension at 72°C for 5 min.

[0108] The two RmproB genes (DNA molecule shown at positions 58 - 1320 of Sequence 1 in the Sequence Listing) were tandemly constructed into an RmproB double-copy cassette using a P2A peptide. The amylase-glucoamylase fusion promoter (Pamy / gla, containing the glucoamylase signal peptide sequence, amplified by PCR from the Aspergillus niger genome, nucleotide sequence being nucleotides 1 - 1785 of Sequence 3 in the Sequence Listing; among which, nucleotides 1732 - 1785 of Sequence 3 in the Sequence Listing are the coding sequence of the glucoamylase signal peptide, and nucleotides 1 - 1731 are the amylase-glucoamylase fusion promoter), the RmproB double-copy cassette (RmproB-P2A-RmproB, nucleotide sequence (CDS) being nucleotides 1786 - 4374 of Sequence 3 in the Sequence Listing), the glucoamylase terminator (Tgla, nucleotide sequence being nucleotides 4375 - 5146 of Sequence 3 in the Sequence Listing), and the hygromycin expression cassette (HygB, nucleotide sequence being nucleotides 5147 - 8318 of Sequence 3 in the Sequence Listing, among which, nucleotides 6227 - 7252 of Sequence 3 in the Sequence Listing are the coding sequence of the hygromycin resistance gene) were successively ligated into the Blunt plasmid, replacing the small fragment between the NotI recognition site and the XhoI recognition site, while keeping the other nucleotide sequences of the Blunt plasmid unchanged. The resulting recombinant expression vector of the RmproB gene was named Blunt-Pamy / gla-HygB-RmproB(P2A), and its plasmid map is shown in Figure 1 。

[0109] Blunt-Pamy / gla-HygB-RmproB(P2A) is a recombinant expression vector of the RmproB gene obtained by replacing the small fragment between the NotI recognition site and the XhoI recognition site of the Blunt plasmid with a nucleotide fragment whose nucleotide sequence is Sequence 3 in the Sequence Listing, while keeping the other nucleotide sequences of the Blunt plasmid unchanged.

[0110] Blunt-Pamy / gla-HygB-RmproB(P2A) was transformed into the cloning host Escherichia coli DH5α, and the positive Escherichia coli transformants were verified by colony PCR and sequenced.

[0111] Extract the recombinant plasmid from E. coli with correct sequencing results. Use NotI endonuclease to linearize the plasmid Blunt-Pamy / gla-HygB-RmproB(P2A) by single enzyme digestion to obtain the linearized plasmid Blunt-Pamy / gla-HygB-RmproB(P2A). The 400 μL enzyme digestion system is as follows: 356 μL of the recombinant plasmid Blunt-Pamy / gla-HygB-RmproB(P2A), 40 μL of Cutsmart buffer, 2 μL of NotI, and digest overnight at 37°C. Recover the linearized plasmid Blunt-Pamy / gla-HygB-RmproB(P2A) by alcohol precipitation.

[0112] II. Expression of Recombinant Aspartic Protease Gene in Aspergillus niger

[0113] Preparation and purification of Aspergillus niger protoplasts: Culture the Aspergillus niger host (A. niger) AT for 3 days at 30°C in a shaking incubator with DPY liquid medium. After suction filtration using a Buchner funnel, collect the bacterial cells to prepare protoplasts. Use the enzyme solution to lyse the mycelia into protoplasts, and wash twice with STC buffer for later use.

[0114] PEG (polyethylene glycol)-mediated transformation of Aspergillus niger protoplasts: Add 50 μg of the linearized Blunt-Pamy / gla-HygB-RmproB(P2A) plasmid to 160 μL of Aspergillus niger protoplasts, then add 60 μL of PEG, gently flick to mix well, and incubate on ice for 30 min, flicking every 10 min. Then add 1 mL of PEG and let it stand at room temperature for 20 min.

[0115] Pour plate culture: Mix 3 mL of STC buffer and 6 mL of CD hypertonic soft agar in a 15 mL centrifuge tube. Transfer the transformed component after PEG incubation to the 15 mL centrifuge tube, and add HygB at a final concentration of 300 μg / mL. Invert the tube up and down to mix well, then pour it onto the CD hypertonic medium plate. After even distribution, culture at 30°C for 5 days.

[0116] Identification of transformants: Extract the genomic DNA of the transformants after two passages of the single colonies grown on the hypertonic plate on CD solid medium. Design verification primers YZ-Pgla-F and YZ-PgpdA-R for amplification and sequencing verification.

[0117] YZ-Pgla-F: 5′-GTTGATGCATGTGCTTCTTCCTTCAG-3′;

[0118] YZ-Tgla-R: 5′-TTGGACAAATGAACGTATCTTATCGAGATCCT-3′.

[0119] The transformants with the correct genotype are the recombinant aspartic protease RmproB Aspergillus niger. After culturing them in a shake flask at 30 °C and 200 rpm for 120 h, the protease activity was detected in the centrifuged fermentation supernatant.

[0120] III. Liquid fermentation of recombinant Aspergillus niger

[0121] Seed culture: The recombinant Aspergillus niger with aspartic protease RmproB obtained in step 2 was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of DPY liquid medium and cultured at 30 °C and 200 rpm for 72 h to obtain a seed solution containing a large number of mycelial pellets.

[0122] Mycelial growth stage: The seed solution was inoculated into a 5 L fermenter (containing 3 L of fermentation medium). During the fermentation process, the temperature was 30 °C, the pH was adjusted to 6.0 with ammonia water and phosphoric acid, and the stirring speed was 300 rpm. As the mycelium grew, the dissolved oxygen (DO) continuously decreased. When the starch hydrolyzate in the fermentation medium was completely consumed (about 60 - 72 h), feeding began.

[0123] Fed-batch fermentation stage: The temperature was 30 °C and the pH was maintained at 6.0. Feeding was carried out with a 50% corn starch hydrolyzate solution (50 g / 100 mL) at a feeding flow rate of 5 mL / h / L. By controlling the air flow rate and stirring speed, the DO was maintained above 5% until the end of fermentation. During the whole fermentation process, the wet cell weight, protein content in the fermentation broth, and protease activity were monitored.

[0124] The fermentation process is shown in Figure 2 , ● is the enzyme activity of protease in the fermentation supernatant (the supernatant obtained by centrifuging the fermentation broth at 10,000 × g for 10 min after fermentation); ■ is the protein content in the fermentation supernatant; ▲ is the wet cell weight. The enzyme activity of the fermentation supernatant reached the highest of 2000 U / mL on the 7th day, and the protein content reached 6.1 mg / mL. The wet cell weight reached the highest on the 5th day, which was 516.5 g / L.

[0125] Example 2. Purification and enzymatic properties of recombinant aspartic protease RmproB

[0126] I. Purification of recombinant aspartic protease RmproB

[0127] After the fermentation in Example 1 was completed, the fermentation broth was centrifuged at 10,000×g for 10 min to obtain the supernatant. 20 mL of the supernatant was taken and dialyzed in a 20 mM phosphate buffer at pH 6.0, and then loaded onto a Q-Sepharose FF chromatography column equilibrated with 20 mM phosphate buffer at pH 6.0. After washing the unbound proteins with 5 column volumes of 20 mM phosphate buffer at pH 6.0, linear elution was performed for 60 min (the NaCl concentration linearly increased from 0 to 500 mM within 60 min) with a buffer containing 0 - 500 mM NaCl (a liquid with a gradient NaCl concentration obtained by adding NaCl to 20 mM phosphate buffer at pH 6.0), and the OD of the eluate was detected. 280nm The flow rate for equilibrating Q-Sepharose with the buffer was 1 mL / min, the flow rate during sample loading was 0.5 mL / min, the flow rate for washing the unbound proteins with 5 column volumes was 1 mL / min, and the linear elution rate of the buffer containing 0 - 500 mM NaCl was 1 mL / min. SDS-PAGE analysis was performed on the elution peaks in different collection tubes, and the protease components were collected according to the molecular weight of the protein electrophoresis.

[0128] The RmproB enzyme solution purified by the Q column was loaded onto a Sephacryl-100 chromatography column equilibrated with 20 mM phosphate buffer at pH 6.0. Elution was performed with 20 mM phosphate buffer at pH 6.0 at a flow rate of 0.3 mL / min, and the OD of the eluate was detected. 280nm The elution peaks were collected for SDS-PAGE analysis. The protease components were collected according to the molecular weight of the protein electrophoresis, and the obtained pure enzyme - recombinant aspartic protease RmproB was combined and reserved for use.

[0129] Recombinant aspartic protease RmproB was purified by two steps of Q-Sepharose anion exchange chromatography and Sephacryl-100 gel filtration to reach electrophoretic purity. The recovery rate of this enzyme was 18.8%, the purification fold was 3.9 times, and the specific enzyme activity of the pure enzyme was 3176.1 U / mg (see Table 2 for details). The SDS-PAGE of the crude enzyme solution and its obtained purified product (RmproB) is as Figure 3 shown, indicating that the size of the recombinant protein RmproB is 49.4 kDa.

[0130] Table 2 Purification table of recombinant aspartic protease RmproB

[0131]

[0132]

[0133] aPrepare the casein substrate (concentration 1%) using pH 2.5 lactate-sodium lactate buffer, and determine the protease activity at 40°C.

[0134] b Determine the protein content using the Lowry method.

[0135] II. Enzymatic properties of recombinant aspartic protease RmproB

[0136] 1) Determination of the optimal pH

[0137] Determine the optimal pH of the enzyme using buffer systems with the following different pH values: potassium chloride-hydrochloric acid (KCl-HCl, 50 mM, pH 1.0 - 2.0); lactate-sodium lactate (50 mM, pH 2.0 - 4.0); citrate-sodium citrate (50 mM, pH 3.5 - 6.5); sodium dihydrogen phosphate-disodium hydrogen phosphate (phosphate, 50 mM, pH 6.5 - 8.0). Then, determine the protease activity at 40°C using the standard method. Taking the highest enzyme activity as 100%, calculate the relative enzyme activities measured at each pH.

[0138] The results are as Figure 4 shown. The optimal pH of recombinant aspartic protease RmproB is 2.5.

[0139] 2) Determination of pH stability

[0140] Dilute the RmproB enzyme solution with the above buffer solutions of different pH values, incubate in a 40°C constant temperature water bath for 30 min, cool in an ice-water bath for 30 min, and determine the protease activity at 40°C and pH 2.5. Using the untreated enzyme solution as a control, calculate the residual enzyme activities of the enzyme solutions after treatment under different acid-base conditions. Calculate the relative enzyme activities as the percentage of the residual enzyme activity to the control enzyme activity.

[0141] The results are as Figure 5 shown. Recombinant aspartic protease RmproB is stable in acidic conditions. When the pH is 1.5 - 6.5, the residual enzyme activity is above 80%.

[0142] 3) Determination of the optimal reaction temperature

[0143] Dilute RmproB appropriately in 50 mM pH 2.5 lactate-sodium lactate buffer, and determine the protease activity at different temperatures from 20 - 60°C. Taking the highest enzyme activity value as 100%, calculate the relative enzyme activities measured at each temperature.

[0144] The results are as Figure 6 shown. The optimal temperature of recombinant aspartic protease RmproB is 40°C.

[0145] 4) Determination of temperature stability

[0146] RmproB was appropriately diluted in 50 mM pH 2.5 lactic acid - sodium lactate buffer, incubated at 25 - 60 °C for 30 min respectively, cooled in an ice - water bath for 30 min, and the protease activity was measured at 40 °C and pH 2.5. The enzyme solution without heat treatment was used as a control, and the residual enzyme activity of the enzyme solution after different heat treatments was calculated respectively. The relative enzyme activity was calculated as the percentage of the residual enzyme activity to the control enzyme activity.

[0147] The results are as Figure 7 shown. The recombinant aspartic protease RmproB remained stable below 45 °C, and the residual enzyme activity was above 80%.

[0148] 5) Determination of substrate specificity

[0149] Using 1% casein, skimmed milk, bovine serum albumin, hemoglobin, soy protein isolate, gelatin, albumin egg, myoglobin, protamine sulfate, and collagen, etc. as substrates respectively, the enzyme activity was measured according to the method in step one. When casein was used as the substrate, the protease activity was set as 100%, and the specific enzyme activity and relative enzyme activity of the protease towards various substrates were calculated respectively.

[0150] The results are shown in Table 3. The recombinant aspartic protease RmproB showed the highest hydrolysis activity towards casein, and had relatively high hydrolysis activities towards hemoglobin (94.8%), myoglobin (80.4%), bovine serum albumin (73.8%), and skimmed milk powder (60.4%); the hydrolysis activities towards albumin egg (43.9%), whey protein (19.9%), and lactoglobulin (5.1%) were relatively weak; there was no hydrolysis effect on soy protein isolate, gelatin, protamine sulfate, and collagen.

[0151] Table 3 Substrate specificity of recombinant aspartic protease RmproB a

[0152] Substrate Specific Enzyme Activity (U / mg) Relative Enzyme Activity (%) Casein 3176.1±67.1 100 Hemoglobin 3011.1±78.2 94.8±2.5 Myoglobin 2552.3±68.8 80.4±2.2 Bovine Serum Albumin 2344.3±79.7 73.8±2.5 Skim Milk Powder 1917.1±84.7 60.4±2.7 Ovalbumin 1393.5±17.0 43.9±0.5 Whey Protein 631.3±81.0 19.9±2.5 Lactoglobulin 161.9±148.2 5.1±4.7 Soybean Protein Isolate 0±0 0±0 Gelatin 0±0 0±0 Protamine 0±0 0±0 Collagen 0±0 0±0

[0153] a In 50 mM pH 2.5 lactic acid - sodium lactate buffer at 40 °C, the protease activity of RmproB towards different substrates was measured. Three parallels were set for each experimental group.

[0154] Example 3, Coagulating Characteristics of Recombinant Aspartic Protease RmproB

[0155] I. Optimal pH for the Coagulating Activity of RmproB

[0156] For the recombinant aspartic protease RmproB purified in Example 2, 10% skim milk powder solution (substrate) was prepared with sodium citrate buffer (50 mM, pH 3.5 - 6.5) at different pH values. The rennet activity was measured by the standard method at 40°C. Taking the highest enzyme activity as 100%, the relative rennet activities at different pH values were calculated respectively. Meanwhile, the ratio of rennet activity to protease activity (CA / PA) was measured under the above pH conditions.

[0157] The results are as Figure 8 shown. The optimal pH value for the coagulating activity of the recombinant aspartic protease RmproB purified in Example 2 is 4.0. At pH 4.0, the CA / PA value is the highest, which is 6.1.

[0158] II. Optimal Calcium Chloride Concentration for the Coagulating Activity of RmproB.

[0159] 10% skim milk powder emulsion was prepared with 50 mM sodium citrate buffer at pH 4.0, and calcium chloride was added to a final concentration of 0 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 80 mM, and 100 mM. Then the rennet activity was measured by the standard method at 40°C. Taking the highest enzyme activity as 100%, the relative rennet activities at different calcium chloride concentrations were calculated respectively.

[0160] The results are as Figure 9 shown. For the recombinant aspartic protease RmproB in Example 2, the rennet activity is the highest at a calcium chloride concentration of 5 mM. The coagulating activity measured under this reaction condition (50 mM sodium citrate buffer at pH 4.0, 5 mM calcium chloride concentration) is 3894.1 SU / mg.

[0161] III. Hydrolysis Characteristics of RmproB on Casein.

[0162] 1% α was prepared using 50 mM sodium citrate buffer at pH 4.0 s-、 β-, and κ-casein solutions. 10 SU of recombinant aspartic protease RmproB was added to 1 mL of different casein solutions and incubated at 40 °C. Samples were taken at 0 min, 5 min, 10 min, and 30 min of the reaction, and the hydrolysis degree of each component casein was analyzed by SDS-PAGE. The κ-casein sample at 10 min of the reaction was taken, and peptide segments were detected using a time-of-flight mass spectrometer (MALDI-TOF-MS) to identify the cleavage sites of RmproB on κ-casein.

[0163] The hydrolysis results of RmproB on casein are as Figure 10 shown. RmproB has different degrees of hydrolysis on the three caseins. Among them, the hydrolysis effect on κ-casein is the strongest, followed by that on α s -casein, and the hydrolysis effect on β-casein is relatively weak. The cleavage sites of RmproB on κ-casein are shown in Table 4, where Lys22-Ile23, Leu33-Ser34, Leu51-Ile52, Lys64-Pro65, and Phe105-Met106 are the main cleavage sites.

[0164] Table 4 Identification of the polypeptide product sequence of recombinant aspartic protease RmproB hydrolyzing κ-casein

[0165]

[0166]

[0167] a The peptide segment sequence of κ-casein hydrolyzed by RmproB identified by MALDI-TOF-MS.

[0168] The aspartic protease involved in the present invention has protease activity and chymosin activity. The gene encoding this enzyme was expressed in Aspergillus niger and liquid fermented. The protease activity reached 2000 U / mL, and the protein content reached 6.1 mg / mL. The optimum pH of this aspartic protease is 2.5 and it remains stable at pH 1.5 - 6.5; the optimum temperature is 40 °C and it maintains a relatively high enzyme activity below 45 °C; its substrate specificity is wide, and it shows high hydrolysis activity towards casein, hemoglobin, myoglobin, bovine serum albumin, and skim milk. This enzyme has a significant coagulation effect, and the chymosin activity is 3894.1 SU / mg, which can specifically hydrolyze the peptide bonds of Lys22-Ile23, Leu33-Ser34, Leu51-Ile52, Lys64-Pro65, and Phe105-Met106 of κ-casein. These properties make it show great application potential in the food industry.

[0169] This engineered bacterium can be industrialized in an enzyme preparation company, and the produced enzyme preparation can be applied in industries such as food and feed, with great economic value and social benefits.

[0170] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims. Sequence Listing <110> China Agricultural University <120> A Rhizomucor miehei aspartic protease with coagulation activity, its preparation method and application <130> GNCSY212005 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 439 <212> PRT <213> Artificial Sequence <400> 1 Met Lys Leu Phe Ala Leu Ser Ala Ala Val Ala Ala Leu Leu Ser Val 1 5 10 15 Ser Asp Ala Ala Leu Thr Arg Ile Pro Ile Lys Lys Thr Ala Glu Thr 20 25 30 Pro Asn Gln Lys Leu Glu Arg Tyr Thr His Thr Gly Glu Tyr Leu Thr 35 40 45 Gln Lys Tyr Phe Gly Ala Gln Arg Ser Gln Gln Asn Ser Val Met Gln 50 55 60 Pro Phe Gln Val Gly Pro Asp Gly Arg Val Glu His Gly Val Pro Ile 65 70 75 80 Ser Asn Tyr Met Asn Ala Gln Tyr Tyr Gly Glu Ile Glu Leu Gly Ser 85 90 95 Pro Pro Gln Thr Phe Ser Val Val Phe Asp Thr Gly Ser Ser Asn Leu 100 105 110 Trp Val Pro Ser Thr His Cys Thr Ser Ile Ala Cys Phe Leu His Arg 115 120 125 Arg Tyr Asp Ser Ser Arg Ser Ser Thr Phe Lys Glu Asn Gly Thr Glu 130 135 140 Phe Ala Ile Gln Tyr Gly Thr Gly Ser Leu Glu Gly Phe Ile Ser Glu 145 150 155 160 Asp Thr Leu His Val Gly Gly Val Lys Val Thr Gly Gln Gly Phe Ala 165 170 175 Glu Ser Val Lys Glu Pro Gly Phe Thr Phe Ala Leu Ala Arg Phe Asp 180 185 190 Gly Ile Phe Gly Leu Gly Tyr Asp Arg Ile Ser Val Lys Gly Val Val 195 200 205 Pro Pro Phe Tyr His Met Val Glu Arg Asp Leu Ile Asp Glu Pro Ile 210 215 220 Phe Ser Phe Trp Leu Asn Gly Asp Leu Asp Asn Glu Glu Asn Gly Gly 225 230 235 240 Glu Leu Val Phe Gly Gly Val Asp Glu Asp His Phe Glu Gly Glu Ile 245 250 255 Ala Trp Ser Pro Val Lys Arg Lys Gly Tyr Trp Glu Ile Glu Leu Glu 260 265 270 Asp Ile Lys Phe Gly Gly Glu Ser Val Asp Leu Asp Pro Val Gly Ala 275 280 285 Ala Ile Asp Thr Gly Ser Ser Leu Leu Val Ala Pro Thr Thr Val Ala 290 295 300 Asp Leu Ile Asn Lys Glu Leu Gly Ala Glu Lys Asn Trp Ala Gly Gln 305 310 315 320 Tyr Val Leu Asp Cys Ser Lys Val Pro Asn Leu Pro Glu Phe Cys Phe 325 330 335 Val Phe Ser Gly Lys Asp Phe Cys Leu Ser Gly Glu Asp Tyr Val Leu 340 345 350 Gln Leu Gln Asp Gln Cys Ile Ser Gly Phe Met Gly Met Asp Ile Pro 355 360 365 Glu Pro Ala Gly Pro Leu Trp Ile Val Gly Asp Val Phe Leu Arg Lys 370 375 380 Phe Tyr Ser Val Tyr Asp Leu Gly Asn Asp Arg Val Gly Leu Ala Lys 385 390 395 400 Ala Asn Ser Pro Pro Pro Ile Pro Ala Tyr Val Tyr His Ala Arg Glu 405 410 415 Arg Arg Cys Val Tyr Phe Leu Ala His Ile Ser Ile His Ser Leu His 420 425 430 Leu His Gln Ile Val Ala Ser 435 <210> 2 <211> 1320 <212> DNA <213> Artificial Sequence <400> 2 atgaaattgt ttgccttgtc cgcagctgtt gctgctctcc tctcagtgtc tgatgctgct 60 ctcaccagaa ttcctatcaa gaagacagca gaaactccca accagaaatt ggagcgctac 120 acacacactg gtgaatacct cacccaaaag tactttggtg cgcagcgcag tcagcagaac 180 agcgttatgc agccattcca agttggcccc gacggccgtg tcgagcatgg cgttccgatt 240 tcgaactata tgaacgcaca atattatggc gaaatagagc ttggttcgcc tcctcaaaca 300 ttctccgttg tctttgatac cggttcatcc aacctctggg tcccctcgac tcactgcacc 360 tctatcgcat gcttccttca ccgccgctac gactctagca ggtcaagcac attcaaagag 420 aatggtaccg agtttgctat ccagtacggt actggctccc ttgaaggttt catcagcgag 480 gatactttgc atgttggtgg agttaaggtt actggccagg gctttgccga atctgtcaag 540 gaacctggct tcacatttgc acttgcccgc ttcgacggca tttttggtct aggctacgac 600 cgtatctctg tcaagggcgt cgtaccgccc ttctaccaca tggtggagcg cgatcttatc 660 gatgaaccaa tcttctcgtt ctggctgaac ggcgatctcg ataatgagga aaacggaggt 720 gagcttgtct ttggcggtgt cgatgaagac cactttgagg gcgagattgc ttggtcgcca 780 gtcaagcgca agggatactg ggaaatcgag ttggaagata tcaaattcgg cggcgaatcc 840 gttgacttgg atcccgttgg tgctgccatt gatacgggtt cctcgcttct cgttgcacct 900 accactgttg ccgacctcat caacaaggag ctcggtgctg agaagaactg ggctggtcaa 960 tatgttcttg attgcagcaa ggtccctaac ttgccagaat tctgctttgt gtttagcggt 1020 aaagatttct gcttaagcgg cgaggactat gttctccagc tccaagatca gtgcatttcc 1080 ggattcatgg gcatggatat cccagagcct gctggtcccc tctggattgt gggtgatgtc 1140 ttcctccgca agttctacag cgtgtatgat ttgggtaacg accgtgtcgg tttggcaaaa 1200 gcaaactccc cacctcctat tcccgcttat gtgtaccatg ctcgagaacg tcgttgtgtt 1260 tattttcttg cacatatatc tatacacagt cttcatctac atcagattgt cgcaagctag 1320 <210> 3 <211> 8318 <212> DNA <213> Artificial Sequence <400> 3 taaatcggct tctaggcgcg ctccatctaa atgttctggc tgtggtgtac aggggcataa 60 aattacgcac tacccgaatc gatagaacta ctcattttta tatagaagtc agaattcatg 120 gtgttttgat cattttaaat ttttatatgg cgggtggtgg gcaactcgct tgcgcgggca 180 actcgcttac cgattacgtt agggctgata tttacgtaaa aatcgtcaag ggatgcaaga 240 ccaaagtact aaaaccccgg agtcaacagc atccaagccc aagtccttca cggagaaacc 300 ccagcgtcca catcacgagc gaaggaccac ctctaggcat cggacgcacc atccaattag 360 aagcagcaaa gcgaaacagc ccaagaaaaa ggtcggcccg tcggcctttt ctgcaacgct 420 gatcacgggc agcgatccaa ccaacaccct ccagagtgac taggggcgga aatttatcgg 480 gattaatttc cactcaacca caaatcacag tcgtccccgg tattgtcctg cagaatgcaa 540 tttaaactct tctgcgaatc gcttggattc cccgcccctg gccgtagagc ttaaagtatg 600 tcccttgtcg atgcgatgta tcacaacata taaatactag caagggatgc catgcttgga 660 ggatagcaac cgacaacatc acatcaagct ctcccttctc tgaacaataa accccacaga 720 aggcatttat gcctctcgta tgcagaggaa atctcccctg atcttccgaa ctggtcgtac 780 ctggcgacct atgactatgg caccccagtt ctggggacct tccacggaag tgacctgctg 840 caggtgttct atgggatcaa gccaaactat gcagctagtt ctagccacac gtactatctg 900 agctttgtgt atacgctgga tccgaactcc aaccgggggg agtacattga gtggccgcag 960 tggaaggaat cgcggcagtt gatgaatttc ggagcgaacg acgccagtct ccttacggat 1020 gatttccgca acgggacata tgagttcatc ctgcagaata ccgcggcgtt ccacatctga 1080 tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 1140 tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 1140 gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 1200 gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 1200 ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 1260 ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 1260 taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 1320 taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 1320 aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 1380 aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 1380 ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 1440 ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 1440 ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 1500 ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 1500 gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 1560 gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 1560 gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc cctcgtgcag atgaggtttg 1620 gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc cctcgtgcag atgaggtttg 1620 gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 1680 gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 1680 agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatgtcgttc 1740 agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatgtcgttc 1740 cgatctctac tcgccctgag cggcctcgtc tgcacagggt tggcagctct caccagaatt 1800 cgatctctac tcgccctgag cggcctcgtc tgcacagggt tggcagctct caccagaatt 1800 cctatcaaga agacagcaga aactcccaac cagaaattgg agcgctacac acacactggt 1860 gaatacctca cccaaaagta ctttggtgcg cagcgcagtc agcagaacag cgttatgcag 1920 ccattccaag ttggccccga cggccgtgtc gagcatggcg ttccgatttc gaactatatg 1980 aacgcacaat attatggcga aatagagctt ggttcgcctc ctcaaacatt ctccgttgtc 2040 tttgataccg gttcatccaa cctctgggtc ccctcgactc actgcacctc tatcgcatgc 2100 ttccttcacc gccgctacga ctctagcagg tcaagcacat tcaaagagaa tggtaccgag 2160 tttgctatcc agtacggtac tggctccctt gaaggtttca tcagcgagga tactttgcat 2220 gttggtggag ttaaggttac tggccagggc tttgccgaat ctgtcaagga acctggcttc 2280 acatttgcac ttgcccgctt cgacggcatt tttggtctag gctacgaccg tatctctgtc 2340 aagggcgtcg taccgccctt ctaccacatg gtggagcgcg atcttatcga tgaaccaatc 2400 ttctcgttct ggctgaacgg cgatctcgat aatgaggaaa acggaggtga gcttgtcttt 2460 ggcggtgtcg atgaagacca ctttgagggc gagattgctt ggtcgccagt caagcgcaag 2520 ggatactggg aaatcgagtt ggaagatatc aaattcggcg gcgaatccgt tgacttggat 2580 cccgttggtg ctgccattga tacgggttcc tcgcttctcg ttgcacctac cactgttgcc 2640 gacctcatca acaaggagct cggtgctgag aagaactggg ctggtcaata tgttcttgat 2700 tgcagcaagg tccctaactt gccagaattc tgctttgtgt ttagcggtaa agatttctgc 2760 ttaagcggcg aggactatgt tctccagctc caagatcagt gcatttccgg attcatgggc 2820 atggatatcc cagagcctgc tggtcccctc tggattgtgg gtgatgtctt cctccgcaag 2880 ttctacagcg tgtatgattt gggtaacgac cgtgtcggtt tggcaaaagc aaactcccca 2940 cctcctattc ccgcttatgt gtaccatgct cgagaacgtc gttgtgttta ttttcttgca 3000 catatatcta tacacagtct tcatctacat cagattgtcg caagcggaag cggagctact 3060 aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc tgctctcacc 3120 agaattccta tcaagaagac agcagaaact cccaaccaga aattggagcg ctacacacac 3180 actggtgaat acctcaccca aaagtacttt ggtgcgcagc gcagtcagca gaacagcgtt 3240 atgcagccat tccaagttgg ccccgacggc cgtgtcgagc atggcgttcc gatttcgaac 3300 tatatgaacg cacaatatta tggcgaaata gagcttggtt cgcctcctca aacattctcc 3360 gttgtctttg ataccggttc atccaacctc tgggtcccct cgactcactg cacctctatc 3420 gcatgcttcc ttcaccgccg ctacgactct agcaggtcaa gcacattcaa agagaatggt 3480 accgagtttg ctatccagta cggtactggc tcccttgaag gtttcatcag cgaggatact 3540 ttgcatgttg gtggagttaa ggttactggc cagggctttg ccgaatctgt caaggaacct 3600 ggcttcacat ttgcacttgc ccgcttcgac ggcatttttg gtctaggcta cgaccgtatc 3660 tctgtcaagg gcgtcgtacc gcccttctac cacatggtgg agcgcgatct tatcgatgaa 3720 ccaatcttct cgttctggct gaacggcgat ctcgataatg aggaaaacgg aggtgagctt 3780 gtctttggcg gtgtcgatga agaccacttt gagggcgaga ttgcttggtc gccagtcaag 3840 cgcaagggat actgggaaat cgagttggaa gatatcaaat tcggcggcga atccgttgac 3900 ttggatcccg ttggtgctgc cattgatacg ggttcctcgc ttctcgttgc acctaccact 3960 gttgccgacc tcatcaacaa ggagctcggt gctgagaaga actgggctgg tcaatatgtt 4020 cttgattgca gcaaggtccc taacttgcca gaattctgct ttgtgtttag cggtaaagat 4080 ttctgcttaa gcggcgagga ctatgttctc cagctccaag atcagtgcat ttccggattc 4140 atgggcatgg atatcccaga gcctgctggt cccctctgga ttgtgggtga tgtcttcctc 4200 cgcaagttct acagcgtgta tgatttgggt aacgaccgtg tcggtttggc aaaagcaaac 4260 tccccacctc ctattcccgc ttatgtgtac catgctcgag aacgtcgttg tgtttatttt 4320 cttgcacata tatctataca cagtcttcat ctacatcaga ttgtcgcaag ctagaactta 4380 acgttactga aatcatcaaa cagcttgacg aatctggata taagatcgtt ggtgtcgatg 4440 tcagctccgg agttgagaca aatggtgttc aggatctcga taagatacgt tcatttgtcc 4500 aagcagcaaa gagtgccttc tagtgattta atagctccat gtcaacaaga ataaaacgcg 4560 tttcgggttt acctcttcca gatacagctc atctgcaatg cattaatgca ttggacctcg 4620 caaccctagt acgcccttca ggctccggcg aagcagaaga atagcttagc agagtctatt 4680 ttcattttcg ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga 4740 ggaatccgct cttggctcca cgcgactata tatttgtctc taattgtact ttgacatgct 4800 cctcttcttt actctgatag cttgactatg aaaattccgt caccagcccc tgggttcgca 4860 aagataattg cactgtttct tccttgaact ctcaagccta caggacacac attcatcgta 4920 ggtataaacc tcgaaaatca ttcctactaa gatgggtata caatagtaac catgcatggt 4980 tgcctagtga atgctccgta acacccaata cgccggccga aactttttta caactctcct 5040 atgagtcgtt tacccagaat gcacaggtac acttgtttag aggtaatcct tctttctaga 5100 agtcctcgtg tactgtgtaa gcgcccactc cacatctcca ctcgaccaga ataagatggt 5160 ggagagctta taccgagctc ccaaatctgt ccagatcatg gttgaccggt gcctggatct 5220 tcctatagaa tcatccttat tcgttgacct agctgattct ggagtgaccc agagggtcat 5280 gacttgagcc taaaatccgc cgcctccacc atttgtagaa aaatgtgacg aactcgtgag 5340 ctctgtacag tgaccggtga ctctttctgg catgcggaga gacggacgga cgcagagaga 5400 agggctgagt aataagcgcc actgcgccag acagctctgg cggctctgag gtgcagtgga 5460 tgattattaa tccgggaccg gccgcccctc cgccccgaag tggaaaggct ggtgtgcccc 5520 tcgttgacca agaatctatt gcatcatcgg agaatatgga gcttcatcga atcaccggca 5580 gtaagcgaag gagaatgtga agccaggggt gtatagccgt cggcgaaata gcatgccatt 5640 aacctaggta cagaagtcca attgcttccg atctggtaaa agattcacga gatagtacct 5700 tctccgaagt aggtagagcg agtacccggc gcgtaagctc cctaattggc ccatccggca 5760 tctgtagggc gtccaaatat cgtgcctctc ctgctttgcc cggtgtatga aaccggaaag 5820 gccgctcagg agctggccag cggcgcagac cgggaacaca agctggcagt cgacccatcc 5880 ggtgctctgc actcgacctg ctgaggtccc tcagtccctg gtaggcagct ttgccccgtc 5940 tgtccgcccg gtgtgtcggc ggggttgaca aggtcgttgc gtcagtccaa catttgttgc 6000 catattttcc tgctctcccc accagctgct cttttctttt ctctttcttt tcccatcttc 6060 agtatattca tcttcccatc caagaacctt tatttcccct aagtaagtac tttgctacat 6120 ccatactcca tccttcccat cccttattcc tttgaacctt tcagttcgag ctttcccact 6180 tcatcgcagc ttgactaaca gctaccccgc ttgagcagac atcaccatga aaaagcctga 6240 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct 6300 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg 6360 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg 6420 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga 6480 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga 6540 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc 6600 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac 6660 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt 6720 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc 6780 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct 6840 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc 6900 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca 6960 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta 7020 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga 7080 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg 7140 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact 7200 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agacaatcaa 7260 tccatttcgc tatagttaaa ggatggggat gagggcaatt ggttatatga tcatgtatgt 7320 agtgggtgtg cataatagta gtgaaatgga agccaagtca tgtgattgta atcgaccgac 7380 ggaattgagg atatccggaa atacagacac cgtgaaagcc atggtctttc cttcgtgtag 7440 aagaccagac agacagtccc tgatttaccc ttgcacaaag cactagaaaa ttagcattcc 7500 atccttctct gcttgctctg ctgatatcac tgtcattcaa tgcatagcca tgagctcatc 7560 ttagatccaa gcacgtaatt ccatagccga ggtccacagt ggagcagcaa cattccccat 7620 cattgctttc cccaggggcc tcccaacgac taaatcaaga gtatatctct accgtccaat 7680 agatcgtctt cgcttcaaaa tctttgacaa ttccaagagg gtccccatcc atcaaaccca 7740 gttcaataat agccgagatg catggtggag tcaattaggc agtattgctg gaatgtcggg 7800 gccagttggc ccggtggtca ttggccgcct gtgatgccat ctgccactaa atccgatcat 7860 tgatccaccg cccacgaggc gcgtctttgc tttttgcgcg gcgtccaggt tcaactctct 7920 ctgcagctcc agtccaacgc tgactgacta gtttacctac tggtctgatc ggctccatca 7980 gagctatggc gttatcccgt gccgttgctg cgcaatcgct atcttgatcg caaccttgaa 8040 ctcactcttg ttttaatagt gatcttggtg acggagtgtc ggtgagtgac aaccaacatc 8100 gtgcaaggga gattgatacg gaattgtcgc tcccatcatg atgttcttgc cggctttgtt 8160 ggccctattc gtgggatgcg atgccctcgc tgtgcagcag caggtactgc tggatgagga 8220 gccatcggtc tctgcacgca aacccaactt cctcttcatt ctcacggatg atcaggatct 8280 ccggatgaat tctccggcgt atatgccgta tacgcagg 8318

Claims

1. A protein, characterized in that: It is a protein as described in any one of A1) - A3) below: A1) A protein whose amino acid sequence is positions 20 to 439 of Sequence 1 in the Sequence Listing; A2) A protein whose amino acid sequence is positions 1 to 439 of Sequence 1 in the Sequence Listing; A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of any one of A1) - A2).

2. A biological material related to the protein according to claim 1, characterized in that: The biological material is any one of the following B1) to B4): B1) A nucleic acid molecule encoding the protein described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1); B4) A recombinant microorganism containing the nucleic acid molecule described in B1).

3. The related biological material according to claim 2, wherein: The nucleic acid molecule described in B1) is a coding gene as shown in any one of C1 - C4 below: C1) A DNA molecule whose coding sequence of the coding strand is nucleotides 58 - 1320 of Sequence 2 in the Sequence Listing; C2) A DNA molecule whose coding sequence of the coding strand is nucleotides 1 - 1320 of Sequence 2 in the Sequence Listing; C3) A DNA molecule having 75% or more identity with the nucleotide sequence defined in any one of C1) - C2) and encoding the protein described in claim 1; C4) A DNA molecule that hybridizes with the nucleotide sequence defined in any one of C1) - C3) under stringent conditions and encodes the protein described in claim 1.

4. The related biological material according to claim 2 or 3, characterized in that: The recombinant microorganism described in B4) is obtained by introducing the coding gene of the protein described in claim 1 into a recipient microorganism to obtain a recombinant microorganism that expresses the protein described in claim 1; the recipient microorganism is a eukaryotic microorganism.

5. The related biological material according to claim 4, characterized in that: The recipient microorganism is a microorganism of the genus Aspergillus.

6. The related biological material according to claim 5, characterized in that: The recipient microorganism is Aspergillus niger.

7. A method for preparing the protein according to claim 1, characterized in that, Expressing a DNA molecule containing the coding gene of the protein described in claim 1 in an organism to obtain the protein described in claim 1; the organism is a microorganism.

8. The method according to claim 7, wherein Expressing a DNA molecule containing the coding gene of the protein described in claim 1 in an organism includes introducing the coding gene of the protein described in claim 1 into a recipient microorganism to obtain a recombinant microorganism that expresses the protein described in claim 1, culturing the recombinant microorganism, and expressing to obtain the protein described in claim 1.

9. A coagulant, characterized in that: The active ingredient of the coagulant contains the protein described in claim 1.

10. Use of the protein according to claim 1 or the biomaterial according to any one of claims 2-6, characterized in that: The application is any one of the following P1 - P3: P1. The application of the biological material described in any one of claims 2 - 6 in the preparation of aspartic protease; P2. The application of the protein described in claim 1 or the biological material described in any one of claims 2 - 6 in the preparation of a coagulant; P3. The application of the protein described in claim 1 or the biological material described in any one of claims 2 - 6 in the preparation of cheese.

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