Acid protease bs2688, gene and application thereof

Abstract

This invention belongs to the field of agricultural biotechnology, specifically relating to a fungal-derived acidic protease Bs2688, its gene, and its applications. The amino acid sequence of the protease of this invention is shown in SEQ ID NO.1 or SEQ ID NO.2. This invention provides a novel protease gene, enabling the production of a high-quality protease using genetic engineering techniques, which can be applied in industries such as feed, food, and medicine.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a fungal-derived acidic protease Bs2688, its gene, and its applications. Background Technology

[0002] Proteases are a class of enzymes that catalyze the hydrolysis of proteins and are widely found in plants, animals, and microorganisms. Compared to proteases from plant and animal sources, microbial proteases are easier to cultivate, simpler to handle, and produce higher yields, making them suitable for large-scale industrial production. Therefore, microbial proteases have become an important source of proteases.

[0003] Proteases can be classified in many ways. Based on their pH level, they can be divided into acidic proteases, basic proteases, and neutral proteases. Acidic proteases are generally stable between pH 2.0 and 6.0, with the optimal pH varying slightly among species, but typically around pH 3.0. For example, the optimal pH for acidic proteases produced by *Aspergillus niger* is 3.0, while that of *Penicillium glaucus* is 3.5, and that of yeast is also around pH 3.0. These enzymes exhibit high sequence similarity and their three-dimensional structure is symmetrical and bilobed. Based on their active site, proteases are classified into serine proteases, aspartic proteases, cysteine ​​proteases, and metalloproteinases.

[0004] Proteases have wide applications in the food, brewing, fur and leather, pharmaceutical, and feed industries. In the dairy industry, proteases participate in cheese production by utilizing the high specificity of rennet for casein. During the fermentation process of baijiu (Chinese liquor), acidic proteases work synergistically to dissolve particles in fermentation raw materials, improving raw material utilization, promoting microbial growth, breaking down proteins to provide aroma precursors and flavor compounds, and decomposing yeast cell proteins. In fur and leather manufacturing, acidic proteases remove interfibrous stroma, making the leather softer and fuller. In the feed industry, the addition of acidic proteases improves protein digestibility, breaking down high-molecular-weight proteins into low-molecular-weight peptides and amino acids, making them easier for livestock and poultry to digest and absorb. This reduces irritation to the digestive tract of young animals, minimizes nutritional deficiencies, improves feed utilization, and promotes livestock and poultry growth.

[0005] Currently, most acidic proteases used in industrial production are fungal acidic proteases. These enzymes have an optimal pH of around 3.0. When the pH increases, the activity of acidic proteases decreases significantly. Furthermore, these enzymes are not heat-resistant and are very unstable at temperatures above 50°C. Therefore, the low activity of acidic proteases, and the difference between their optimal operating conditions and the environmental conditions they catalyze (such as pH and temperature), leads to reduced catalytic efficiency, thus limiting their industrial application. Summary of the Invention

[0006] To address the problems of low enzyme activity and low catalytic efficiency of existing acidic proteases, this invention provides a fungal-derived acidic protease Bs2688, which features good pH stability, high temperature resistance, and ease of production and fermentation.

[0007] The purpose of this invention is to provide an acidic protease Bs2688.

[0008] Another object of the present invention is to provide the gene for the above-mentioned protease.

[0009] Another object of the present invention is to provide a recombinant vector comprising the above-described protease.

[0010] Another object of the present invention is to provide a recombinant strain containing the above-mentioned protease gene.

[0011] Another object of the present invention is to provide a method for preparing proteases.

[0012] Another object of the present invention is to provide applications of the above-mentioned protease.

[0013] According to a specific embodiment of the present invention, the amino acid sequence of the acidic protease Bs2688 is shown in SEQ ID NO.1:

[0014]

[0015] The enzyme is 407 amino acids in length, with the N-terminal 19 amino acids forming a signal peptide sequence, namely "MHSFVTAAALVASASLTLA". Therefore, the theoretical molecular weight of the protease Bs2688 is 40.3 kDa, and its amino acid sequence is shown in SEQ ID NO.2.

[0016]

[0017]

[0018] This invention also provides a gene encoding the aforementioned protease. Full-length DNA sequence analysis revealed that the structural gene for the acidic protease Bs2688 is 1330 bp in length, contains two introns, and its genomic sequence is shown in SEQ ID NO. 3.

[0019]

[0020] This invention uses cDNA as a template to isolate and clone the protease encoding gene Bs2688 by PCR. The full-length cDNA of protease Bs2688 is 1224 bp, and its sequence is shown in SEQ ID NO.4:

[0021]

[0022]

[0023] The base sequence of the signal peptide is as follows:

[0024] "ATGCATTCAT TCGTTACGGC CGCGGCCCTT GTGGCCTCGG CCTCCCTCAC CCTCGCG"

[0025] Therefore, the coding sequence of the mature protease is shown in SEQ ID NO.5:

[0026]

[0027] This invention also provides a recombinant vector containing the above-mentioned protease gene, preferably pPIC9-Bs2688. The protease gene of this invention is inserted between suitable restriction enzyme sites on the expression vector, making its nucleotide sequence operably linked to the expression regulatory sequence. As a most preferred embodiment of this invention, the protease gene is preferably inserted between the SnaB I and Avr II restriction enzyme sites on the plasmid pPIC9, so that the nucleotide sequence is downstream of and regulated by the AOX1 promoter, yielding the recombinant yeast expression plasmid pPIC9-Bs2688.

[0028] The present invention also provides a recombinant strain containing the above-mentioned protease gene, preferably the recombinant strain GS115 / Bs2688.

[0029] The present invention also provides a method for preparing a protease, comprising the following steps:

[0030] 1) Transform host cells using the above recombinant vector to obtain recombinant bacterial strains;

[0031] 2) Culture recombinant strains and induce the expression of recombinant proteases;

[0032] 3) Recover and purify the expressed protease.

[0033] Preferably, the host cell is Pichia pastoris cell, Saccharomyces cerevisiae cell, or Hansenula polymorpha cell. Preferably, the recombinant yeast expression plasmid is transformed into Pichia pastoris GS115 cells to obtain the recombinant strain GS115 / Bs2688.

[0034] This invention also provides applications of the aforementioned protease. Genetic engineering techniques are used to industrially produce the protease, which breaks down casein, and then applies it in fields such as animal feed, food, or medicine.

[0035] This invention provides a novel protease, Bs2688, with an optimal pH of 3.0. Within a pH range of 1-7, this enzyme maintains over 80% of its activity. The optimal temperature for Bs2688 is 75°C, and it retains over 60% activity even at 80°C. This invention enables the production of high-quality proteases using genetic engineering techniques, and their application in industries such as feed, food, and pharmaceuticals. Attached Figure Description

[0036] Figure 1 This indicates the optimal pH value for the acidic protease Bs2688;

[0037] Figure 2 The pH stability of acidic protease Bs2688 is shown;

[0038] Figure 3 This indicates the optimal reaction temperature for acidic protease Bs2688;

[0039] Figure 4 The thermostability of acidic protease Bs2688 is shown. Detailed Implementation

[0040] Experimental materials and reagents

[0041] 1. Strains and vectors: Pichia pastoris GS115 and Pichia pastoris expression vector pPIC9.

[0042] 2. Enzymes and other biochemical reagents: endonucleases, ligases.

[0043] 3. Culture medium:

[0044] (1) Enzyme-producing culture medium: 30 g / L wheat bran, 30 g / L corn cob powder, 30 g / L soybean meal, 5 g / L barley glucan, 5 g / L (NH4)SO4, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·7H2O, 0.2 g / L CaCl2 in 1 L deionized water, sterilized at 121℃ and 15 lbs for 20 min.

[0045] (2) Escherichia coli culture medium LB (126 peptone, 0.5% yeast extract, 126 NaCl, pH 7.0).

[0046] (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.000049 <Biotin, 1% glycerol (v / v).

[0047] (4) BMMY medium: same components as BMGY except that 0.5% methanol is used instead of glycerol, pH 4.0.

[0048] Note: For the molecular biology experimental methods not specifically described in the following examples, they are all carried out according to the specific methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook, or according to the kit and product instructions.

[0049] Example 1 Preparation of protease Bs2688 mutant

[0050] 1. Cloning of protease-encoding gene Bs2688

[0051] The fungus Bispora sp. MEY-1 cultured in liquid for 3 days was centrifuged at 12,000 rpm for 10 min. The collected mycelia were added to a sterilized mortar and quickly ground to powder with liquid nitrogen. Then the ground mycelia were transferred to a new 50 mL centrifuge tube containing 15 mL of CTAB lysis buffer, gently inverted up and down to mix evenly, and incubated in a 65 °C water bath for 3 h. Every 20 min, gently invert up and down to mix evenly to fully lyse the mycelia. Centrifuge at 4 °C, 12,000 rpm for 10 min, aspirate the supernatant into a new centrifuge tube, add an equal volume of chloroform for extraction, and let it stand at room temperature for 5 min. Centrifuge at 4 °C, 12,000 rpm for 10 min. Take the supernatant and add an equal volume of phenol / chloroform for extraction, and let it stand at room temperature for 5 min. Centrifuge at 4 °C, 12,000 rpm for 10 min. To remove as much impurity protein as possible, then take the supernatant and add an equal volume of isopropanol, let it stand at room temperature for 5 min, and then centrifuge at 10,000 rpm for 10 min at 4 °C. Discard the supernatant, wash the precipitate twice with 70% ethanol, dry it under vacuum, add an appropriate amount of dd H2O to dissolve, and store it at -20 °C for later use.

[0052] Design cloning primers Bs2688F and Bs2688R, and perform PCR amplification with the genomic DNA of Bispora sp. MEY-1 as the template to obtain a fragment of about 1300 bp. The genomic sequence with the nucleotide sequence shown in SEQ ID NO. 3 was obtained by splicing multiple fragments.

[0053] Table 1 Primers required for PCR amplification

[0054]

[0055] 2. Obtaining of protease cDNA

[0056] Total RNA was extracted from Bispora sp. MEY-1 and analyzed using Oligo(dT) assay. 20 One strand of cDNA was obtained by reverse transcriptase. Then, primers Bs2688F and Bs2688R were designed to amplify the open reading frame. The specific sequences are shown in Table 1. The single-stranded cDNA was amplified to obtain the cDNA sequence of the protease. The amplified product was recovered and sequenced.

[0057] After comparing the genomic sequence and cDNA sequence of the protease, it was found that the gene contains two introns. The cDNA is 1224 bp long and encodes 407 amino acids. The N-terminal 19 amino acids are its signal peptide sequence. The gene Bs2688, which encodes the protease and was isolated and cloned from Bisporasp.MEY-1, was identified as a new gene.

[0058] 3. Construction of engineered protease strains

[0059] (1) Constructing expression vectors

[0060] Using the correctly sequenced cDNA of the protease Bs2688 as a template, primers F and R, containing SnaB I and Avr II restriction sites (as shown in Table 1), were synthesized to amplify the coding region of the mature Bs2688 protein. The PCR product was then digested with SnaB I and AvrII and ligated into the expression vector pPIC9. The sequence of the mature Bs2688 protein was inserted downstream of the signal peptide sequence in the expression vector, forming the correct reading frame, thus constructing the yeast expression vector pPIC9-Bs2688. This vector was then transformed into *E. coli* competent cells (Trans1). Positive transformants were sequenced. The expression plasmid vector DNA was linearized using the restriction endonuclease BglII, and then electroporated into yeast GS115 competent cells. After culturing at 30°C for 2-3 days, transformants grown on MD plates were picked for further expression experiments.

[0061] For mutants with correct sequencing, recombinant plasmids were prepared, and the expression plasmid vector DNA was linearized using the restriction endonuclease Bgl II. The DNA was then transformed into yeast GS115 competent cells by electroporation and cultured at 30°C for 2-3 days. Transformants grown on MD plates were selected for further expression experiments.

[0062] A recombinant expression vector was constructed from the coding region of the complete signal peptide protein of Bs2688 in the same manner.

[0063] (2) Screening of transformants with high protease activity

[0064] Single colonies were picked from MD plates containing transformants and spotted onto the plates according to their numbers. The MD plates were then incubated at 30°C for 1–2 days until colonies grew. Transformants were picked from the MD plates according to their numbers and inoculated into centrifuge tubes containing 3 mL of BMGY medium. The tubes were incubated at 30°C and 220 rpm for 48 h on a shaker. The culture was then centrifuged at 3,000 × g for 15 min, the supernatant was discarded, and 1 mL of BMMY medium containing 0.5% methanol was added to the centrifuge tubes. The tubes were then incubated at 30°C and 220 rpm for induced culture. After 48 h of induction culture, the tubes were centrifuged at 3,000 × g for 5 min, and the supernatant was used for enzyme activity detection to screen for transformants with high protease activity.

[0065] 4. Preparation of recombinant protease

[0066] (1) Large-scale expression of protease gene Bs2688 in Pichia pastoris at shake-flask level

[0067] Transformants with high enzyme activity were screened and inoculated into 1L Erlenmeyer flasks containing 300mL BMGY liquid medium. The flasks were shaken at 30℃ and 220rpm for 48h. The cells were centrifuged at 5,000rpm for 5min, the supernatant was gently discarded, and 100mL of BMMY liquid medium containing 0.5% methanol was added to the cells. The cells were then induced at 30℃ and 220rpm for 72h. During the induction culture, methanol solution was added every 24h to compensate for the loss of methanol and keep the methanol concentration at about 0.5%. (3) The cells were centrifuged at 12,000×g for 10min, the supernatant fermentation broth was collected, the enzyme activity was detected, and SDS-PAGE protein electrophoresis analysis was performed.

[0068] (2) Purification of recombinant protease

[0069] The supernatant of the recombinant protease expressed in shake flasks was collected, concentrated using a 10 kDa membrane, and the culture medium was replaced with low-salt buffer. Further concentration was then achieved using a 10 kDa ultrafiltration tube. The concentrated recombinant protease Bs2688, diluted to a certain factor, was purified by ion-exchange chromatography. Enzyme activity and protein concentration were measured in the collected eluent.

[0070] Example 2. Verification of the enzymatic properties of the recombinant protease

[0071] The activity of the protease of this invention was analyzed using the Folin-Ciocalteu reagent colorimetric method. The specific method is as follows: Under pH 3.0 and 55°C conditions, a 1 mL reaction system consisted of 500 μL of appropriately diluted enzyme solution and 500 μL of substrate. The reaction was allowed to proceed for 10 min, and then 1 mL of trichloroacetic acid (0.4 mol / L) was added to terminate the reaction. The reaction system was then centrifuged at 12000 rpm for 3 min, and 500 μL of the supernatant was aspirated and 2.5 mL of sodium carbonate (0.4 mol / L) was added. Then, 500 μL of Folin-Ciocalteu reagent was added, and the mixture was incubated at 40°C for 20 min. After cooling, the OD value was measured at 680 nm. The protease activity unit is defined as the amount of enzyme required to break down casein substrate to produce 1 μmol of tyrosine per minute under certain conditions.

[0072] 1. Optimal pH and pH stability of protease Bs2688

[0073] The optimal pH was determined by performing enzymatic reactions of the purified protease Bs2688 at different pH values. The buffers used were glycine-hydrochloric acid buffer (pH 1.0–3.0), citrate-disodium hydrogen phosphate buffer (pH 3.0–8.0), and Tris-HCl buffer (pH 8.0–10.0).

[0074] like Figure 1 As shown, at 55°C, the optimal pH for protease Bs2688 is 3.0, and within the pH range of 2.5-3.5, the enzyme can maintain more than 70% of its activity.

[0075] The enzyme solution was treated in buffer solutions of different pH values ​​at 37°C for 60 min, and then the enzyme activity was measured to study the pH stability of the enzyme. Figure 2 As shown, the experimental results indicate that the protease Bs2688 can maintain more than 80% of its enzyme activity between pH 1.0 and pH 7.0, demonstrating that the enzyme has excellent pH stability.

[0076] 2. Optimal reaction temperature and thermal stability of protease Bs2688

[0077] The enzyme activity of the protease was determined at pH 3.0 and at 30-90°C.

[0078] like Figure 3 As shown, the optimal reaction temperature of the protease Bs2688 of the present invention is 75°C, and it still retains more than 60% enzyme activity at 80°C.

[0079] The protein Bs2688 was treated at different temperatures for different times, and then its activity was tested at 75°C to determine its thermal stability.

[0080] like Figure 4As shown, the experimental results indicate that after treatment at 70℃ for 60 min, the remaining enzyme activity is above 60%, and even after treatment at 75℃ for 30 min, the enzyme still retains 40% of its activity, which shows that the enzyme has good stability. sequence list <110> Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences <120> Acidic protease Bs2688, its gene, and its applications <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 407 <212> PRT <213> Thermophilic fungus MEY‑1 (Bispora sp. MEY‑1) <400> 1 Met His Ser Phe Val Thr Ala Ala Ala Leu Val Ala Ser Ala Ser Leu 1 5 10 15 Thr Leu Ala Ala Pro Ala Gln Ile Val Gly Arg Ser Thr Phe Gln Ile 20 25 30 Asp Gln Val Ala Ser Gly Lys Val Tyr Lys Asn Gly Pro Met Ala Met 35 40 45 Met Gln Thr Tyr Asn Lys Tyr Ala His Val Gly Ala Val Ala Pro Ala 50 55 60 Ala Val Val Ala Ala Ala Ala Ala Ala Gln Thr Gly Glu Val Ser Ala 65 70 75 80 Asn Pro Glu Gln Tyr Asp Glu Ser Tyr Leu Cys Pro Val Thr Ile Gly 85 90 95 Asp Gln Thr Leu Asn Leu Asp Phe Asp Thr Gly Ser Ala Asp Leu Trp 100 105 110 Val Phe Ser Thr Leu Thr Pro Ser Ser Glu Ser Thr Gly His Thr Leu 115 120 125 Tyr Asn Pro Ala Asp Ser Gly Thr Glu Lys Gln Gly Tyr Thr Trp Asn 130 135 140 Ile Thr Tyr Gly Asp Gly Ser Gly Ala Ala Gly Val Val Tyr Ala Asp 145 150 155 160 Lys Val Val Val Gly Gly Val Thr Ala Thr Ser Gln Ala Val Glu Ala 165 170 175 Ala Thr Ser Val Ser Ser Glu Phe Thr Gln Asp Thr Lys Asn Asp Gly 180 185 190 Leu Leu Gly Leu Ala Phe Ser Ser Ile Asn Thr Val Gln Pro Val Gln 195 200 205 Gln Thr Thr Phe Phe Asp Thr Val Lys Asp Thr Leu Ala Lys Lys Leu 210 215 220 Phe Thr Ala Asp Leu Lys Lys Gly Ala Ala Gly Ser Tyr Gly Phe Gly 225 230 235 240 Tyr Ile Asp Ser Ser Lys Tyr Thr Gly Thr Ile Thr Tyr Val Pro Val 245 250 255 Asn Asn Glu Asn Gly Phe Trp Gln Phe Thr Ala Gly Gly Tyr Ser Ile 260 265 270 Gly Gly Gly Asn Gly Thr Ser Gly Ser Asn Ala Thr Thr Gly Ser Ile 275 280 285 Gly Thr Ser Ile Ala Asp Thr Gly Thr Thr Leu Leu Tyr Leu Pro Ser 290 295 300 Asn Val Val Thr Ala Tyr Tyr Lys Gln Val Ser Gly Ala Ser Tyr Asn 305 310 315 320 Ser Ala Gln Gly Gly Tyr Thr Tyr Pro Cys Gly Ala Thr Leu Pro Asp 325 330 335 Phe Asn Val Ala Ile Gly Gly Lys Thr Phe Val Val Pro Gly Thr Asp 340 345 350 Leu Asn Tyr Ala Pro Ile Asn Ser Ala Gly Thr Thr Cys Phe Gly Gly 355 360 365 Ile Gln Ala Asn Thr Gly Ile Gly Phe Asn Ile Phe Gly Asp Ile Phe 370 375 380 Leu Lys Ser Val Tyr Ala Val Phe Asp Gln Thr Gln Ser Ser Pro Arg 385 390 395 400 Leu Gly Phe Ala Glu Gln Ser 405 <210> 2 <211> 388 <212> PRT <213> Thermophilic fungus MEY-1 (Bispora sp. MEY-1) <400> 2 Ala Pro Ala Gln Ile Val Gly Arg Ser Thr Phe Gln Ile Asp Gln Val 1 5 10 15 Ala Ser Gly Lys Val Tyr Lys Asn Gly Pro Met Ala Met Met Gln Thr 20 25 30 Tyr Asn Lys Tyr Ala His Val Gly Ala Val Ala Pro Ala Ala Val Val 35 40 45 Ala Ala Ala Ala Ala Ala Gln Thr Gly Glu Val Ser Ala Asn Pro Glu 50 55 60 Gln Tyr Asp Glu Ser Tyr Leu Cys Pro Val Thr Ile Gly Asp Gln Thr 65 70 75 80 Leu Asn Leu Asp Phe Asp Thr Gly Ser Ala Asp Leu Trp Val Phe Ser 85 90 95 Thr Leu Thr Pro Ser Ser Glu Ser Thr Gly His Thr Leu Tyr Asn Pro 100 105 110 Ala Asp Ser Gly Thr Glu Lys Gln Gly Tyr Thr Trp Asn Ile Thr Tyr 115 120 125 Gly Asp Gly Ser Gly Ala Ala Gly Val Val Tyr Ala Asp Lys Val Val 130 135 140 Val Gly Gly Val Thr Ala Thr Ser Gln Ala Val Glu Ala Ala Thr Ser 145 150 155 160 Val Ser Ser Glu Phe Thr Gln Asp Thr Lys Asn Asp Gly Leu Leu Gly 165 170 175 Leu Ala Phe Ser Ser Ile Asn Thr Val Gln Pro Val Gln Gln Thr Thr 180 185 190 Phe Phe Asp Thr Val Lys Asp Thr Leu Ala Lys Lys Leu Phe Thr Ala 195 200 205 Asp Leu Lys Lys Gly Ala Ala Gly Ser Tyr Gly Phe Gly Tyr Ile Asp 210 215 220 Ser Ser Lys Tyr Thr Gly Thr Ile Thr Tyr Val Pro Val Asn Asn Glu 225 230 235 240 Asn Gly Phe Trp Gln Phe Thr Ala Gly Gly Tyr Ser Ile Gly Gly Gly 245 250 255 Asn Gly Thr Ser Gly Ser Asn Ala Thr Thr Gly Ser Ile Gly Thr Ser 260 265 270 Ile Ala Asp Thr Gly Thr Thr Leu Leu Tyr Leu Pro Ser Asn Val Val 275 280 285 Thr Ala Tyr Tyr Lys Gln Val Ser Gly Ala Ser Tyr Asn Ser Ala Gln 290 295 300 Gly Gly Tyr Thr Tyr Pro Cys Gly Ala Thr Leu Pro Asp Phe Asn Val 305 310 315 320 Ala Ile Gly Gly Lys Thr Phe Val Val Pro Gly Thr Asp Leu Asn Tyr 325 330 335 Ala Pro Ile Asn Ser Ala Gly Thr Thr Cys Phe Gly Gly Ile Gln Ala 340 345 350 Asn Thr Gly Ile Gly Phe Asn Ile Phe Gly Asp Ile Phe Leu Lys Ser 355 360 365 Val Tyr Ala Val Phe Asp Gln Thr Gln Ser Ser Pro Arg Leu Gly Phe 370 375 380 Ala Glu Gln Ser [[ID=२३]]385 <210> 3 <211> 1330 <212> DNA <213> Thermophilic fungus MEY-¹ (Bispora sp. MEY-¹) <400> 3 atgcattcat tcgttacggc cgcggccctt gtggcctcgg cctccctcac cctcgcggct 60 ccggcccaga ttgtcggccg cagcaccttt cagatcgatc aagtggcctc tggtaaggtc 120 tacaagaacg gccctatggc catgatgcag acatacaaca agtacgcgca cgtaggcgcc 180 It should be noted that in the above translation, there is a possible error in the original text where "MEY‑1" is written as "MEY-¹" in the translation of item . It should be kept consistent with the original as "MEY‑1". Also, the "२३" in item [[ID=२३]] seems to be an incorrect encoding and should be "23". After correcting these, the translation would be more accurate. The corrected translation for item would be "<213> Thermophilic fungus MEY-1 (Bispora sp. MEY-1)" and for item would be "385".gtcgcgccg ctgccgttgt ggccgcgcg gccgcgcgc agactggcga ggtgtcgca 240 aatcccgagc agtacgacga aagctacctt tgtcctgtca ctattgggga tcagaccttg 300 aacttggact tcgacacggg cagcgcggac ctgtgagcag tttctatcga aaacctcatt 360 gcttcaggac tgattgcaga tacagttggg tgttttcaac cctcactccg tcaagcgagt 420 caacaggcca cacgttgtat aaccccgccg actctggcac ggagaagcag ggctatacct 480 ggaacatcac ctacggcgac ggctcgggcg cagccggtgt ggtgtacgcc gataaggtgg 540 tcgttggcgg ggtcaccgcg acctcgcagg cggtggaggc ggcgacatcg gtctccagcg 600 aattcacaca ggacaccaag aacgatggcc tgctcggctt ggcgttcagc tcgatcaata 660 ccgttcagcc ggtgcaacag actactttct tcgatacggt caaggacacg ctggccaaga 720 agctcttcac tgccgatctc aagaaggggg ctgccggcag ctatggtttt gggtgagtcg 780 gattagactg gtttctgata gacaaggatt aacggtcgac tccagttaca tcgacagctc 840 caaatacacc ggcaccatca cctatgtgcc cgtgaacaat gagaacggct tctggcagtt 900 caccgcaggc ggctactcca tcggtggcgg caacggcacg tcaggcagca acgcgaccac 960 aggcagcatt ggcacctcca tcgcggacac cggcaccacc ctcctctact tgcccagcaa 1020 cgtagtcacg gcttactaca agcaagtctc gggcgcttct tataactcgg cgcaaggcgg 1080 ttacacttac ccgtgcggtg ccactctgcc cgacttcaac gtggccattg gcggcaagac 1140 tttcgtcgtc cccggcaccg atctcaatta cgcgcctatc aacagcgcgg gcaccacgtg 1200 cttcggcggg attcaagcta acacgggcat cggattcaac atcttcggcg acattttcct 1260 aaagagcgtc tacgccgtct tcgaccagac tcagagctcg ccgcgcctcg gctttgccga 1320 gcaatcgtaa 1330 <210> 4 <211> 1224 <212> DNA <213> Thermophilic fungus MEY-1 (Bispora sp. MEY-1) <400> 4 atgcattcat tcgttacggc cgcggccctt gtggcctcgg cctccctcac cctcgcggct 60 ccggcccaga ttgtcggccg cagcaccttt cagatcgatc aagtggcctc tggtaaggtc 120 tacaagaacg gccctatggc catgatgcag acatacaaca agtacgcgca cgtaggcgcc 180 gtcgcgcccg ctgccgttgt ggccgccgcg gccgccgcgc agactggcga ggtgtccgca 240 aatcccgagc agtacgacga aagctacctt tgtcctgtca ctattgggga tcagaccttg 300 aacttggact tcgacacggg cagcgcggac ctttgggtgt tttcaaccct cactccgtca 360 agcgagtcaa caggccacac gttgtataac cccgccgact ctggcacgga gaagcagggc 420 tatacctgga acatcaccta cggcgacggc tcgggcgcag ccggtgtggt gtacgccgat 480 aaggtggtcg ttggcggggt caccgcgacc tcgcaggcgg tggaggcggc gacatcggtc 540 tccagcgaat tcacacagga caccaagaac gatggcctgc tcggcttggc gttcagctcg 600 atcaataccg ttcagccggt gcaacagact actttcttcg atacggtcaa ggacacgctg 660 gccaagaagc tcttcactgc cgatctcaag aagggggctg ccggcagcta tggttttggt 720 tacatcgaca gctccaaata caccggcacc atcacctatg tgcccgtgaa caatgagaac 780 ggcttctggc agttcaccgc aggcggctac tccatcggtg gcggcaacgg cacgtcaggc 840 agcaacgcga ccacaggcag cattggcacc tccatcgcgg acaccggcac caccctctc 900 tacttgccca gcaacgtagt cacggcttac tacaagcaag tctcgggcgc ttcttataac 960 tcggcgcaag gcggttacac ttacccgtgc ggtgccactc tgcccgactt caacgtggcc 1020 attggcggca agactttcgt cgtccccggc accgatctca attacgcgcc tatcaacagc 1080 gcgggcacca cgtgcttcgg cgggattcaa gctaacacgg gcatcggatt caacatcttc 1140 ggcgacattt tcctaaagag cgtctacgcc gtcttcgacc agactcagag ctcgccgcgc 1200 ctcggctttg ccgagcaatc gtaa 1224 <210> 5 <211> 1167 <212> DNA <213> Thermophilic fungus MEY-1 (Bispora sp. MEY-1) <400> 5 gctccggccc agattgtcgg ccgcagcacc tttcagatcg atcaagtggc ctctggtaag 60 gtctacaaga acggccctat ggccatgatg cagacataca acaagtacgc gcacgtaggc 120 gccgtcgcgc ccgctgccgt tgtggccgcc gcggccgccg cgcagactgg cgaggtgtcc 180 gcaaatcccg agcagtacga cgaaagctac ctttgtcctg tcactattgg ggatcagacc 240 ttgaacttgg acttcgacac gggcagcgcg gacctttggg tgttttcaac cctcactccg 300 tcaagcgagt caacaggcca cacgttgtat aaccccgccg actctggcac ggagaagcag 360 ggctatacct ggaacatcac ctacggcgac ggctcgggcg cagccggtgt ggtgtacgcc 420 gataaggtgg tcgttggcgg ggtcaccgcg acctcgcagg cggtggaggc ggcgacatcg 480 gtctccagcg aattcacaca ggacaccaag aacgatggcc tgctcggctt ggcgttcagc 540 tcgatcaata ccgttcagcc ggtgcaacag actactttct tcgatacggt caaggacacg 600 ctggccaaga agctcttcac tgccgatctc aagaaggggg ctgccggcag ctatggtttt 660 ggttacatcg acagctccaa atacaccggc accatcacct atgtgcccgt gaacaatgag 720 aacggcttct ggcagttcac cgcaggcggc tactccatcg gtggcggcaa cggcacgtca 780 ggcagcaacg cgaccacagg cagcattggc acctccatcg cggacaccgg caccaccctc 840 ctctacttgc ccagcaacgt agtcacggct tactacaagc aagtctcggg cgcttcttat 900 aactcggcgc aaggcggtta cacttacccg tgcggtgcca ctctgcccga cttcaacgtg 960 gccattggcg gcaagacttt cgtcgtcccc ggcaccgatc tcaattacgc gcctatcaac 1020 agcgcgggca ccacgtgctt cggcgggatt caagctaaca cgggcatcgg attcaacatc 1080 ttcggcgaca ttttcctaaa gagcgtctac gccgtcttcg accagactca gagctcgccg 1140 cgcctcggct ttgccgagca atcgtaa 1167

Claims

1. Acidic protease Bs2688, characterized in that, The amino acid sequence of the acidic protease Bs2688 is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The acidic protease Bs2688 gene, characterized in that, The acidic protease Bs2688 of claim 1 is encoded.

3. The acidic protease Bs2688 gene according to claim 2, characterized in that, The nucleotide sequence of the acidic protease Bs2688 gene is shown in SEQ ID NO.3 or SEQ ID NO.

4.

4. A recombinant expression vector comprising the acidic protease Bs2688 gene as described in claim 2.

5. A recombinant strain containing the acidic protease Bs2688 gene as described in claim 2.

6. A method for preparing acidic protease Bs2688, characterized in that, The method includes the following steps: (1) Transform host cells with a recombinant vector containing the acidic protease Bs2688 gene as described in claim 2 to obtain a recombinant strain; (2) Cultivate recombinant strains and induce expression of acidic protease Bs2688; (3) The acidic protease Bs2688 obtained by isolation and purification.

7. The use of proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.2 as acidic proteases for hydrolyzing casein.

8. Application of proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.2 as feed additives.

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