Acidic protease mutant with improved stability and its product and application

Abstract

This invention relates to the fields of genetic engineering and enzyme engineering, specifically to a mutant acidic protease with improved stability, its products, and applications. More specifically, it relates to a heat-resistant acidic protease mutant IAIC6M and its encoding gene. This invention utilizes the amino acid sequence shown in SEQ ID NO.1. iaic6 The sequence was mutated by D86I, G168A, Q185I, and Q339C to obtain the mutant IAIC6M. Compared with IAIC, this mutant can still maintain more than 60% of its enzyme activity at 75°C for 3 minutes, while IAIC can only maintain 20% of its enzyme activity at 75°C for 3 minutes. The acidic protease mutant IAIC6M of the present invention has the following properties: optimal pH 3.0, and still has high enzyme activity under high temperature conditions, and has wide application value in industry, food, and medicine.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and enzyme engineering, and in particular to an acidic protease mutant with improved stability and a product and application thereof. Background Art

[0002] Acidic proteases, also known as aspartic proteases, are mainly derived from animal viscera and microbial metabolites. They are enzymes that hydrolyze animal and plant proteins into amino acids and small molecular peptides under acidic conditions. The active center is composed of two aspartic acid residues. Acidic proteases include pepsin, chymosin, and some microbial proteases. At present, it has been found that many microorganisms can produce acidic proteases, mainly molds, such as Aspergillus niger, Aspergillus usami, Aspergillus saito, Aspergillus awamori, and Mucor microsporus.

[0003] Since acid proteases have good acid resistance and are not prone to protein corruption caused by bacterial growth during protein hydrolysis, they are widely used in food, animal husbandry, aquatic products, medicine, leather processing and other fields. In the food industry, acid proteases are added to alcohol fermentation to promote protein hydrolysis in raw materials, promote yeast growth, and increase the rate of alcohol fermentation; in animal husbandry, acid proteases are added to feed to decompose crude protein in feed into amino acids and small molecular peptides, promote the absorption and conversion of nutrients in livestock and poultry, and thus reduce breeding costs; in the leather industry, acid proteases promote leather depilation and maintain leather gloss, making the leather softer and improving leather quality.

[0004] At present, most of the acid protease products on the market have unstable enzymatic properties and low enzyme activity, which to a certain extent restricts the industrial application of acid protease. Most of the acid proteases used in industrial production are not heat-resistant. When the temperature is higher than 50°C, the enzyme activity will drop sharply or become inactivated. In addition, the optimal pH of this type of enzyme is around 3. When the pH rises, the enzyme activity decreases, which also affects the industrial application scope of acid protease.

[0005] Traditional acid protease fermentation production has the disadvantages of low yield, poor heat resistance, low enzyme activity, and high production cost. The conventional breeding methods are very limited in improving its yield and heat resistance. Therefore, cultivating engineered bacteria through genetic engineering technology is an effective way to obtain high-yield strains of thermostable acid protease. Summary of the invention

[0006] In order to solve the above problems, the present invention provides an acidic protease mutant, which has the advantages of high temperature resistance and high pH stability.

[0007] In one aspect, the present invention provides a thermostable acidic protease mutant IAIC6M, whose amino acid sequence is shown in SEQ ID NO.2.

[0008] In yet another aspect, the present invention provides a nucleic acid encoding the aforementioned acidic protease mutant IAIC6M.

[0009] Specifically, the sequence of the nucleic acid may be SEQ ID NO. 3 or a sequence having 65% sequence identity with SEQ ID NO. 3.

[0010] Preferably, specifically, the sequence of the nucleic acid may be SEQ ID NO.3 or a sequence having 80% sequence identity with SEQ ID NO.3.

[0011] Further preferably, specifically, the sequence of the nucleic acid may be SEQ ID NO.3 or a sequence having 95% sequence identity with SEQ ID NO.3.

[0012] More preferably, specifically, the sequence of the nucleic acid may be SEQ ID NO.3.

[0013] In yet another aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid.

[0014] Specifically, the recombinant vector includes but is not limited to: a plasmid, a phage or a virus.

[0015] Preferably, the recombinant vector may be a plasmid.

[0016] More preferably, the vector is pPIC9K Plasmid.

[0017] Specifically, the nucleic acid is inserted into a plasmid pPIC9K on Eco RI and Not I restriction enzyme cutting site, so that the nucleotide sequence is located downstream of the AOX1 promoter and is regulated by it.

[0018] In yet another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or recombinant vector.

[0019] Specifically, the host cell can be a eukaryotic cell or a prokaryotic cell.

[0020] More specifically, the host cell may be Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Bacillus or Lactobacillus.

[0021] Preferably, the cell may be Pichia pastoris, Saccharomyces cerevisiae or Hansenula polymorpha.

[0022] More preferably, the cell is Pichia pastoris.

[0023] More preferably, the cell is Pichia pastoris GS115 .

[0024] In another aspect, the present invention provides a method for preparing the aforementioned acidic protease mutant IAIC6M, comprising the following steps:

[0025] S1. transforming host cells with the aforementioned recombinant vector to obtain a recombinant strain;

[0026] S2, culturing the recombinant strain to induce the expression of the acidic protease mutant IAIC6M;

[0027] S3. Recover and purify the expressed acidic protease mutant IAIC6M.

[0028] In yet another aspect, the present invention provides an enzyme preparation, comprising the aforementioned acidic protease mutant IAIC6M.

[0029] Specifically, the enzyme preparation also includes food raw materials and / or secondary additives.

[0030] In yet another aspect, the present invention provides a drug comprising the aforementioned acidic protease mutant IAIC6M.

[0031] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0032] In yet another aspect, the present invention provides a fermentation culture, comprising the aforementioned acid protease mutant IAIC6M.

[0033] In another aspect, the present invention provides use of the aforementioned acidic protease mutant IAIC6M or enzyme preparation in the preparation of a drug.

[0034] In another aspect, the present invention provides the use of the aforementioned acidic protease mutant IAIC6M or enzyme preparation in preparing feed.

[0035] The technical effects achieved by the present invention are:

[0036] The thermostable acidic protease of the present invention has an optimum pH of 3.0, has high enzyme activity at pH 2.0-3.0, has good thermal stability, and has a high temperature resistance that broadens its application range. Acidic protease-producing bacteria are generally fungi such as molds and yeasts, and there are fewer strains of bacteria that produce acidic proteases. Acidic protease has good acid resistance and can be widely used in food, medicine and feed processing industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the optimal pH of the recombinant acidic protease mutant.

[0038] Figure 2 pH stability of recombinant acidic protease mutants.

[0039] Figure 3 is the optimal temperature of the recombinant acidic protease mutant.

[0040] Figure 4 The thermal stability of recombinant acidic protease mutants. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0042] Experimental materials and reagents

[0043] 1. Strains and vectors: Acidic protease mutant gene of the present invention IAIC6M Synthesized by Beijing Ruibo Xingke Biotechnology Co., Ltd., Pichia pastoris expression vector pPIC9K and strains GS115 Purchased from Invitrogen.

[0044] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from TaKaRa, ligases were purchased from TaKaRa, mannan was purchased from Sigma, and the others were domestic reagents (all available from common biochemical reagent companies).

[0045] 3. Culture medium:

[0046] (1) Yeast medium YPD: 1%wt peptone, 0.5%wt yeast extract, 1%wt glucose, 2%wt agar, pH 7.0.

[0047] (2) Escherichia coli culture medium LB: 1%wt peptone, 0.5%wt yeast extract, 1%wtNaCl, pH 7.0.

[0048] (3) BMGY medium: 1%wt yeast extract, 2%wt peptone, 1.34%wt YNB, 0.00004%wt biotin, 1% glycerol (V / V).

[0049] (4) BMMY medium: except for 0.5%wt methanol instead of glycerol, the rest of the ingredients are the same as BMGY.

[0050] Note: The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd Edition) by J. Sambrook, or according to the kits and product instructions.

[0051] Example 1 Aspergillus niger ( Aspergillus niger ) Acid protease mutant encoding gene iAi Synthesis

[0052] The present invention uses the acid protease from Aspergillus niger iaic6 The gene (amino acid sequence as shown in SEQ ID NO.1) was used as a reference, and the following mutations were made to its sequence (D86I, G168A, Q185I, Q339C), and the 5' and 3' ends of the mutated sequence were added respectively. EcoR I and Not I restriction enzyme cutting site, and sent the sequence to Beijing Ruibo Xingke Biotechnology Co., Ltd. for artificial gene synthesis. The amino acid sequence of the artificially synthesized acidic protease mutant is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.3.

[0053] SEQ ID NO.1:

[0054] APAPTRKGFTINQIARPANKTRTINLPGMYARSLAKFGGTVPQSVKEAASKGSAVTTPQNNDEEYLTPVTVGKSTLHLDFDTGSADLWVFSDELPSSEQTGHDLYTPSSSATKLSGYTWDISYGDGSSASGDVYRDTVTVGGVTTNKQAVEAASKISSEFVQNTANDGLLGLAFSSINTVQPKAQTT FFDTVKSQLDSPLFAVQLKHDAPGVYDFGYIDDSKYTGSITYTDADSSQGYWGFSTDGYSIGDGSSSSGFSAIADTGTTLILLDDEIVSAYYEQVSGAQESEEAGGYVFSCSTNPPDFTVVIGDYKAVVPGKYINYAPISTGSSTCFGGIQSNSGLGLSILGDVFLKSQYVVFNSEGPKLGFAAQA;

[0055] SEQ ID NO.2:

[0056] APAPTRKGFTINQIARPANKTRTINLPGMYARSLAKFGGTVPQSVKEAASKGSAVTTPQNNDEEYLTPVTVGKSTLHLDFDTGSAILVFSDELPSSEQTGHDLYTPSSSATKLSGYTWDISYGDGSSASGDVYRDTVGGVTTNKQAVEAASKISSEFVQNTANDALLGLAFSSINTVQPCAITT FFDTVKSQLDSPLFAVQLKHDAPGVYDFGYIDDSKYTGSITYTDADSSQGYWGFSTDGYSIGDGSSSSGFSAIADDGTTLILLDDEIVSAYYEQVSGAQESEEAGGYVFSCSTNPPDFTVVIGDYKAVVPGKYINYAPISTGSSTCFGGICSNSSLGLSLIGDVFLKSQYVVFNSEGPKLGFAAQA;

[0057] SEQ ID NO.3:

[0058]

[0059] Example 2 Thermoresistant acidic protease gene iAi Clone

[0060] The synthesized gene vector was stored in the form of puncture bacteria. The puncture bacteria were picked up with a sterile toothpick in the clean bench and placed in an LB shaking tube containing Amp antibiotic (working concentration: 100 μg / mL). The tube was cultured overnight at 37°C and 220 rpm. The next day, the gene-containing vector was extracted according to the instructions of the PurePlasmid Mini Kit (CW0500).

[0061] According to the acid protease gene sequence, the following primers were designed and synthesized:

[0062] P1 (SEQ ID NO.4):5'-CCGGAATTCCGGGTCCAGCCCCAACC-3';

[0063] P2 (SEQ ID NO. 5): 5'-TTGCGGCCGCAAAGCCTGAGCGGCGAA-3'.

[0064] The extracted vector was used as a template for PCR amplification. The PCR reaction parameters were: 94℃ denaturation for 5 min; then 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 2 min, and 72℃ incubation for 10 min after 30 cycles. A fragment of about 1146 bp was obtained, which was recovered and connected to the pMD19 vector and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 39.3 kDa.

[0065] According to the nucleotide sequence obtained by sequencing, the obtained nucleotide sequence was compared with iAi The sequences were compared and confirmed to be correct.

[0066] Example 3 Preparation of recombinant thermostable acidic protease IAIC6M

[0067] The expression vector pPIC9K Double enzyme digestion ( Eco R I+ Not I), and at the same time, the gene encoding the thermostable acidic protease iAi Enzyme digestion ( Eco R I+ Not I), enzymatically cut out the gene fragment encoding the mature thermostable acidic protease and the expression vector pPIC9K Connect to obtain the gene containing the thermostable acidic protease iAi Recombinant plasmid pPIC9K-iaic6M Pichia pastoris GS115, obtain recombinant Pichia pastoris strains GS115 / IAIC6M .

[0068] Take the recombinant plasmid GS115 strains and control strains (i.e. strains without mutations) GS115 / iaic6 ), inoculated in 300 mL BMGY culture medium, cultured at 30℃ 200 rpm for 48 hours, and then centrifuged to collect the bacteria. Then resuspended in 150 mL BMMY medium, cultured at 30℃ 200 rpm. After 72 hours of induction, the supernatant was collected by centrifugation to determine the activity of the thermostable acidic protease.

[0069] Example 4 Activity Analysis of Thermoresistant Acidic Protease IAIC6M

[0070] Acidic protease activity determination: The specific method is as follows: under pH 3.0, 40℃, add 1 mL of appropriately diluted enzyme solution, 1 mL of substrate, react for 10 min, add 2 mL of trichloroacetic acid solution to terminate the reaction, add sodium carbonate solution and Folin reagent to the filtrate, water bath for 20 min, and measure the OD value at 680 nm. 1 enzyme activity unit (U) is defined as 1 g of solid enzyme powder (or 1 mL of liquid enzyme) hydrolyzes casein to produce 1 ug of tyrosine in 1 min under certain temperature and pH conditions, which is 1 enzyme activity unit, expressed in μ / g (μ / mL).

[0071] Example 5 Determination of properties of thermostable acidic protease IAIC6M

[0072] The enzymatic properties of the recombinant thermostable acidic protease IAIC6M and the unmutated acidic protease IAIC6 were measured and compared. At the same time, the mutant recombinant acidic protease IAIC6M2 was added for enzymatic property comparison. The sequence of IAIC6M2 was S122A and T230V mutated on the basis of SEQ ID NO.1. The specific preparation methods of the IAIC6M mutant and the IAIC6M2 recombinant enzyme are as described in the above examples.

[0073] 1. The optimum pH and pH stability of recombinant acidic protease IAIC6 were determined as follows:

[0074] The purified acidic proteases IAIC6, IAIC6M, and IAIC6M2 were subjected to enzymatic reactions at different pH values ​​to determine their optimum pH values. The appropriately diluted enzyme solution was subjected to enzymatic reactions at different pH values ​​(1.0-10.0) to determine its optimum reaction pH value. The buffers used were as follows: pH 1.0-3.0: 0.1 mol / L glycine-hydrochloric acid buffer; pH 4.0-7.0: 0.1 mol / L citric acid-disodium hydrogen phosphate buffer; pH 8.0-10.0: 0.1 mol / L glycine-sodium hydroxide buffer. The pH adaptability results of the acidic protease IAIC6 in buffer systems of different pH values ​​at 40°C ( Figure 1 ) showed that the optimum pH of the three was 3.0, but the recombinant acidic protease IAIC6M was able to maintain more than 80% of its enzyme activity in the pH range of 2.0-3.0, indicating that the pH application range of the recombinant enzyme IAIC6M has been improved.

[0075] The purified acidic proteases IAIC6, IAIC6M, and IAIC6M2 were treated in the above-mentioned buffers of different pH values ​​at 40°C for 180 min, and then the enzyme activity was measured in a pH 3.0 buffer system at 40°C to study the pH tolerance of the enzyme. Figure 2 ) showed that the recombinant acidic protease IAIC6M was very stable between pH 1.0 and 8.0. After treatment for 180 min in this pH range, the remaining enzyme activity was above 60%, and the relative enzyme activity was significantly improved compared with that of IAIC6 and IAIC6M2, indicating that the recombinant enzyme had good pH stability.

[0076] 2. The optimum temperature and thermal stability determination method of recombinant acid protease IAIC6 is as follows:

[0077] The optimum temperature of acid protease was determined by performing enzymatic reaction in pH 3.0 buffer system and different temperatures (30-85°C). The temperature resistance was determined by treating acid protease at different temperatures for different time periods and then measuring the enzyme activity at 40°C. The optimum temperature of recombinant acid protease IAIC6M was determined ( Figure 3 ) showed that its optimum temperature was 50°C, and it still had more than 45% enzyme activity at 40-60°C.

[0078] The enzyme thermal stability test showed that ( Figure 4 ), IAIC6M has good thermal stability and can maintain more than 60% enzyme activity after incubation at 75℃ for 3 minutes. The remaining enzyme activity of IAIC6 is only 20%, and that of IAIC6M2 is only 41%. Compared with the two, IAIC6M has significantly improved thermal stability and good heat resistance.

Claims

1. A thermostable acidic protease mutant IAIC6M, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. A nucleic acid encoding the acidic protease mutant IAIC6M according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The sequence of the nucleic acid is shown in SEQ ID NO.

3.

4. A recombinant vector, characterized in that: Comprising the nucleic acid of claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector includes plasmid, bacteriophage or virus.

6. A host cell, characterized in that It comprises the nucleic acid according to any one of claims 2-3 or the recombinant vector according to any one of claims 4-5.

7. The host cell according to claim 6, characterized in that The host cell is a eukaryotic cell or a prokaryotic cell.

8. The method for preparing the acidic protease mutant IAIC6M according to claim 1, characterized in that: The following steps are involved: S1. Transform a host cell with the recombinant vector described in any one of 4-5 to obtain a recombinant strain; S2, culturing the recombinant strain to induce the expression of the acidic protease mutant IAIC6M; S3. Recover and purify the expressed acidic protease mutant IAIC6M.

9. An enzyme preparation, characterized in that The enzyme preparation comprises the acidic protease mutant IAIC6M according to claim 1.

10. Use of the acidic protease mutant IAIC6M according to claim 1 or the enzyme preparation according to claim 9 in preparing feed.

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