CN107384900B - A fungus-derived acid protease 6749 and its gene and application - Google Patents

Abstract

A fungus-derived acid protease 6749 and its gene and application. The invention relates to the field of genetic engineering. Specifically, the present invention relates to an acid protease 6749 derived from fungi and its gene and application, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2. The acid protease of the invention has good properties and can be used in industries such as food, feed, medicine and the like. According to the technical scheme of the present invention, it is possible to realize the production of proteases with good properties and suitable for industrial application by means of genetic engineering.

Description

A fungus-derived acid protease 6749 and its gene and application

technical field

The invention relates to the field of genetic engineering. Specifically, the present invention relates to an acid protease 6749 derived from fungi and its gene and application.

Background technique

Protease is a kind of enzyme that catalyzes the hydrolysis of protein, and is widely used in industries such as food, washing, and tanning. Compared with animal and plant-derived proteases, microbial-derived proteases have the characteristics of convenient cultivation, simple operation and high enzyme production, which are convenient for industrial batch production and can be used in large-scale production. Therefore, microbial-derived proteases have become an important source of current proteases.

Proteases are classified into acidic proteases, alkaline proteases and neutral proteases according to the pH at which they act. According to the active center, proteases can be divided into four categories: serine proteases, aspartic proteases, cysteine proteases and metalloproteases. Aspartic proteases are a class of proteolytic enzymes active at acidic pH. Acid protease is usually stable between pH 2.0 and 6.0, and the optimum pH value varies slightly with different species, but it is usually around pH 3.0. For example, the optimum pH of acid protease produced by Aspergillus niger is 3.0, Penicillium grisea is at pH 3.5, and yeast is also at pH 3.0. The active center of aspartic proteases contains two aspartic acid residues, the catalytic residues are located within the conserved region Asp-Thr/Ser-Gly (DT/SG) motif and form the enzyme active site.

Proteases are widely used in industries such as food, brewing, fur and leather, medicine and feed. The addition of acid protease in the feed can improve the digestibility of protein, degrade high-molecular protein into low-molecular peptides and amino acids, which is easy for livestock and poultry to digest and absorb, can reduce the stimulation of feed on the digestive tract of pups, reduce nutritional barriers, and improve Improve feed utilization and promote the growth of livestock and poultry.

At present, the properties of most acid proteases are unsatisfactory, and the enzyme activity is not high, which brings great waste to industrial production and food processing, and also limits its application range to a certain extent. Due to the difference between the optimal action conditions of the enzyme itself and the catalyzed environmental conditions (such as pH, temperature, etc.), the catalytic efficiency of the enzyme is reduced, and its industrial application is limited. Most of the acid proteases used in industrial production are fungal acid proteases. The optimum pH value of such enzymes is about 3.0. When the pH value increases, the enzyme activity of acid proteases will decrease significantly, and these enzymes are not heat-resistant. It is very unstable when the temperature reaches above 50°C, thus limiting the application of acid protease.

Contents of the invention

The object of the present invention is to provide an acid protease 6749.

Another object of the present invention is to provide a gene encoding the above protease 6749.

Another object of the present invention is to provide a recombinant vector comprising the above protease 6749 gene.

Another object of the present invention is to provide a recombinant strain comprising the above protease 6749 gene.

Another object of the present invention is to provide a method for preparing protease.

Another object of the present invention is to provide the application of the above-mentioned protease.

The first technical problem to be solved in the present invention is to overcome the deficiencies in the prior art and provide a new protease with excellent properties and suitable for use in industries such as food, feed, and medicine. Its amino acid sequence is as SEQ ID NO.1:

Among them, the full length of the enzyme is 394 amino acids, and the N-terminal 20 amino acids are the signal peptide sequence, namely "MVVFSKVTAVLAGLSAVASA".

Therefore, the theoretical molecular weight of mature protease 6749 is 40kDa, and its amino acid sequence is as SEQ ID NO.2:

The optimum pH of the protease is 3.0, and within the range of pH 2.5 to pH 3.5, the enzyme can maintain more than 70% of its enzyme activity; the optimum temperature is 55°C, and it still has more than 80% of its enzyme activity at 60°C.

The present invention also provides the gene encoding the above-mentioned protease. The present invention isolates and clones the protease gene 6749 by means of PCR. The full-length cDNA of the protease 6749 is 1185bp, and its cDNA sequence is shown in SEQ ID NO.4:

Wherein, the base sequence of the signal peptide is:

"ATGGTTGTTT TCAGCAAGGT CACGGCCGTC CTGGCCGGTC TCTCTGCCGT TGCGTCGGC"

Therefore, the coding sequence of the mature gene is

Shown in SEQ ID NO.5:

The theoretical molecular weight of the mature protein is 39.7kDa, and the enzyme belongs to aspartic acid protease. The amino acid sequence of protease gene 6749 was compared with GenBank by BLAST, and 6749 was determined to be a new protease.

The present invention also provides a recombinant vector comprising the above protease gene, preferably pPIC9-6749. The protease gene of the present invention is inserted between suitable restriction sites of the expression vector, so that its nucleotide sequence is operably linked with the expression control sequence. As a most preferred embodiment of the present invention, it is preferred that the protease gene is inserted between EcoR I and the Not I restriction enzyme site on the plasmid pPIC9, so that the nucleotide sequence is positioned at the downstream of the AOX1 promoter and is subject to It is regulated to obtain the recombinant yeast expression plasmid pPIC9-6749.

The present invention also provides a recombinant strain comprising the above protease gene, preferably the recombinant strain GS115/6749.

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

1) Transforming host cells with the above-mentioned recombinant vectors to obtain recombinant strains;

2) cultivating the recombinant strain to induce the expression of the recombinant protease; and

3) Recover and purify the expressed protease.

Wherein, preferably, the host cell is a Pichia pastoris cell, a Saccharomyces cerevisiae cell or a Hansenula polymorpha cell, and the recombinant yeast expression plasmid is preferably transformed into a Pichia pastoris cell (Pichia pastoris cell). ) GS115 to obtain the recombinant strain GS115/6749.

The present invention also provides the application of the above protease. The use of genetic engineering means to industrialize the production of protease.

The present invention has obtained a new acid protease gene from Thermoascus crustaceus JCM 12803 bacterial strain, and the protease encoded by it has the following advantages: higher activity under acidic conditions, higher reaction temperature, stable in a wider pH range . All these advantages mean that the newly invented protease can be used in industries such as feed, food, medicine, etc., and the technical scheme of the present invention can realize the production of protease with good properties and suitable for industrial application by means of genetic engineering.

Description of drawings

Fig. 1 shows the optimum pH value of recombinant protease according to a specific embodiment of the present invention.

Figure 2 shows the pH stability of recombinant proteases according to specific embodiments of the present invention.

Figure 3 shows the optimum reaction temperature of recombinant proteases according to specific embodiments of the present invention.

Figure 4 shows the thermal stability of recombinant proteases according to specific embodiments of the present invention.

Detailed ways

Test materials and reagents

1. Strains and vectors: Pichia pastoris GS115 was preserved in our laboratory; Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.

2. Enzymes and other biochemical reagents: endonucleases were purchased from TaKaRa Company, ligases were purchased from Invitrogen Company, and the others were domestic reagents (all of which can be purchased from common biochemical reagent companies).

3. Medium:

(I) Enzyme production medium: 30g/L wheat bran, 30g/L corncob powder, 30g/L soybean meal, 5g/L barley dextran, 5g/L (NH ₄ )SO ₄ , 1g/L KH ₂ PO ₄ , 0.5g/L MgSO ₄ 7H ₂ O, 0.01g/L FeSO ₄ 7H ₂ O, 0.2g/L CaCl ₂ in 1L deionized water, sterilized at 121℃, 15 pounds for 20min

(2) Escherichia coli medium LB (126 peptone, 0.5% yeast extract, 126NaCI, pH 7.0).

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

(4) BMMY medium: replace glycerin with 0.5% methanol, and the rest of the ingredients are the same as BMGY, pH 4.0.

Explanation: For the molecular biology experimental methods not specifically described in the following examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or follow the kit and product manual.

Cloning of embodiment 1 protease coding gene 6749

Thermoascus crustaceus genomic DNA was extracted and stored at -20°C for later use.

Synthetic amplification primers were designed, and the total DNA of Thermoascus crustaceus was used as template for PCR amplification. The PCR reaction parameters are: 95°C for 5min; 35 cycles of 94°C for 30sec, 60°C for 30sec, 72°C for 2min, and 72°C for 10min. A fragment of about 1800bp was obtained, which was recovered and sequenced.

Table 1 Primers required for this experiment

Extract the total RNA of Thermoascus crustaceus, utilize Oligo (dT) ₂₀ and reverse transcriptase to obtain a strand of cDNA, then design primers F and R (see Table 1) for amplifying the open reading frame, and amplify the single-stranded cDNA to obtain The cDNA sequence of the protease is amplified and sequenced after the product is recovered.

The full-length cDNA sequence of acid protease 6749 is 1185bp, encoding 394 amino acids and a stop codon, and 19 amino acids at the N-terminal are its signal peptide sequence. The gene encoding protease isolated and cloned from Thermoascus crustaceus is proved to be a new gene by comparison .

The construction of embodiment 2 protease engineering strains

(1) Construction of expression vector and expression in yeast

Using the correctly sequenced cDNA of protease 6749 as a template, primers F and R with EcoR I and Not I restriction sites were designed and synthesized (see Table 1) to amplify the coding region of the mature protein of 6749. And use EcoR I and NotI to digest the PCR product, connect it into the expression vector pPIC9 (Invitrogen, SanDiego), the sequence of the mature protein of protease 6749 is inserted into the downstream of the signal peptide sequence of the above expression vector, and form a correct reading frame with the signal peptide, construct The yeast expression vector pPIC9-6749 was transformed into Escherichia coli competent cell Trans1. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale preparation of recombinant plasmids. Linearize expression plasmid vector DNA with restriction endonuclease Bgl II, transform yeast GS115 competent cells by electroporation, culture at 30°C for 2-3 days, pick transformants grown on MD plates for further expression experiments, specific operations Please refer to the Pichia expression manual.

In the same way, an expression vector containing cDNA of the 6749 signal peptide sequence was constructed and transformed.

(2) Screening of transformants with high protease activity

Use a sterilized toothpick to pick a single colony from the MD plate with transformants, spot it on the MD plate according to the number, and place the MD plate in a 30°C incubator for 1 to 2 days until the colony grows. Pick the transformant from the MD plate according to the number and inoculate it in a centrifuge tube containing 3mL of BMGY medium, culture it on a shaker at 30°C and 220rpm for 48h; centrifuge the bacterial solution cultured on a shaker for 48h at 3,000×g for 15min, remove the supernatant, Add 1 mL of BMMY medium containing 0.5% methanol to the centrifuge tube, induce culture at 30°C and 220 rpm; after induction culture for 48 hours, centrifuge at 3,000×g for 5 minutes, take the supernatant for enzyme activity detection, and screen out those with high protease activity. For specific operations, please refer to the Pichia pastoris expression manual.

The preparation of embodiment 3 recombinant proteases

(1) Mass expression of protease gene 6749 at shake flask level in Pichia pastoris

The transformant with high enzyme activity was screened out, inoculated into a 1L Erlenmeyer flask with 300mL of BMGY liquid medium, cultured on a shaking table at 30°C at 220rpm for 48h; centrifuged at 5,000rpm for 5min, discarded the supernatant gently, and then added 100mL containing 0.5% methanol BMMY liquid medium, 30°C, 220rpm induction culture for 72h. During the induction culture period, add methanol solution once every 24 hours to compensate for the loss of methanol, and keep the methanol concentration at about 0.5%; (3) Centrifuge at 12,000×g for 10 minutes, collect the supernatant fermentation liquid, detect the enzyme activity and perform SDS-PAGE protein Electrophoretic analysis.

(2) Purification of recombinant protease

The recombinant protease supernatant expressed in the shake flask was collected, concentrated through a 10kDa membrane bag, and at the same time the culture medium was replaced with a low-salt buffer, and then further concentrated with a 10kDa ultrafiltration tube. Concentrate the recombinant 6749 which can be diluted to a certain fold, and purify by ion exchange chromatography. Specifically, take 2.0 mL of the 6749 concentrated solution and pass it through the HiTrap Q Sepharose XL anion column that has been equilibrated with 20 mM Tris-HCl (pH 7.5), and then use 0.1 mol/L NaCl for linear gradient elution. Remove liquid to detect enzyme activity and determine protein concentration.

Embodiment 4 recombinant protease partial property analysis

The activity analysis of the protease of the present invention is carried out by using Folin's phenol reagent chromogenic method. The specific method is as follows: at pH 3.0 and 55°C, 1 mL of reaction system includes 500 μL of appropriate diluted enzyme solution, 500 μL of substrate, react for 10 min, add 1 mL of trichloroacetic acid (0.4 mol/L) to terminate the reaction; The system was centrifuged at 12000 rpm for 3 minutes, 500 μL of supernatant was absorbed, 2.5 mL of sodium carbonate (0.4 mol/L) was added, and 500 μL of Folin’s phenol reagent was added, the color was developed at 40°C for 20 minutes and the OD value was measured at 680 nm after cooling. Definition of protease activity unit: Under certain conditions, the amount of enzyme required to decompose the substrate casein to generate 1 μmol of tyrosine per minute is 1 activity unit (U). (1) Optimum pH and pH stability of protease 6749

Purified (Example 3) expressed protease 6749 was subjected to enzymatic reactions at different pHs to determine its optimum pH. The buffers used are glycine-hydrochloric acid buffer with pH 1.0-3.0, citric acid-disodium hydrogen phosphate buffer with pH 3.0-8.0 and Tris-HCl buffer with pH 8.0-10.0. The pH suitability results of the purified protease 6749 measured in different pH buffer systems at 55°C (Figure 1) show that the optimum pH of 6749 is 3.0, and the enzyme can maintain 70% of its above enzyme activity.

The enzyme solution was treated at 30°C for 60 min in buffer solutions with different pH values, and then the enzyme activity was measured to study the pH stability of the enzyme. The results showed ( FIG. 2 ). The analysis results showed that more than 80% of the enzyme activity could be maintained between pH2.0-pH7.0, indicating that the enzyme had excellent pH stability.

(2) Protease 6749 reaction optimum temperature and thermal stability

Purified protease was tested for enzyme activity at different temperatures (30-70°C) under the condition of pH 3.0. The analysis results showed that the optimum reaction temperature of the enzyme was 55°C, and it still had 50% enzyme activity at 65°C. Vitality (Figure 3). The temperature resistance was measured by treating the protease at different temperatures for different times, and then measuring the enzyme activity at 60°C. The thermal stability experiment showed that the protease was poorly thermally stable, and 50% of the enzyme activity remained after being treated at 30° C. for 10 minutes ( FIG. 4 ).

<110> Institute of Feed, Chinese Academy of Agricultural Sciences

<120> A fungus-derived acid protease 6749 and its gene and application

<160>6

<210> 1

<211> 394

<212> PRT

<213> Thermoascus crustaceus JCM 12803

<400> 1

MVVFSKVTAV LAGLSAVASA VPTIKPRIGF SVQQVSKQVT PKTINLPAIY ANSLNKFGGT 60

VPQNVKAAAE TGSAITTPEA NDIAYLTPVN IGGSTLNLDI DTGSADLWVF STELPQQQSA 120

GHDIYKPSSN ATKLQGYTWS ISYGDGSSAS GDVYKDTVSV GNVVAHNQAV EAAKRISRQF 180

TQDQDNDGLL GLAFSSINTV KPKAQTTFFD TVKSQLDSPL FAVTLKHNAP GSYDFGYIDN 240

KKYTGKITYT DVDSSQGFWG FTASGYGVGD GEVNSNPIKG IADTGTSLLL VPNDIVEAYY 300

SQVQGAQNSA QLGGYVFNCN TQLPSFTVAI EGYKAVIPGD LIKYAPVTDG SPICFGGIQG 360

NEDLGFSIFG DIFLKSQYVV FSADGPKLGF APQA 394

<210> 2

<211> 374

<212> PRT

<213> Thermoascus crustaceus JCM 12803

<400> 2

VPTIKPRIGF SVQQVSKQVT PKTINLPAIY ANSLNKFGGT VPQNVKAAAE TGSAITTPEA 60

NDIAYLTPVN IGGSTLNLDI DTGSADLWVF STELPQQQSA GHDIYKPSSN ATKLQGYTWS 120

ISYGDGSSAS GDVYKDTVSV GNVVAHNQAV EAAKRISRQF TQDQDNDGLL GLAFSSINTV 180

KPKAQTTFFD TVKSQLDSPL FAVTLKHNAP GSYDFGYIDN KKYTGKITYT DVDSSQGFWG 240

FTASGYGVGD GEVNSNPIKG IADTGTSLLL VPNDIVEAYY SQVQGAQNSA QLGGYVFNCN 300

TQLPSFTVAI EGYKAVIPGD LIKYAPVTDG SPICFGGIQG NEDLGFSIFG DIFLKSQYVV 360

FSADGPKLGF APQA 374

<210> 3

<211> 20

<212> PRT

<213> Thermoascus crustaceus JCM 12803

<400> 3

MVVFSKVTAV LAGLSAVASA 20

<210> 4

<211> 1185

<212> DNA

<213> Thermoascus crustaceus JCM 12803

<400> 4

atggttgttt tcagcaaggt cacggccgtc ctggccggtc tctctgccgt tgcgtcggct 60

gttccccacca tcaagcctcg cattggtttc tctgtccagc aggtttccaa gcaggtcacc 120

ccgaagacta tcaacctccc agctatctac gccaacagtc tcaacaagtt tggaggcacg 180

gtgcctcaaa atgtgaaggc ggctgctgag acaggcagcg ctatcacaac cccagaggcc 240

aacgacattg cctacctcac tccagtgaac atcggcggtt ccaccctgaa cctcgatatt 300

gacaccggct ctgcggatct gtgggtgttc tcgaccgaac tgcctcagca acagagtgct 360

ggacatgata tctacaagcc gtcgtccaac gcgacaaagc tgcaaggata cacctggagc 420

atctcctacg gtgacggcag ctctgctagc ggcgacgtct acaaggacac cgtcagcgtt 480

ggcaatgtgg tagcccacaa ccaggcagtt gaggccgcca aaaggatcag ccgtcaattc 540

accccaggacc aggacaatga cggcctgctg ggcctggctt ttagctccat caacactgtc 600

aagcctaagg ctcagactac tttctttgac accgtcaagt cgcagcttga ctctccgctc 660

tttgcagtta ccttgaagca taacgcccct ggtagctacg actttggcta catcgacaac 720

aagaagtaca ccggcaagat cacctacacc gatgtcgact cttcccaggg cttctggggc 780

ttcaccgcca gcggctacgg cgttggagat ggagaggtca actccaaccc gatcaagggc 840

attgctgaca ccggtaccag cctgctcctc gtgcccaacg acatcgtcga agcctattac 900

agccaagtcc agggcgccca gaacagcgcg cagcttggag gatacgtttt caactgcaac 960

acccagctcc cgtccttcac tgtcgccatc gaaggctaca aagccgtcat tcccggtgac 1020

ctcatcaagt acgcccccgt cacggacggc agcccgatct gcttcggcgg catccagggc 1080

aacgaggacc tcggtttctc catcttcgga gacatcttcc tgaagagcca gtatgtcgtc 1140

ttcagcgctg acggccctaa gctgggtttc gccccgcagg cttag 1185

<210> 5

<211> 1125

<212> DNA

<213> Thermoascus crustaceus JCM 12803

<400> 5

gttccccacca tcaagcctcg cattggtttc tctgtccagc aggtttccaa gcaggtcacc 60

ccgaagacta tcaacctccc agctatctac gccaacagtc tcaacaagtt tggaggcacg 120

gtgcctcaaa atgtgaaggc ggctgctgag acaggcagcg ctatcacaac cccagaggcc 180

aacgacattg cctacctcac tccagtgaac atcggcggtt ccaccctgaa cctcgatatt 240

gacaccggct ctgcggatct gtgggtgttc tcgaccgaac tgcctcagca acagagtgct 300

ggacatgata tctacaagcc gtcgtccaac gcgacaaagc tgcaaggata cacctggagc 360

atctcctacg gtgacggcag ctctgctagc ggcgacgtct acaaggacac cgtcagcgtt 420

ggcaatgtgg tagcccacaa ccaggcagtt gaggccgcca aaaggatcag ccgtcaattc 480

accccaggacc aggacaatga cggcctgctg ggcctggctt ttagctccat caacactgtc 540

aagcctaagg ctcagactac tttctttgac accgtcaagt cgcagcttga ctctccgctc 600

tttgcagtta ccttgaagca taacgcccct ggtagctacg actttggcta catcgacaac 660

aagaagtaca ccggcaagat cacctacacc gatgtcgact cttcccaggg cttctggggc 720

ttcaccgcca gcggctacgg cgttggagat ggagaggtca actccaaccc gatcaagggc 780

attgctgaca ccggtaccag cctgctcctc gtgcccaacg acatcgtcga agcctattac 840

agccaagtcc agggcgccca gaacagcgcg cagcttggag gatacgtttt caactgcaac 900

acccagctcc cgtccttcac tgtcgccatc gaaggctaca aagccgtcat tcccggtgac 960

ctcatcaagt acgcccccgt cacggacggc agcccgatct gcttcggcgg catccagggc 1020

aacgaggacc tcggtttctc catcttcgga gacatcttcc tgaagagcca gtatgtcgtc 1080

ttcagcgctg acggccctaa gctgggtttc gccccgcagg cttag 1125

<210> 6

<211> 60

<212> DNA

<213> Thermoascus crustaceus JCM 12803

<400> 4

atggttgttt tcagcaaggt cacggccgtc ctggccggtc tctctgccgt tgcgtcggct 60

Claims (9)

1. An acid protease, characterized in that its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2. 2. An acid protease gene, characterized in that, the acid protease according to claim 1 is encoded. 3. The acid protease gene according to claim 2, characterized in that its nucleotide sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5. 4. comprise the recombinant expression vector of acid protease gene described in claim 2. 5. comprise the recombinant expression vector pPIC9-6749 of acid protease gene described in claim 2, wherein, the acid protease gene of nucleotide sequence as shown in SEQ ID NO.5 is inserted into the restriction enzyme cutting site on the plasmid pPIC9 Between, make this nucleotide sequence be positioned at the downstream of AOX1 promoter and be regulated by it, obtain recombinant expression vector pPIC9-6749. 6. comprising the recombinant strain of acid protease gene described in claim 2. 7. The recombinant strain GS115/6749 comprising the acid protease gene according to claim 2, wherein the recombinant expression vector pPIC9-6749 according to claim 5 is transformed into Pichia pastoris cell GS115 to obtain the recombinant strain GS115/6749. 8. a method for preparing acid protease according to claim 1, is characterized in that, comprises the following steps: (1) transforming host cells with the recombinant expression vector according to claim 4; (2) cultivating host cells; (3) Separation and purification to obtain acid protease. 9. the acid protease described in claim 1 is used for the application of hydrolyzing protein.