CN107384900A

CN107384900A - The acid protease 6749 and its gene of a kind of originated from fungus and application

Info

Publication number: CN107384900A
Application number: CN201710645685.9A
Authority: CN
Inventors: 姚斌; 罗会颖; 郭玉杰; 涂涛; 王苑; 黄火清; 柏映国; 苏小运; 王亚茹; 孟昆
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Animal Science of CAAS
Priority date: 2017-08-01
Filing date: 2017-08-01
Publication date: 2017-11-24
Anticipated expiration: 2037-08-01
Also published as: CN107384900B

Abstract

The acid protease 6749 and its gene of a kind of originated from fungus and application.The present invention relates to genetic engineering field.In particular it relates to a kind of from the acid protease 6749 and its gene of fungi and application, its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.The acid protease of the present invention has good property, can make to be applied to the industry such as food, feed, medicine.Technique according to the invention scheme can realizes the protease using the excellent suitable commercial Application of genetic engineering means nature of production.

Description

The acid protease 6749 and its gene of a kind of originated from fungus and application

Technical field

The present invention relates to genetic engineering field.In particular it relates to a kind of acid protease from fungi 6749 and its gene and application.

Background technology

Protease is the class of enzymes of catalytic proteins hydrolysis, is all widely used in the industry such as food, washing, process hides.Phase Than in the protease of plant and animal material, microbial protein enzyme, having and cultivating the characteristics of convenient, simple to operate and yield of enzyme is high, It is easy to industrialized mass production, is able to large-scale production and application.Therefore, microbial protein enzyme becomes current protease Important sources.

Protease is divided into acid protease, alkali protease and neutral proteinase according to its pH acted on.By activity Protease can be divided into four classes by center：Serine protease, aspartic protease, cysteine proteinase and metalloprotein Enzyme.Aspartic protease is a kind of proteolytic enzyme active at acidic.Acid protease generally pH 2.0~ It is stable between 6.0, Optimun pH is slightly different with kind difference, but generally all in pH 3.0 or so, such as aspergillus niger The most suitable p H of produced acid protease are 3.0, and Penicillum glaucum is pH 3.5, and saccharomycete is also in pH 3.0.Aspartic protease Activated centre include two asparagicacid residues, the catalytic residue is located at conserved region Asp-Thr/Ser-Gly (DT/SG) motif It is interior and form enzyme active sites.

Protease in food, brewage, be widely used in the industry such as fur and leather, medicine and feed.It is sour in feed The addition of property protease can improve the digestibility of protein, and it is low molecular peptide and amino to make high molecular protein degradation Acid, it is easy to livestock and poultry and digests and assimilates, feed can be reduced to the gastral stimulation of young baby, reduce dystrophia, improves efficiency of feed utilization, Promote growth of animals or poultry.

The property of current most of acid proteases is not fully up to expectations, and enzyme activity is not high, to industrialized production and food processing Great waste is brought, also limits its application to a certain extent.By enzyme itself optimal condition is with being catalyzed Environmental condition between difference (such as pH, temperature), cause the catalytic efficiency of enzyme to reduce, be restricted its commercial Application. Acid protease for industrialized production is mostly mould acid protease, and the Optimun pH of this fermentoid is 3.0 or so, When the ph is increased, the enzyme activity of acid protease can be reduced substantially, and this fermentoid is thermo-labile, when temperature reaches more than 50 DEG C very It is unstable, so as to limit the application of acid protease.

The content of the invention

It is an object of the invention to provide a kind of acid protease 6749.

Another object of the present invention is to provide the gene for encoding above-mentioned protease 6749.

Another object of the present invention is to provide the recombinant vector for including the above-mentioned gene of protease 6749.

Another object of the present invention is to provide the recombinant bacterial strain for including the above-mentioned gene of protease 6749.

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

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

Present invention technical problem to be solved first is overcome the deficiencies in the prior art, there is provided a kind of good properties, It is suitable for the new protease applied in the industries such as food, feed, medicine, its amino acid sequence such as SEQ ID NO.1：

Wherein, 394 amino acid of the enzyme total length, 20 amino acid of N-terminal are signal peptide sequence, i.e. " MVVFSKVTAV LAGLSAVASA”。

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

The optimal pH of the protease be 3.0, in the range of 2.5~pH of pH 3.5, the enzyme be able to maintain that its more than 70% Enzyme activity；55 DEG C of optimum temperature, still there is more than 80% enzyme activity at 60 DEG C.

Present invention also offers the gene for encoding above-mentioned protease.The present invention has cloned this by PCR method separation Protease gene 6749, the cDNA total lengths of protease 6749 are 1185bp, and its cDNA sequence is as shown in SEQ IDNO.4：

Wherein, the base sequence of signal peptide is：

“ATGGTTGTTT TCAGCAAGGT CACGGCCGTC CTGGCCGGTC TCTCTGCCGT TGCGTCGGC”

Therefore, the coded sequence of ripe gene is

Shown in SEQ ID NO.5：

Maturation protein theoretical molecular is 39.7kDa, and the enzyme belongs to aspartic protease.By protease gene 6749 Amino acid sequence carries out BLAST in GenBank and compares discovery, and it is a kind of new protease to determine 6749.

Present invention also offers the recombinant vector for including above-mentioned protease gene, preferably pPIC9-6749.By the present invention Protease gene be inserted between the suitable restriction enzyme site of expression vector, make its nucleotide sequence exercisable and table It is connected up to regulating and controlling sequence.As the most preferred embodiment of the present invention, protease gene is preferably inserted into matter Between EcoR I and Not I restriction enzyme sites on grain pPIC9, the nucleotide sequence is set to be located at the downstream of AOXl promoters And regulated and controled by it, obtain expression of recombinant yeast plasmid pPIC9-6749.

Present invention also offers the recombinant bacterial strain for including above-mentioned protease gene, preferably recombinant bacterial strain GS115/6749.

Present invention also offers a kind of method for preparing protease, comprise the following steps：

1) host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；

2) recombinant bacterial strain is cultivated, induces the expression of recombinant protease；And

3) reclaim and purify expressed protease.

Wherein, preferably described host cell is Pichia pastoris (Pichia pastoris) cell, brewer's yeast (Saccharomyces cerevisiae) cell or Hansenula polymorpha (Hansenula polymorpha) cell, preferably will Expression of recombinant yeast plasmid converts Pichia pastoris (Pichic pastoris) GS115, obtains recombinant bacterial strain GS115/ 6749。

Present invention also offers the application of above-mentioned protease.Carry out industrialization production protease with genetic engineering means.

The present invention has obtained a new acidic protein from the bacterial strains of Thermoascus crustaceus JCM 12803 Enzyme gene, its protease encoded have following advantage：Have in acid condition greater activity, higher reaction temperature, It is stable in the range of wider pH.All these advantages can mean that neoteric protease in industries such as feed, food, medicine In, technique according to the invention scheme can realizes the egg using the excellent suitable commercial Application of genetic engineering means nature of production White enzyme.

Brief description of the drawings

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

Fig. 2 shows the pH stability according to the recombinant protease of the specific embodiment of the invention.

Fig. 3 shows the optimal reactive temperature of the recombinant protease according to the specific embodiment of the invention.

Fig. 4 shows the recombinant protein enzyme heat stability according to the specific embodiment of the invention.

Embodiment

Test material and reagent

1st, bacterial strain and carrier：Pichia pastoris (Pichia pastoris GS115) preserves for this laboratory；Pichia pastoris table Invitrogen companies are purchased from up to carrier pPIC9 and bacterial strain GS115.

2nd, enzyme and other biochemical reagents：Restriction endonuclease is purchased from TaKaRa companies, and ligase is purchased from Invitrogen companies, its It is all domestic reagent (can be commercially available from common biochemical Reagent Company).

3rd, culture medium：

(I) culture medium：30g/L wheat bran, 30g/L maize cob meals, 30g/L dregs of beans, 5g/L barleys, 5g/L (NH₄)SO₄, 1g/L KH₂PO₄, 0.5g/L MgSO₄·7H₂O, 0.01g/L FeSO₄·7H₂O, 0.2g/L CaCl₂In 1L go from In sub- water, sterilization treatment 20min under the conditions of 121 DEG C, 15 pounds

(2) Escherichia coli culture medium LB (126 peptones, 0.5% yeast extract, 126NaCI, pH7.O).

(3) BMGY culture mediums；1% yeast extract, 2% peptone, 1.34%YNB, 0.000049<Biotin, 1% is sweet Oily (v/v).

(4) BMMY culture mediums：Divided by 0.5% methanol replace glycerine, remaining composition is identical with BMGY, pH4.0.

Explanation：Do not make the experimental methods of molecular biology illustrated, equal reference in following examples《Molecular Cloning: A Laboratory Guide》Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or according to kit and product description Carry out.

The clone of the proteinase encoding genes 6749 of embodiment 1

Thermoascus crustaceus extracting genome DNAs, be placed in -20 DEG C it is standby.

Design synthesis amplimer, enter performing PCR amplification by template of Thermoascus crustaceus STb genes.PCR is anti- The parameter is answered to be：95℃5min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 2min, 35 circulations, 72 DEG C of 10min.Obtain one about 1800bp fragments, it is sequenced after the fragment is reclaimed.

Primer needed for 1 experiment of table

Thermoascus crustaceus total serum IgEs are extracted, are utilized Oligo (dT)₂₀The one of cDNA is obtained with reverse transcriptase The primers F and R (being shown in Table 1) of bar chain, then design amplification ORFs, expand the single-stranded cDNA, obtain protease CDNA sequence, amplification are sequenced after obtaining product recovery.

The cDNA sequence total length 1185bp of acid protease 6749, encode 394 amino acid and a terminator codon, N It is its signal peptide sequence to hold 19 amino acid, proves that separation clones what is obtained from Thermoascus crustaceus through comparing The gene of encoding proteins enzyme is new gene.

The structure of the protease engineered strain of embodiment 2

(1) structure of expression vector and the expression in yeast

So that the cDNA of correct protease 6749 is sequenced as template, design has synthesized restricted with EcoR I and Not I The primers F and R (being shown in Table 1) of restriction enzyme site, are expanded to the code area of 6749 maturation protein.And utilize EcoR I and Not I digestion PCR primers, connection enter expression vector pPIC9 (Invitrogen, SanDiego), the maturation protein of protease 6749 Sequence is inserted into the downstream of the signal peptide sequence of above-mentioned expression vector, forms correct reading frame with signal peptide, is built into ferment Female expression vector pPIC9-6749, conversion competent escherichia coli cell Trans1.Positive transformant carries out DNA sequencing, sequencing Show that the correct transformant of sequence is used for a large amount of Prepare restructuring plasmids.Linearisation expression matter is carried out with restriction enzyme Bgl II Grain carrier DNA, electroporated yeast GS115 competent cells, 30 DEG C are cultivated 2-3 days, the conversion that picking grows on MD flat boards Son carries out further expression experiment, and concrete operations refer to Pichia anomala expression operation manual.

CDNA of the structure containing 6749 signal peptide sequences expression vector in the same way, and convert.

(2) screening of high protein enzymatic activity transformant

With sterilized toothpick from picking single bacterium colony on the MD plates with transformant, first put according to numbering on MD flat boards, MD flat boards are placed in 30 DEG C of incubators and cultivated 1~2 day, are grown to bacterium colony.It is inoculated with by number from picking transformant on MD flat boards In the centrifuge tube equipped with 3mL BMGY culture mediums, 30 DEG C, 220rpm shaking table cultures 48h；By shaking table culture 48h bacterium solution 3, 000 × g centrifuge 15min, remove supernatant, the BMMY culture mediums that 1mL contains 0.5% methanol added in centrifuge tube, 30 DEG C, 220rpm Fiber differentiations；After Fiber differentiation 48h, 3,000 × g centrifugation 5min, take supernatant to be used for Enzyme assay, therefrom filter out The transformant of high protein enzymatic activity, concrete operations refer to Pichia anomala expression operation manual.

The preparation of the recombinant protease of embodiment 3

(1) the horizontal great expression of shaking flask in Pichia pastoris of protease gene 6749

The higher transformant of enzyme activity is filtered out, is inoculated in the 1L triangular flasks of 300mL BMGY fluid nutrient mediums, 30 DEG C, 220rpm shaking table shaken cultivations 48h；5,000rpm centrifugation 5min, supernatant is softly abandoned, then add 100mL to thalline and contain 0.5% The BMMY fluid nutrient mediums of methanol, 30 DEG C, 220rpm Fiber differentiations 72h.During Fiber differentiation, interval 24h adds a methanol Solution makes methanol concentration be maintained at 0.5% or so to compensate the loss of methanol；(3) 12,000 × g centrifuge 10min, collect supernatant Zymotic fluid, detect enzymatic activity and carry out SDS-PAGE protein electrophoresis analyses.

(2) purifying of recombinant protease

The recombinant protease supernatant of shaking flask expression is collected, is concentrated by 10kDa film bags, while use low salt buffer Culture medium therein is replaced, is then further concentrated with 10kDa super filter tubes.Concentration can be diluted to the restructuring of certain multiple 6749, purified by ion-exchange chromatography.Specifically, 6749 concentrate 2.0mL are taken through using 20mM Tris-HCl in advance (pH 7.5) equilibrated HiTrap Q Sepharose XL anion columns, then carry out linear ladder with 0.1mol/L NaCl Degree elution, enzymatic activity is detected to the eluent of Fraction collection and carries out the measure of protein concentration.

The recombinant protease some properties of embodiment 4 are analyzed

Activity analysis is carried out to the protease of the present invention using forint phenol reagent development process.Specific method is as follows： PH3.0, under the conditions of 55 DEG C, 1mL reaction system includes the appropriate dilution enzyme liquids of 500 μ L, 500 μ L substrates, reacts 10min, adds Enter 1mL trichloroacetic acids (0.4mol/L) terminating reaction；Reaction system 12000rpm is centrifuged into 3min, 500 μ L of supernatant liquid is inhaled and adds Enter 2.5mL sodium carbonate (0.4mol/L), add 500 μ L forint phenol reagents, 680nm measure OD after 40 DEG C of colour developing 20min coolings Value.Proteinase activity unit definition：Under certain condition, decomposition substrate casein per minute is generated needed for l μm of ol tyrosine Enzyme amount is 1 active unit (U).(1) optimal pH of protease 6749 and pH stability

It is most suitable to determine it that the protease 6749 of purified (embodiment 3) expression carries out enzymatic reaction under different pH pH.Buffer solution used is that pH 1.0~3.0 is glycine-HCI buffer solution, the citrate-phosphate disodium hydrogen system of pH3.0~8.0 8.0~l0.0 of row buffer solution and pH Tris-HCl series of buffer.Buffer body of the protease 6749 of purifying in different pH The pH adaptive result (Fig. 1) for be, being determined at 55 DEG C shows：6749 optimal pH is 3.0, should in the range of pH 2.5-pH 3.5 Enzyme is able to maintain that its more than 70% enzyme activity.

Enzyme liquid is handled into 60min in the buffer solution of different pH value at 30 DEG C, then determines enzymatic activity with the pH of studying enzyme Stability.As a result show (Fig. 2), analysis result shows to be able to maintain that more than 80% enzyme activity, explanation between pH2.0-pH7.0 The enzyme has excellent pH stability.

(2) protease 6749 reacts optimum temperature and heat endurance

The protease of purifying determines the enzymatic activity under different temperatures (30-70 DEG C), analysis experiment knot under the conditions of pH 3.0 Fruit shows to show, the optimal reactive temperature of the enzyme is 55 DEG C, still has 50% enzyme activity (Fig. 3) at 65 DEG C.Temperature tolerance It is determined as protease and handles different time at different temperatures, then enzyme assay is carried out at 60 DEG C.Heat endurance tests table It is bright：It is poor under the protease thermostabilization that 10min, remaining 50% enzyme activity (Fig. 4) are handled at 30 DEG C.

<110>Institute of Feeds,China Academy of Agriculture Sciences

<120>The acid protease 6749 and its gene of a kind of originated from fungus 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

gttcccacca 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

acccaggacc 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

gttcccacca 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

acccaggacc 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

1. a kind of acid protease, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.

A kind of 2. acid protease gene, it is characterised in that the acid protease described in coding claim 1.

3. acid protease gene according to claim 2, it is characterised in that its nucleotide sequence such as SEQ ID NO.3, Shown in SEQ ID NO.4 or SEQ ID NO.5.

4. include the recombinant expression carrier of acid protease gene described in claim 2.

5. include the recombinant expression carrier pPIC9-6749 of acid protease gene described in claim 2.

6. include the recombinant bacterial strain of acid protease gene described in claim 2.

7. include the recombinant bacterial strain GS115/6749 of acid protease gene described in claim 2.

A kind of 8. method for preparing acid protease described in claim 1, it is characterised in that comprise the following steps：

(1) host cell is converted with the recombinant expression carrier described in claim 4；

(2) host cell is cultivated；

(3) acquisition acid protease is isolated and purified.

9. the application of acid protease described in claim 1.