CN110343688A - Alkali protease PA3 and its encoding gene and application - Google Patents

Abstract

The invention belongs to agricultural biological technical fields, and in particular to alkali protease PA3 and its encoding gene and application.The present invention provides the alkali protease PA3 in the source Torrubiella hemipterigena, its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2, it can be applied to the multiple fields such as food, feed, process hides, washing, medicine with good property.Alkali protease PA3 excellent using genetic engineering means nature of production, being suitble to industrial application may be implemented according to the technique and scheme of the present invention.

Description

Alkali protease PA3 and its encoding gene and application

Technical field

The invention belongs to agricultural biological technical fields, and in particular to alkali protease PA3 and its encoding gene and application.

Background technique

Protease refers to the class of enzymes that protein hydrolysis is realized by cutting peptide bonds, and source is very extensive, is largely distributed in Animal, plant, in microorganism.Compared to the protease of plant and animal material, microbial protein enzyme, which has, cultivates convenient, operation letter Single and yield of enzyme height etc. is convenient for the characteristics of industrialized batch production, and therefore, microbial protein enzyme becomes current albumen The important sources of enzyme.Protease plays vital effect in cellular process, and a large amount of physiological activities rely on albumen Enzyme executes, this exoproteinase also has industrial application characteristic, as one of most important industrial enzyme preparation, is widely used in each A field, the market demand is big, and economic value is high.

Using optimal pH as standard, protease is divided into acid protease, alkali protease and neutral proteinase.Its neutral and alkali egg White enzyme accounting in protease preparation total sales volume is maximum.The optimal pH of alkali protease is distributed between pH7.5-11.0, And keep stablizing usually in meta-alkali environment, a small number of alkali proteases also have certain tolerance in acid condition.Alkaline egg The molecular weight of albumen of white enzyme is usually in 18-34kDa.Industrial alkali protease is mainly derived from Bacillus, such as withered grass gemma Bacillus, bacillus amyloliquefaciens etc..The alkali protease having now been found that is essentially serine protease, since activated centre is deposited In a serine, when there are inhibitor such as phenylmethylsulfonyl fluoride (PMSF), diisopropyl phosphoryl fluorides (DFP), active quilt Inhibit.The fermentoid is usually to the peptide bond preferential interaction for being located at aromatic amino acid c-terminus.Common alkali protease has pancreas egg White enzyme, hay bacillus albuminoid enzyme etc..

Directly using wild strain fermentation production of alkaline protease, that there are producing enzyme levels is low, property is unstable, isolates and purifies The disadvantages of trouble.With the development of technique for gene engineering, many good expression systems are established the height to realize target protein Effect expression.Successfully building protein expression system has escherichia expression system, yeast expression system, bacillus subtilis at present Expression system, insect expression system and mammalian expression systems etc..Wherein pichia yeast expression system expresses limitation with it It is small, yield is high, controllability is strong, product less investment, the features such as product is easily separated and purification effect is good is as the expression by favor System has the function of perfect posttranslational modification especially for the destination protein of originated from fungus.Utilize Pichia anomala expression The alkali protease of originated from fungus is conducive to its industrialization large-scale production, preferably plays application value convenient for it.Using finish When red Yeast expression foreign gene, codon optimization is carried out according to Pichia pastoris Preference to gene, although currently, the prior art Codon preference in Pichia pastoris is calculated according to different methods and proposes preferred codons and rare codon, but It is the result of the preferred codons of partial amino-acid to be had differences, therefore in selection, the specific of foreign gene need to be combined Situation and host cell species, are made a concrete analysis of.It is close except rare codon is substituted for preference in codon optimisation process Outside numeral, notes also the adjustment big AT content of gene local, avoid making premature transcription termination；And also need to improve translation eventually Only efficiency, meanwhile, removal is unfavorable for mRNA stable sequence and secondary structure, to improve whole translation efficiency, promotes expression. When therefore, using Pichia anomala expression foreign gene, expected results can not be obtained by only carrying out preferred codons replacement, need to integrate Consider the factors such as gene, host cell.

Summary of the invention

The purpose of the present invention is to provide a kind of alkali protease PA3.

A further object of the present invention is to provide the encoding genes of above-mentioned alkali protease PA3.

A further object of the present invention is to provide the recombinant expression carriers containing said gene.

A further object of the present invention is to provide contain said gene recombinant bacterial strain.

A further object of the present invention is to provide the preparation methods of above-mentioned alkali protease PA3.

A further object of the present invention is to provide the applications of above-mentioned alkali protease PA3.

The alkali protease PA3 of specific embodiment according to the present invention, amino acid sequence is as shown in SEQ ID NO.1:

Wherein, 387 amino acid of the enzyme overall length, wherein 1-15 amino acids constitute signal peptide, i.e. " MRLAKFL ALLPLAAG ", 16-106 amino acids constitute leader peptide, i.e. " APSLARREEPAPLLEARGAQAIPGKFIVKLREGSPLAA LQQAMSLLGGKADHVFQNVFSGFAASMNPAVIELMRNHPDVEYIEQDGKVNIN”。

The alkali protease PA3 of specific embodiment according to the present invention, the amino acid sequence of maturation protein such as SEQ ID Shown in NO.2:

Wherein, 281 amino acid of enzyme maturation protein overall length, theoretical molecular weight 28.2kDa, isoelectric point 8.74.

The alkaline protease gene pa3 of specific embodiment according to the present invention encodes above-mentioned alkali protease PA3.

The alkaline protease gene pa3 of specific embodiment according to the present invention, above-mentioned alkali protease is in addition to signal peptide The corresponding nucleic acid sequence of 372 amino acid as shown in SEQ ID NO.3:

The alkaline protease gene pa3 of specific embodiment according to the present invention, above-mentioned alkali protease include that signal peptide exists The interior corresponding nucleic acid sequence of 387 amino acid is as shown in SEQ ID NO.4:

The alkali protease PA3 of specific embodiment according to the present invention, corresponding to encoding gene it is excellent without secret son Nucleic acid sequence in the case of change is as shown in SEQ ID NO.5:

The recombinant expression carrier of the alkaline protease gene of specific embodiment, preferably pPIC9r- according to the present invention pa3.Alkaline protease gene of the invention is inserted between suitable restriction enzyme cleavage sites of the expression vector, its nucleotide is made Sequence is operable to be linked to the expression control sequence.As the most preferred embodiment of the invention, preferably by egg White enzyme gene is inserted between EcoR I and Not the I restriction enzyme site on plasmid pPIC9r, makes the nucleotide sequence position In AOXl promoter downstream and regulated and controled by it, obtain expression of recombinant yeast plasmid pPIC9r-pa3.

The recombinant bacterial strain of the alkaline protease gene of specific embodiment according to the present invention, preferably recombinant bacterial strain GS115/ PA3.Wherein, the preferably described host cell is Pichia pastoris (Pichia pastoris) cell, preferably by expression of recombinant yeast matter Grain conversion Pichia pastoris (Pichic pastoris) GS115, obtains recombinant bacterial strain GS115/PA3.

The method for preparing alkali protease PA3 of specific embodiment according to the present invention, the described method comprises the following steps:

(1) host cell is converted with the recombinant expression carrier comprising alkaline protease gene pa3, obtains recombinant bacterial strain；

(2) recombinant bacterial strain, inducing expression alkali protease PA3 are cultivated；

(3) it separates and purifies alkali protease PA3.

The present invention also provides the applications of above-mentioned protease, carry out industrialization production protease with genetic engineering means.

Beneficial effects of the present invention:

The present invention provides the alkali protease PA3 and its base in the source fungi (Torrubiella hemipterigena) Cause can be applied to the multiple fields such as food, feed, process hides, washing, medicine.It can realize according to the technique and scheme of the present invention Utilize the alkali protease of the excellent suitable industrial application of genetic engineering means nature of production.

Detailed description of the invention

Fig. 1 shows the optimum pH of recombinant protease PA3 of the present invention；

Fig. 2 shows the pH stability of recombinant protease PA3 of the present invention；

Fig. 3 shows recombinant protease PA3 optimal reactive temperature of the present invention；

Fig. 4 shows recombinant protease PA3 thermal stability of the present invention.

Specific embodiment

Test material and reagent

1, bacterial strain and carrier: expressive host Pichia pastoris GS115, expression plasmid carrier pPIC9r.

2, biochemical reagents: restriction enzyme EcoR I, Not I, Bgl II, Fastpfu archaeal dna polymerase, Assembly Kit kit, casein.

3, culture medium:

LB culture medium: 0.5% yeast extract, 1% peptone, 1%NaCl, pH 7.0

YPD culture medium: 1% yeast extract, 2% peptone, 2% glucose

MD solid medium: 2% glucose, 1.5% agarose, 1.34%YNB, 0.00004%Biotin

Milk bilayer screening and culturing medium:

Upper layer: 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 0.5% methanol (V/ V), 1.5% agarose；

Lower layer: 4% milk powder, 1.5% agarose.

BMGY culture medium: 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34%YNB, 0.00004% Biotin。

BMMY culture medium: 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 0.5% first Alcohol (V/V).

Illustrate: not doing the experimental methods of molecular biology illustrated in detail in the present embodiment, referring to " molecular cloning is real Test guide " specific method listed in book of (third edition) J. Pehanorm Brooker one carries out, or according to kit and the description of product Book carries out.

Embodiment 1 obtains alkaline protease gene pa3 and construction of expression vector

According to the protein sequence (Serial No.: CEJ93023.1) announced in Genbank, codon optimization is carried out to it, Chemical synthesis optimized after gene, number pa3.

Gene pa3 is connected on pUC57 carrier, the present invention by the method for homologous recombination realize target gene pa3 with The building of expression vector is completed in the connection of expression vector pPIC9r.

Firstly, introducing homology segment into target gene by specific primer by the method for PCR.

1 pa3 specific primer of table

Note: underscore is labeled as the overlap homologous with carrier pPIC9r

Using recombinant plasmid pUC57-pa3 as template, pcr amplification reaction is carried out by specific primer；Meanwhile utilizing limit Property restriction endonuclease EcoRI, NotI processed carries out digestion to expression vector pPIC9, and subsequent 1% agarose gel electrophoresis simultaneously recycles PCR Product and digestion products.Expression vector pPIC9r after the recovery and the target fragment for carrying homology segment pass through Assembly Kit Kit is attached.

Connect 15min under the conditions of 50 DEG C, be placed in the cooled on ice several seconds after reaction, after can recombinant products be directly used in Convert or be stored in -20 DEG C.

Connection product is transformed into E.coli Trans-T1 competence, and transformed bacteria solution is coated on LB plate (containing 100 μ g/mL Ampicillin) upper 37 DEG C of incubators are incubated overnight, and picked clones colony polymerase chain reaction (PCR) method is identified and is sequenced, and sequencing is correct Recombinant plasmid is named as pPIC9r-pa3.

The building of 2 alkali protease PA3 engineered strain of embodiment

It is a large amount of to extract recombination pPIC9-pa3 plasmid, linearization process, line are carried out to it using restriction enzyme BglII Property system.

After 37 DEG C of digestion 2h, takes 10 μ L digestion products to carry out agarose gel electrophoresis, be confirmed whether that digestion is complete, it is rear to utilize Omega Gel Extraction kit kit is recycled and is purified to digestion products.The linearisation product electricity of purification and recovery is turned Enter in Pichia pastoris competence (electric converter parameter: Fungus, PIC).Bacterium solution after electricity turns is coated on MD plate, 30 DEG C of trainings Support 48h.After growing transformant on MD plate, with the toothpick after sterilizing from 36 monoclonals of picking on MD plate in milk double deck screen It selects on plate, cultivates 72h under the conditions of 30 DEG C.According to whether occurring hydrolysis circle around transformant and the successive and size that goes too far, judge Whether transformant expresses proteinase activity, and measures its activity height, is screened out from it the transformant of high protein enzymatic activity.

3 preparation and reorganization alkali protease PA3 of embodiment

(1) shaking flask horizontal expression recombinant basic protease P A3

The higher transformant of the enzyme activity that will be singled out is inoculated in 30mL YPD fluid nutrient medium, and in 30 DEG C, 200rpm is shaken 48h is cultivated in bed, obtains seed liquor.Then seed liquor is transferred in 300mL BMGY culture medium according to 1% inoculum concentration, in 30 DEG C, 48h is cultivated in 200rpm shaking table, thallus is transferred in 200mL BMMY culture medium later, in 30 DEG C, 200rpm shaking table Middle culture 48h, during which every for 24 hours, adding 0.5% (V/V) methanol.

(2) purifying of recombinant basic protease P A3

After fermentation process, bacterium solution 12000rpm is centrifuged 10min, removes thallus and other insoluble matters, collects supernatant, It both is crude enzyme liquid.Crude enzyme liquid is concentrated with the film packet (Vivascience, Hannover, Germany) of 5kDa size, and Desalting processing is carried out to crude enzyme liquid by dialysis (molecular cut off: 3kDa), is replaced buffer A (pH6.5,10mM phosphorus Sour disodium hydrogen-citrate buffer solution) in.Enzyme solution after desalination passes through ion exchange column (cation exchange column HiTrap SP HP It 5mL) is purified, (adds 1M NaCl on the basis of buffer A using buffer B, and adjust pH and guarantee itself and buffer A is identical) gradient (0-1.0M NaCl) elution is carried out, the component with enzyme activity eluted is collected, is utilized SDS-PAGE verifies the purity and molecular size range of its albumen, and carries out liquid chromatogram-electro-spray ionization-mass spectrometry (liquid chromatography-electrospray ionization-tandem mass spectrometry, LC- ESI-MS it) identifies.

The 4 basic characterization analysis of recombinant basic protease P A3 of embodiment

Activity analysis is carried out to alkali protease of the invention using forint phenol reagent development process.The specific method is as follows: 500 μ L dilution enzyme solution appropriate and 500 μ L substrates (1%W/V casein), react 20min under the conditions of 9.5,60 DEG C of pH, rear to be added 1mL trichloroacetic acid (0.4mol/L) terminates reaction.After reaction system 12000rpm after termination is centrifuged 3min, supernatant is taken to carry out Colour developing.Coloring reaction system is as follows: 2.5mL 0.4mol/L sodium carbonate liquor, 500 μ L supernatant, 500 μ L forint phenol reagent, and 40 It is reacted 20 minutes at DEG C, the light absorption value of its wavelength 680nm is read after it is cooled to room temperature.All reactions are all provided with that there are three parallel And blank control.

The enzyme activity (U) of protease is defined as: under prescribed conditions, enzyme amount needed for discharging 1 μ g tyrosine per minute is one A enzyme-activity unit.

It is pure that enzyme solution used in zymologic property research reaches electrophoresis.Buffer system used in the present invention is as follows: lactic acid-sodium lactate is slow It rushes system (50mmol/L, pH3.0-5.0), phosphate buffer (200mmol/L, pH5.0-8.0), borax-sodium hydroxide Buffer system (100mmol/L, pH8.0-12.0).

(1) optimal pH of recombinant basic protease P A3 and pH stability

Enzyme solution after purification is diluted into suitable multiple using the buffer of pH5.0-12.0, under optimum temperature, and is in The substrate reactions 20min of corresponding pH value measures its activity, to determine the optimal pH of recombinase.

As a result as shown in Figure 1, the optimal pH of PA3 after purification be 9.5, pH8.0-10.0 keep 80% or more it is opposite Enzyme activity.After reaction condition is more than pH10.0, the enzyme activity of PA3 is decreased obviously, and under the conditions of pH11.0, opposite enzyme activity is about Under the conditions of 30%, pH12, opposite enzyme activity is less than 10%.From the point of view of overall trend, PA3 belongs to alkali protease, but in slant acidity Under conditions of, PA3 still keeps certain activity, and when pH6.0, PA3 can play about 50% activity.

By enzyme solution after purification at 37 DEG C, 1h is pre-processed under the conditions of pH3.0-12.0, enzyme solution carries out suitably to treated Dilution measures remaining enzyme activity, under optimum temperature and pH to determine the pH stability of recombinase.

As a result as shown in Fig. 2, PA3 is also showed preferably in terms of pH stability, after handling 1h under the conditions of pH4.0-11.0, The remaining enzyme activity of PA3 can keep 90% or more, this shows that PA3 can keep stable pH range more wide in range.

(2) optimum temperature and thermal stability of recombinant basic protease P A3

The corresponding buffer system of enzyme solution optimal pH after purification is diluted into suitable multiple, is measured at 20 DEG C -80 DEG C not Enzyme activity under synthermal, to determine recombinase optimum temperature.

As a result as shown in figure 3, the optimum temperature of PA3 is between 60 DEG C, -65 DEG C of temperature 50 C, opposite enzyme activity is maintained at 60% or more, it with respect to enzyme activity is about 30% at 40 DEG C there are also 55% or more opposite enzyme activity at 50 DEG C.65 DEG C to 70 DEG C areas Section, enzyme activity decline is obvious, and at 70 DEG C, opposite enzyme activity is about 14%.

Enzyme solution after purification is diluted to 50 μ g/mL with the corresponding buffer system of optimal pH, respectively in 50 DEG C, 55 DEG C, 60 It is heat-treated at DEG C, the processing time is respectively 2min, 5min, 10min, 20min, 30min and 60min, treated sample It is placed on ice to terminate the heat loss of enzyme, dilutes suitable multiple after cooling, measure remaining enzyme activity, under optimum reaction conditions with true Its fixed thermal stability, the activity surveyed with untreated enzyme solution is 100%.

As a result as shown in figure 4, the thermal stability of PA3 is good in the performance of middle low-temperature zone, 1h is handled almost under the conditions of 50 DEG C There is no enzyme activity loss；But when treatment temperature reaches 55 DEG C, enzyme activity declines, after handling 1h, 50% or more activity of loss；60 DEG C when processing 20min after be nearly no detectable activity.

Sequence table

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

<120>alkali protease PA3 and its encoding gene and application

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<170> SIPOSequenceListing 1.0

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<211> 387

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<213>fungi (Torrubiella hemipterigena)

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Met Arg Leu Ala Lys Phe Leu Ala Leu Leu Pro Leu Ala Ala Gly Ala

1 5 10 15

Pro Ser Leu Ala Arg Arg Glu Glu Pro Ala Pro Leu Leu Glu Ala Arg

20 25 30

Gly Ala Gln Ala Ile Pro Gly Lys Phe Ile Val Lys Leu Arg Glu Gly

35 40 45

Ser Pro Leu Ala Ala Leu Gln Gln Ala Met Ser Leu Leu Gly Gly Lys

50 55 60

Ala Asp His Val Phe Gln Asn Val Phe Ser Gly Phe Ala Ala Ser Met

65 70 75 80

Asn Pro Ala Val Ile Glu Leu Met Arg Asn His Pro Asp Val Glu Tyr

85 90 95

Ile Glu Gln Asp Gly Lys Val Asn Ile Asn Ala Tyr Thr Thr Gln Thr

100 105 110

Gly Ala Pro Trp Gly Leu Gly Arg Ile Ser His Arg Ala Lys Gly Ser

115 120 125

Thr Ser Tyr Thr Tyr Asp Thr Ser Ala Gly Glu Gly Thr Cys Val Tyr

130 135 140

Val Ile Asp Thr Gly Val Glu Asp Thr His Pro Glu Phe Glu Gly Arg

145 150 155 160

Ala Lys Leu Ile Lys Thr Tyr Tyr Gly Asn Arg Asp Gly His Gly His

165 170 175

Gly Thr His Cys Ser Gly Thr Ile Gly Ser Lys Thr Tyr Gly Val Ala

180 185 190

Lys Lys Thr Lys Ile Tyr Gly Val Lys Val Leu Asp Asp Asn Gly Ser

195 200 205

Gly Thr Phe Ser Asn Ile Ile Ala Gly Val Asp Phe Val Ala Asn Asp

210 215 220

Tyr Lys Thr Arg Gly Cys Pro Lys Gly Ala Val Ala Ser Met Ser Leu

225 230 235 240

Gly Gly Gly Lys Thr Gln Ala Val Asn Asp Ala Val Ala Arg Leu Gln

245 250 255

Arg Ala Gly Val Phe Val Ala Val Ala Ala Gly Asn Asp Asn Thr Asp

260 265 270

Ala Ala Asn Thr Ser Pro Ala Ser Glu Pro Ser Val Cys Thr Val Gly

275 280 285

Ala Ser Asp Lys Asp Asp Val Arg Ser Thr Phe Ser Asn Tyr Gly Ser

290 295 300

Val Val Asp Ile Phe Ala Pro Gly Thr Ala Ile Leu Ser Thr Trp Ile

305 310 315 320

Gly Gly Arg Thr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His

325 330 335

Ile Ala Gly Leu Ala Ala Tyr Leu Met Gly Lys Asp Gly Ala Val Ala

340 345 350

Ala Gly Leu Cys Ala Lys Ile Ala Gln Thr Ala Thr Arg Asn Val Leu

355 360 365

Arg Asn Ile Pro Ala Gly Thr Ile Asn Ala Leu Ala Phe Asn Gly Asn

370 375 380

Pro Ser Gly

385

<210> 2

<211> 281

<212> PRT

<213>fungi (Torrubiella hemipterigena)

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Ala Tyr Thr Thr Gln Thr Gly Ala Pro Trp Gly Leu Gly Arg Ile Ser

1 5 10 15

His Arg Ala Lys Gly Ser Thr Ser Tyr Thr Tyr Asp Thr Ser Ala Gly

20 25 30

Glu Gly Thr Cys Val Tyr Val Ile Asp Thr Gly Val Glu Asp Thr His

35 40 45

Pro Glu Phe Glu Gly Arg Ala Lys Leu Ile Lys Thr Tyr Tyr Gly Asn

50 55 60

Arg Asp Gly His Gly His Gly Thr His Cys Ser Gly Thr Ile Gly Ser

65 70 75 80

Lys Thr Tyr Gly Val Ala Lys Lys Thr Lys Ile Tyr Gly Val Lys Val

85 90 95

Leu Asp Asp Asn Gly Ser Gly Thr Phe Ser Asn Ile Ile Ala Gly Val

100 105 110

Asp Phe Val Ala Asn Asp Tyr Lys Thr Arg Gly Cys Pro Lys Gly Ala

115 120 125

Val Ala Ser Met Ser Leu Gly Gly Gly Lys Thr Gln Ala Val Asn Asp

130 135 140

Ala Val Ala Arg Leu Gln Arg Ala Gly Val Phe Val Ala Val Ala Ala

145 150 155 160

Gly Asn Asp Asn Thr Asp Ala Ala Asn Thr Ser Pro Ala Ser Glu Pro

165 170 175

Ser Val Cys Thr Val Gly Ala Ser Asp Lys Asp Asp Val Arg Ser Thr

180 185 190

Phe Ser Asn Tyr Gly Ser Val Val Asp Ile Phe Ala Pro Gly Thr Ala

195 200 205

Ile Leu Ser Thr Trp Ile Gly Gly Arg Thr Asn Thr Ile Ser Gly Thr

210 215 220

Ser Met Ala Thr Pro His Ile Ala Gly Leu Ala Ala Tyr Leu Met Gly

225 230 235 240

Lys Asp Gly Ala Val Ala Ala Gly Leu Cys Ala Lys Ile Ala Gln Thr

245 250 255

Ala Thr Arg Asn Val Leu Arg Asn Ile Pro Ala Gly Thr Ile Asn Ala

260 265 270

Leu Ala Phe Asn Gly Asn Pro Ser Gly

275 280

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<212> DNA

<213>fungi (Torrubiella hemipterigena)

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gctatcccag gtaagttcat cgtcaagttg agagagggtt ctccattggc tgcattgcaa 120

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tacattgagc aggacggtaa ggttaacatc aacgcctaca ctactcagac tggtgctcca 300

tggggtttgg gtagaatttc tcatagagct aagggttcca cctcctacac ttacgatact 360

tccgctggtg agggtacttg tgtttacgtt atcgacactg gtgtcgagga cactcaccca 420

gaatttgagg gtagagccaa gctgatcaag acctactacg gtaacagaga tggtcacggt 480

catggtactc actgttccgg tactattggt tccaagactt acggtgtcgc caaaaagacc 540

aaaatctacg gtgtcaaggt cctggacgat aacggttctg gtactttctc caacattatc 600

gccggtgttg acttcgttgc caacgactac aagactagag gttgtccaaa gggtgctgtt 660

gcctctatgt ctcttggtgg tggaaagact caagctgtta acgacgctgt tgctagattg 720

caacgtgccg gtgtttttgt tgctgttgct gctggtaacg acaacactga tgctgctaat 780

acttctccag cttctgagcc atccgtctgt actgttggtg cttctgataa ggacgacgtc 840

agatccacct tctctaacta cggttccgtt gttgacatct tcgctccagg tactgctatc 900

ttgtccactt ggattggtgg taggactaac accatctccg gtacttctat ggctactcca 960

cacattgctg gtttggctgc ttacctgatg ggtaaagatg gtgcagttgc tgcaggtttg 1020

tgtgctaaga ttgctcagac tgccaccaga aacgtcctga gaaatattcc agctggtact 1080

atcaacgccc tggcctttaa cggtaatcca tctggttaa 1119

<210> 4

<211> 1164

<212> DNA

<213>fungi (Torrubiella hemipterigena)

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atgcgtctgg ctaagttcct tgctctactc cctcttgccg ctggcgctcc atccttggct 60

agaagagaag aaccagctcc tttgttggaa gctagaggtg ctcaagctat cccaggtaag 120

ttcatcgtca agttgagaga gggttctcca ttggctgcat tgcaacaagc tatgtccttg 180

cttggtggta aggctgacca cgttttccag aacgttttct ctggtttcgc cgcctctatg 240

aacccagctg ttattgagtt gatgagaaac catccagacg tcgagtacat tgagcaggac 300

ggtaaggtta acatcaacgc ctacactact cagactggtg ctccatgggg tttgggtaga 360

atttctcata gagctaaggg ttccacctcc tacacttacg atacttccgc tggtgagggt 420

acttgtgttt acgttatcga cactggtgtc gaggacactc acccagaatt tgagggtaga 480

gccaagctga tcaagaccta ctacggtaac agagatggtc acggtcatgg tactcactgt 540

tccggtacta ttggttccaa gacttacggt gtcgccaaaa agaccaaaat ctacggtgtc 600

aaggtcctgg acgataacgg ttctggtact ttctccaaca ttatcgccgg tgttgacttc 660

gttgccaacg actacaagac tagaggttgt ccaaagggtg ctgttgcctc tatgtctctt 720

ggtggtggaa agactcaagc tgttaacgac gctgttgcta gattgcaacg tgccggtgtt 780

tttgttgctg ttgctgctgg taacgacaac actgatgctg ctaatacttc tccagcttct 840

gagccatccg tctgtactgt tggtgcttct gataaggacg acgtcagatc caccttctct 900

aactacggtt ccgttgttga catcttcgct ccaggtactg ctatcttgtc cacttggatt 960

ggtggtagga ctaacaccat ctccggtact tctatggcta ctccacacat tgctggtttg 1020

gctgcttacc tgatgggtaa agatggtgca gttgctgcag gtttgtgtgc taagattgct 1080

cagactgcca ccagaaacgt cctgagaaat attccagctg gtactatcaa cgccctggcc 1140

tttaacggta atccatctgg ttaa 1164

<210> 5

<211> 1164

<212> DNA

<213>fungi (Torrubiella hemipterigena)

<400> 5

atgcgtctgg ctaagttcct tgctctactc cctcttgccg ctggcgctcc tagcctcgct 60

agacgagagg agcctgctcc ccttttggag gctcgcggtg ctcaggcaat tcctggaaaa 120

ttcattgtca agctgcgaga aggcagtcca cttgctgctc tacagcaggc catgtcgctc 180

ttgggcggca aagcagacca cgtcttccag aacgtctttt ctggtttcgc tgcatccatg 240

aatccagccg tcattgagtt gatgcgcaac catcctgatg tcgagtatat cgaacaggat 300

ggcaaggtga acatcaacgc ttacaccacg cagaccggag ctccatgggg acttggtcga 360

atttctcaca gggcaaaggg tagcacttcc tacacctatg acacttcagc tggcgaggga 420

acctgtgtct atgtcatcga tactggtgtg gaggacaccc atcctgaatt tgaaggccgt 480

gccaagctca tcaagaccta ttacggcaac agggatggcc atggccatgg cactcactgc 540

tctggaacca ttggctccaa gacctatggc gttgccaaaa agaccaagat ttacggtgtc 600

aaggtgttgg acgataatgg atcgggaacc ttctccaaca tcattgctgg cgtggacttt 660

gttgccaacg actacaagac ccgcggctgc cccaagggtg ctgtagcatc catgtctctt 720

ggtggtggta agacacaggc agtcaacgat gctgttgcac gtctccagcg agctggtgtt 780

tttgttgctg tcgccgctgg aaacgacaac accgatgccg ccaacacgtc tccagcctcg 840

gagccatctg tctgcaccgt tggtgctagc gacaaggacg atgtgcgatc taccttttcc 900

aactatggat ctgttgtaga catcttcgcg ccaggcactg ctatcctgtc tacttggatc 960

ggcggccgca ctaacaccat ctccggtaca tccatggcca ctccccatat tgctggtctg 1020

gctgcctatc tcatgggcaa ggacggagct gttgccgctg gtttgtgtgc caagattgcg 1080

caaactgcca ctagaaatgt tctccgcaac atccccgcag gcaccatcaa tgcccttgca 1140

ttcaatggta accctagcgg gtaa 1164

Claims (10)

1. alkali protease PA3, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.

2. alkaline protease gene pa3, which is characterized in that encode alkali protease PA3 described in claim 1.

3. alkaline protease gene pa3 according to claim 2, which is characterized in that its nucleotide sequence such as SEQ ID Shown in NO.3 or SEQ ID NO.4.

4. the recombinant expression carrier comprising alkaline protease gene described in claim 2.

5. the recombinant expression carrier pPIC9r-pa3 comprising alkaline protease gene described in claim 2.

6. the recombinant bacterial strain comprising alkaline protease gene described in claim 2.

7. the recombinant bacterial strain GS115/PA3 comprising alkaline protease gene described in claim 2.

8. the method for preparing alkali protease PA3, which is characterized in that the described method comprises the following steps:

(1) host cell is converted with the recombinant expression carrier comprising alkaline protease gene pa3 as claimed in claim 2, obtained Recombinant bacterial strain；

(2) recombinant bacterial strain, inducing expression alkali protease PA3 are cultivated；

(3) it separates and purifies alkali protease PA3.

9. the method according to claim 8 for preparing alkali protease PA3, which is characterized in that the host cell is complete Red yeast cells.

10. the application of alkali protease PA3 described in claim 1.