CN109810967A - The acid protease Bs2688 mutant Y282L and its gene and application that thermal stability improves - Google Patents

Abstract

The invention belongs to agricultural biological technical fields, and in particular to the acid protease Bs2688 mutant and its gene of a kind of originated from fungus and application.The amino acid sequence of protease of the present invention is as shown in SEQ ID NO.3.The present invention provides a new protease genes, using the excellent protease of genetic engineering means nature of production, and are applied to the industry such as feed, food, medicine.

Description

Thermal stability improve acid protease Bs2688 mutant Y282L and its gene and Using

Technical field

The invention belongs to agricultural biological technical fields, and in particular to a kind of acid egg that the thermal stability of originated from fungus improves White enzyme Bs2688 mutant and its gene and application.

Background technique

Protease is the class of enzymes of catalytic proteins hydrolysis, is widely present in plant, animal and microorganism.Phase Than in the protease of plant and animal material, microbial protein enzyme has the characteristics that culture is convenient, easy to operate and yield of enzyme is high, Large-scale production and application are able to convenient for industrialized batch production.Therefore, microbial protein enzyme becomes current protease Important sources.

The mode classification of protease has very much, according to the pH of albumen enzyme effect, is divided into acid protease, basic protein Enzyme and neutral proteinase.Acid protease be usually between pH 2.0~6.0 it is stable, optimum pH with kind difference slightly Difference, but usually all in pH 3.0 or so, such as optimal pH for acid protease that aspergillus niger produces is 3.0, Penicillum glaucum pH 3.5, saccharomycete is also in pH 3.0.The fermentoid possesses higher sequence similarity, and three-dimensional structure is in symmetrical bifolium.By work The difference at property center, protease are divided into: serine protease, aspartic protease, cysteine proteinase and metal egg White enzyme.

Protease is widely used in the industries such as food, brewing, fur and leather, medicine and feed.Dairy industry In, using renin to the high specificity of casein, protease can participate in the manufacturing process of cheese.The fermentation of brewed spirit In the process, synergistic effect is played using acid protease, dissolves the particle of fermentation raw material, improve the utilization rate of raw material, promoted micro- Biological growth, decomposing protein provide raw fragrant precursor substance and flavor substance, decompose yeast mycoprotein.In fur, leather In manufacturing process, acid protease removes interfibrillar substance, keeps cortex more soft, plentiful.In feedstuff industry, acid protease Addition the digestibility of protein can be improved, make the low molecular peptide of high molecular protein degradation and amino acid, be easy to raise Fowl digests and assimilates, and can reduce feed to the gastral stimulation of young baby, reduce dystrophia, improve efficiency of feed utilization, promote livestock and poultry Growth.

Acid protease currently used for industrialized production is mostly mould acid protease, the most suitable action pH of this fermentoid Value is 3.0 or so, and when the ph is increased, the enzyme activity of acid protease can be substantially reduced, and this fermentoid is thermo-labile, when temperature reaches It is very unstable at 50 DEG C or more.Therefore, the enzyme activity of acid protease is not high, enzyme itself optimal condition and the environment being catalyzed Between condition pH, in terms of difference, cause the catalytic efficiency of enzyme to reduce, to limit answering for acid protease With.

Summary of the invention

Enzyme activity in order to solve the problems, such as existing acid protease is not high, catalytic efficiency is low, and the present invention provides one kind The acid protease Bs2688 mutant that the thermal stability of originated from fungus improves, with acidproof, high temperature resistant, is readily produced fermentation The characteristics of.

The object of the present invention is to provide the acid protease Bs2688 mutant that a kind of thermostabilization improves.

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

Another object of the present invention is to provide the recombinant vector comprising above-mentioned proteinase encoding genes.

Another object of the present invention is to provide the recombinant bacterial strain comprising above-mentioned proteinase encoding genes.

Another object of the present invention is to provide a kind of method for preparing Cathepsin B s2688 mutant.

Another object of the present invention is to provide the application of above-mentioned Cathepsin B s2688 mutant.

Specific embodiment according to the present invention, the amino acid sequence of acid protease Bs2688 such as SEQ ID No.1 institute Show:

Wherein, 407 amino acid of the enzyme overall length, 19 amino acid of N-terminal are signal peptide sequence, i.e., "MHSFVTAAALVASASLTLA".Therefore, the theoretical molecular weight of proproteinase Bs2688 is 40.3kDa, and amino acid sequence is such as Shown in SEQ ID No.2:

For the thermal stability for further increasing acid protease Bs2688, mutation transformation has been carried out to it, has obtained mutant Y282L.After handling 5min at 75 DEG C, the enzyme activity residue 60% of wild type, and the remaining enzyme activity of mutant Y282L is 82%, compared with Wild type has increased significantly.

Specific embodiment according to the present invention, the ammonia for the acid protease Bs2688 mutant Y282L that heat resistance improves Base acid sequence is as shown in SEQ ID No.3:

The present invention also provides the gene of the mutant Y282L of coding SEQ ID No.3 amino acid sequence, nucleotides sequences Column are as shown in SEQ ID No.4:

The present invention also provides the recombinant vectors comprising above-mentioned protease gene, preferably pPIC9-Bs2688.This is sent out Bright protease gene is inserted between suitable restriction enzyme cleavage sites of the expression vector, make its nucleotide sequence it is operable with Expression regulation sequence is connected.As the most preferred embodiment of the invention, preferably protease gene is inserted into Between SnaB I and Avr II restriction enzyme site on plasmid pPIC9, the nucleotide sequence is made to be located at AOXl promoter Downstream is simultaneously regulated and controled by it, obtains expression of recombinant yeast plasmid pPIC9-Bs2688.

The present invention also provides the recombinant bacterial strains comprising above-mentioned protease gene, preferably recombinant bacterial strain GS115/ Bs2688。

The present invention also provides a kind of methods for preparing protease mutant, comprising the following steps:

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

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

3) it recycles and purifies expressed protease.

Wherein, the 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/ Bs2688。

The present invention also provides above-mentioned amino acid sequence albumen as shown in SEQ ID No.3 to hydrolyze junket as protease again Application in terms of albumen.

The present invention provides the mutant Y282L using the excellent protease of genetic engineering means nature of production, at 75 DEG C After managing 5min, the enzyme activity residue 60% of wild type, and the remaining enzyme activity of mutant Y282L of the present invention is 82%, is had compared with wild type It is greatly improved.Protease of the invention can be applied to the industry such as feed, food, medicine.

Detailed description of the invention

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

Fig. 2 shows the pH steadiness of protease mutant of the present invention；

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

Fig. 4 shows the enzyme activity situation of protease mutant of the present invention and wild type after 75 DEG C of processing 5min.

Specific embodiment

Test material and reagent

1, bacterial strain and carrier: Pichia pastoris (Pichia pastoris GS115) and yeast expression vector pPIC9.

2, enzyme and other biochemical reagents: restriction endonuclease, ligase.

3, culture medium:

(1) culture medium: 30g/L wheat bran, 30g/L maize cob meal, 30g/L dregs of beans, 5g/L barley, 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, 126NaCL, pH7.0).

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

(4) BMMY culture medium: glycerol is replaced divided by 0.5% methanol, remaining composition is identical as BMGY, pH4.0.

Embodiment 1 prepares Cathepsin B s2688 mutant

1. cloned proteins enzyme coding gene Bs2688

By Liquid Culture 3 days fungi Bispora sp.MEY-1,12,000rpm were centrifuged 10min, and the mycelium of collection adds Enter in the mortar of high-temperature sterilization, be ground to powder rapidly with liquid nitrogen, ground thallus is then transferred to a new, dress Having in 15ml CTAB lysate 50mL centrifuge tube, soft turned upside down mixes, 65 DEG C of water-bath heat preservation 3h are placed in, every 20min, turned upside down softly mix once, sufficiently to crack thallus.4 DEG C, 12,000rpm centrifugation 10min, draw supernatant extremely In new centrifuge tube, isometric chloroform is added, is placed at room temperature for 5min.4 DEG C, 12,000rpm centrifugation 10min.Take supernatant Isometric phenol/chloroform is added, 5min is placed at room temperature for.4 DEG C, 12,000rpm centrifugation 10min.To remove impurity elimination as far as possible Albumen, then take supernatant that isometric isopropanol is added, after being stored at room temperature 5min, l0000rpm is centrifuged l0min at 4 DEG C.Supernatant is abandoned, Precipitating with 70% ethanol washing twice, be dried in vacuo, appropriate dd H is added₂O dissolution, be placed in -20 DEG C it is spare.

Cloning primer Bs2688F and Bs2688R are designed, carries out PCR by template of Bispora sp.MEY-1 genomic DNA Amplification, obtains an about 1800bp segment.

Primer needed for 1 PCR amplification of table

2. constructing mutant

The total serum IgE for extracting Bispora sp.MEY-1, utilizes Oligo (dT)₂₀A chain of cDNA is obtained with reverse transcriptase, Then the primer Bs2688F and Bs2688R of design amplification open reading frame, particular sequence is as shown in table 1, and it is single-stranded to expand this CDNA, obtains the cDNA sequence of protease, and amplification is sequenced after obtaining product recycling.

It is found after being compared by genome sequence to protease and cDNA sequence in the gene containing 2 intrones, CDNA long 1224bp encodes 407 amino acid, and 19 amino acid of N-terminal are its signal peptide sequence, is compared from Bispora The gene Bs2688 for the coding protease that separation clone obtains in sp.MEY-1 is new gene.

Wild plasmid to build introduces mutating alkali yl, mutational formats Y282L, by above-mentioned gained as template Recombinant plasmid sends to sequencing, verifies the correctness of sequence, and mutant is named as Bs2688-Y282L.

3. constructing protease engineered strain

(1) construction of expression vector

Using the cDNA that correct Cathepsin B s2688 is sequenced as template, designs and synthesized with SnaB I and Avr II limitation Property restriction enzyme site primers F and R the code area of the maturation protein of Bs2688 is expanded as shown in table 1, and utilize SnaB I and Avr II digestion PCR product, connection enter expression vector pPIC9, and the sequence of Cathepsin B s2688 maturation protein is inserted into The downstream for stating the signal peptide sequence of expression vector forms correct reading frame with signal peptide, is built into Yeast expression carrier PPIC9-Bs2688 converts competent escherichia coli cell Trans1.Positive transformant carries out DNA sequencing.Use restriction enzyme Enzyme Bgl II carries out linearisation expression plasmid carrier DNA, and electroporated yeast GS115 competent cell, 30 DEG C are cultivated 2-3 days, The transformant that picking is grown on MD plate carries out further expression experiment.

For correct mutant preparation and reorganization plasmid is sequenced, linearisation expression matter is carried out with restriction enzyme Bgl II Grain carrier DNA, electroporated yeast GS115 competent cell, 30 DEG C are cultivated 2-3 days, the conversion that picking is grown on MD plate Son carries out further expression experiment.

In the same way to the code area structure of the code area of the intact proteins containing signal peptide of Bs2688 and mutant Y282L Build recombinant expression carrier.

(2) screening of high protein enzymatic activity transformant

MD plate is placed in 30 DEG C of trainings according on Kind of Coded Points Used to MD plate by picking single colonie on the MD plate with transformant It supports and is cultivated 1~2 day in case, until bacterium colony is grown.It is inoculated in from picking transformant on MD plate and is cultivated equipped with 3mL BMGY by number In the centrifuge tube of base, 30 DEG C, 220rpm shaking table culture 48h；By the bacterium solution 3 of shaking table culture 48h, 000 × g is centrifuged 15min, goes Clearly, the BMMY culture medium that 1mL contains 0.5% methanol is added in centrifuge tube, in 30 DEG C, 220rpm Fiber differentiation；Fiber differentiation After 48h, 3,000 × g is centrifuged 5min, takes supernatant for Enzyme assay, is screened out from it the transformant of high protein enzymatic activity.

4. preparation and reorganization protease

(1) great expression of protease gene Bs2688 mutant shaking flask level in Pichia pastoris

The higher transformant of enzyme activity is filtered out, is inoculated in the 1L triangular flask of 300mL BMGY fluid nutrient medium, 30 DEG C, 220rpm shaking table shaken cultivation 48h；5,000rpm centrifugation 5min softly abandon supernatant, then 100mL are added to thallus and contains 0.5% The BMMY fluid nutrient medium of methanol, 30 DEG C, 220rpm Fiber differentiation 72h.During Fiber differentiation, a methanol is added at interval for 24 hours Solution makes methanol concentration be maintained at 0.5% or so to compensate the loss of methanol；(3) 12,000 × g are centrifuged 10min, collect supernatant Fermentation liquid detects enzymatic activity and carries out SDS-PAGE protein electrophoresis analysis.

(2) purifying of recombinant protease

The recombinant protease supernatant for collecting shaking flask expression, is concentrated, while using low salt buffer by 10kDa film packet Culture medium therein is replaced, is then further concentrated with 10kDa super filter tube.Concentration can be diluted to the recombinant protein of certain multiple Enzyme Bs2688 mutant, is purified by ion-exchange chromatography, dense to the eluent detection enzymatic activity and progress albumen of collection The measurement of degree.

The zymetology performance of the verifying recombinant protease of embodiment 2.

Activity analysis is carried out to protease of the invention using forint phenol reagent development process.The specific method is as follows: PH3.0, under the conditions of 55 DEG C, the reaction system of 1mL includes 500 μ L dilution enzyme solution appropriate, and 500 μ L substrates react 10min, add Enter 1mL trichloroacetic acid (0.4mol/L) and terminates reaction；Reaction system 12000rpm is centrifuged 3min, 500 μ L supernatants is inhaled and adds Enter 2.5mL sodium carbonate (0.4mol/L), adds 500 μ L forint phenol reagents, 680nm measures OD after 40 DEG C of colour developing 20min are cooling Value.Proteinase activity unit definition: under certain condition, substrate casein is decomposed per minute and is generated needed for l μm of ol tyrosine Enzyme amount is 1 active unit (U).

1. the optimal pH and pH stability of Cathepsin B s2688 mutant

It is anti-that the Cathepsin B s2688 and mutant Bs2688-Y282L of purified expression carry out enzymatic at different pH It should be to measure its optimal pH.Buffer used is 1.0~3.0 glycine-HCI buffer of pH, the citric acid of pH3.0~8.0 One disodium hydrogen phosphate series of buffer and 8.0~l0.0 of pH are Tris-HCl series of buffer.

As shown in Figure 1, the optimal pH of the mutant Bs2688-Y282L measured at 75 DEG C is 3.0, in pH2.5-pH In 3.5 ranges, which is able to maintain that its 70% or more enzyme activity.

Enzyme solution is handled into 60min in the buffer of different pH value at 37 DEG C, then measures enzymatic activity with the pH of studying enzyme Stability.As shown in Fig. 2, the experimental results showed that, mutant Bs2688-Y282L is able to maintain that between pH 1.0-pH 6.0 80% or more enzyme activity.

Under similarity condition, the optimal pH of Cathepsin B s2688 is 3.0, and in 3.5 range of pH 2.5-pH, which can Maintain its 70% or more enzyme activity.The enzyme solution of Cathepsin B s2688 is handled at 37 DEG C in the buffer of different pH value 60min, Cathepsin B s2688 are able to maintain that 80% or more enzyme activity between pH 1.0-pH 7.0.

2. Cathepsin B s2688 reacts optimum temperature and thermal stability

Under the conditions of 3.0 pH, enzymatic activity of the protease at 30-90 DEG C is measured.

As shown in figure 3, the optimal reactive temperature of protease mutant Bs2688-Y282L of the invention is 75 DEG C, 80 DEG C when still have 60% or more enzyme activity.Meanwhile the optimal reactive temperature of wild-type protease Bs2688 of the invention is 75 DEG C, at 80 DEG C with 60% or more enzyme activity.

In order to measure the thermal stability of wild type and mutant, Cathepsin B s2688 and mutant Bs2688-Y282L are existed 5min is handled at 75 DEG C, then measures the remaining enzyme activity of protease.As shown in Figure 4, the results showed that, Cathepsin B s2688 is in 75 DEG C of items The enzyme activity of the residue 53% of 5min is handled under part；Mutant Bs2688-Y282L treated remaining 69% enzyme activity.Cause This, for mutant Bs2688-Y282L compared with wild type Bs2688, the remaining enzyme activity after 5min is handled under the conditions of 75 DEG C is bright It is aobvious to improve.

Sequence table

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

<120>the acid protease Bs2688 mutant Y282L and its gene and application that thermal stability improves

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 407

<212> PRT

<213>thermophilic fungal MEY-1 (Bispora sp. MEY-1)

<400> 1

Met His Ser Phe Val Thr Ala Ala Ala Leu Val Ala Ser Ala Ser Leu

1 5 10 15

Thr Leu Ala Ala Pro Ala Gln Ile Val Gly Arg Ser Thr Phe Gln Ile

20 25 30

Asp Gln Val Ala Ser Gly Lys Val Tyr Lys Asn Gly Pro Met Ala Met

35 40 45

Met Gln Thr Tyr Asn Lys Tyr Ala His Val Gly Ala Val Ala Pro Ala

50 55 60

Ala Val Val Ala Ala Ala Ala Ala Ala Gln Thr Gly Glu Val Ser Ala

65 70 75 80

Asn Pro Glu Gln Tyr Asp Glu Ser Tyr Leu Cys Pro Val Thr Ile Gly

85 90 95

Asp Gln Thr Leu Asn Leu Asp Phe Asp Thr Gly Ser Ala Asp Leu Trp

100 105 110

Val Phe Ser Thr Leu Thr Pro Ser Ser Glu Ser Thr Gly His Thr Leu

115 120 125

Tyr Asn Pro Ala Asp Ser Gly Thr Glu Lys Gln Gly Tyr Thr Trp Asn

130 135 140

Ile Thr Tyr Gly Asp Gly Ser Gly Ala Ala Gly Val Val Tyr Ala Asp

145 150 155 160

Lys Val Val Val Gly Gly Val Thr Ala Thr Ser Gln Ala Val Glu Ala

165 170 175

Ala Thr Ser Val Ser Ser Glu Phe Thr Gln Asp Thr Lys Asn Asp Gly

180 185 190

Leu Leu Gly Leu Ala Phe Ser Ser Ile Asn Thr Val Gln Pro Val Gln

195 200 205

Gln Thr Thr Phe Phe Asp Thr Val Lys Asp Thr Leu Ala Lys Lys Leu

210 215 220

Phe Thr Ala Asp Leu Lys Lys Gly Ala Ala Gly Ser Tyr Gly Phe Gly

225 230 235 240

Tyr Ile Asp Ser Ser Lys Tyr Thr Gly Thr Ile Thr Tyr Val Pro Val

245 250 255

Asn Asn Glu Asn Gly Phe Trp Gln Phe Thr Ala Gly Gly Tyr Ser Ile

260 265 270

Gly Gly Gly Asn Gly Thr Ser Gly Ser Asn Ala Thr Thr Gly Ser Ile

275 280 285

Gly Thr Ser Ile Ala Asp Thr Gly Thr Thr Leu Leu Tyr Leu Pro Ser

290 295 300

Asn Val Val Thr Ala Tyr Tyr Lys Gln Val Ser Gly Ala Ser Tyr Asn

305 310 315 320

Ser Ala Gln Gly Gly Tyr Thr Tyr Pro Cys Gly Ala Thr Leu Pro Asp

325 330 335

Phe Asn Val Ala Ile Gly Gly Lys Thr Phe Val Val Pro Gly Thr Asp

340 345 350

Leu Asn Tyr Ala Pro Ile Asn Ser Ala Gly Thr Thr Cys Phe Gly Gly

355 360 365

Ile Gln Ala Asn Thr Gly Ile Gly Phe Asn Ile Phe Gly Asp Ile Phe

370 375 380

Leu Lys Ser Val Tyr Ala Val Phe Asp Gln Thr Gln Ser Ser Pro Arg

385 390 395 400

Leu Gly Phe Ala Glu Gln Ser

405

<210> 2

<211> 388

<212> PRT

<213>thermophilic fungal MEY-1 (Bispora sp. MEY-1)

<400> 2

Ala Pro Ala Gln Ile Val Gly Arg Ser Thr Phe Gln Ile Asp Gln Val

1 5 10 15

Ala Ser Gly Lys Val Tyr Lys Asn Gly Pro Met Ala Met Met Gln Thr

20 25 30

Tyr Asn Lys Tyr Ala His Val Gly Ala Val Ala Pro Ala Ala Val Val

35 40 45

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

50 55 60

Gln Tyr Asp Glu Ser Tyr Leu Cys Pro Val Thr Ile Gly Asp Gln Thr

65 70 75 80

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

85 90 95

Thr Leu Thr Pro Ser Ser Glu Ser Thr Gly His Thr Leu Tyr Asn Pro

100 105 110

Ala Asp Ser Gly Thr Glu Lys Gln Gly Tyr Thr Trp Asn Ile Thr Tyr

115 120 125

Gly Asp Gly Ser Gly Ala Ala Gly Val Val Tyr Ala Asp Lys Val Val

130 135 140

Val Gly Gly Val Thr Ala Thr Ser Gln Ala Val Glu Ala Ala Thr Ser

145 150 155 160

Val Ser Ser Glu Phe Thr Gln Asp Thr Lys Asn Asp Gly Leu Leu Gly

165 170 175

Leu Ala Phe Ser Ser Ile Asn Thr Val Gln Pro Val Gln Gln Thr Thr

180 185 190

Phe Phe Asp Thr Val Lys Asp Thr Leu Ala Lys Lys Leu Phe Thr Ala

195 200 205

Asp Leu Lys Lys Gly Ala Ala Gly Ser Tyr Gly Phe Gly Tyr Ile Asp

210 215 220

Ser Ser Lys Tyr Thr Gly Thr Ile Thr Tyr Val Pro Val Asn Asn Glu

225 230 235 240

Asn Gly Phe Trp Gln Phe Thr Ala Gly Gly Tyr Ser Ile Gly Gly Gly

245 250 255

Asn Gly Thr Ser Gly Ser Asn Ala Thr Thr Gly Ser Ile Gly Thr Ser

260 265 270

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

275 280 285

Thr Ala Tyr Tyr Lys Gln Val Ser Gly Ala Ser Tyr Asn Ser Ala Gln

290 295 300

Gly Gly Tyr Thr Tyr Pro Cys Gly Ala Thr Leu Pro Asp Phe Asn Val

305 310 315 320

Ala Ile Gly Gly Lys Thr Phe Val Val Pro Gly Thr Asp Leu Asn Tyr

325 330 335

Ala Pro Ile Asn Ser Ala Gly Thr Thr Cys Phe Gly Gly Ile Gln Ala

340 345 350

Asn Thr Gly Ile Gly Phe Asn Ile Phe Gly Asp Ile Phe Leu Lys Ser

355 360 365

Val Tyr Ala Val Phe Asp Gln Thr Gln Ser Ser Pro Arg Leu Gly Phe

370 375 380

Ala Glu Gln Ser

385

<210> 3

<211> 388

<212> PRT

<213>thermophilic fungal MEY-1 (Bispora sp. MEY-1)

<400> 3

Ala Pro Ala Gln Ile Val Gly Arg Ser Thr Phe Gln Ile Asp Gln Val

1 5 10 15

Ala Ser Gly Lys Val Tyr Lys Asn Gly Pro Met Ala Met Met Gln Thr

20 25 30

Tyr Asn Lys Tyr Ala His Val Gly Ala Val Ala Pro Ala Ala Val Val

35 40 45

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

50 55 60

Gln Tyr Asp Glu Ser Tyr Leu Cys Pro Val Thr Ile Gly Asp Gln Thr

65 70 75 80

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

85 90 95

Thr Leu Thr Pro Ser Ser Glu Ser Thr Gly His Thr Leu Tyr Asn Pro

100 105 110

Ala Asp Ser Gly Thr Glu Lys Gln Gly Tyr Thr Trp Asn Ile Thr Tyr

115 120 125

Gly Asp Gly Ser Gly Ala Ala Gly Val Val Tyr Ala Asp Lys Val Val

130 135 140

Val Gly Gly Val Thr Ala Thr Ser Gln Ala Val Glu Ala Ala Thr Ser

145 150 155 160

Val Ser Ser Glu Phe Thr Gln Asp Thr Lys Asn Asp Gly Leu Leu Gly

165 170 175

Leu Ala Phe Ser Ser Ile Asn Thr Val Gln Pro Val Gln Gln Thr Thr

180 185 190

Phe Phe Asp Thr Val Lys Asp Thr Leu Ala Lys Lys Leu Phe Thr Ala

195 200 205

Asp Leu Lys Lys Gly Ala Ala Gly Ser Tyr Gly Phe Gly Tyr Ile Asp

210 215 220

Ser Ser Lys Tyr Thr Gly Thr Ile Thr Tyr Val Pro Val Asn Asn Glu

225 230 235 240

Asn Gly Phe Trp Gln Phe Thr Ala Gly Gly Tyr Ser Ile Gly Gly Gly

245 250 255

Asn Gly Thr Ser Gly Ser Asn Ala Thr Thr Gly Ser Ile Gly Thr Ser

260 265 270

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

275 280 285

Thr Ala Tyr Tyr Lys Gln Val Ser Gly Ala Ser Tyr Asn Ser Ala Gln

290 295 300

Gly Gly Tyr Thr Tyr Pro Cys Gly Ala Thr Leu Pro Asp Phe Asn Val

305 310 315 320

Ala Ile Gly Gly Lys Thr Phe Val Val Pro Gly Thr Asp Leu Asn Tyr

325 330 335

Ala Pro Ile Asn Ser Ala Gly Thr Thr Cys Phe Gly Gly Ile Gln Ala

340 345 350

Asn Thr Gly Ile Gly Phe Asn Ile Phe Gly Asp Ile Phe Leu Lys Ser

355 360 365

Val Tyr Ala Val Phe Asp Gln Thr Gln Ser Ser Pro Arg Leu Gly Phe

370 375 380

Ala Glu Gln Ser

385

<210> 4

<211> 1167

<212> DNA

<213>thermophilic fungal MEY-1 (Bispora sp. MEY-1)

<400> 4

gctccggccc agattgtcgg ccgcagcacc tttcagatcg atcaagtggc ctctggtaag 60

gtctacaaga acggccctat ggccatgatg cagacataca acaagtacgc gcacgtaggc 120

gccgtcgcgc ccgctgccgt tgtggccgcc gcggccgccg cgcagactgg cgaggtgtcc 180

gcaaatcccg agcagtacga cgaaagctac ctttgtcctg tcactattgg ggatcagacc 240

ttgaacttgg acttcgacac gggcagcgcg gacctttggg tgttttcaac cctcactccg 300

tcaagcgagt caacaggcca cacgttgtat aaccccgccg actctggcac ggagaagcag 360

ggctatacct ggaacatcac ctacggcgac ggctcgggcg cagccggtgt ggtgtacgcc 420

gataaggtgg tcgttggcgg ggtcaccgcg acctcgcagg cggtggaggc ggcgacatcg 480

gtctccagcg aattcacaca ggacaccaag aacgatggcc tgctcggctt ggcgttcagc 540

tcgatcaata ccgttcagcc ggtgcaacag actactttct tcgatacggt caaggacacg 600

ctggccaaga agctcttcac tgccgatctc aagaaggggg ctgccggcag ctatggtttt 660

ggttacatcg acagctccaa atacaccggc accatcacct atgtgcccgt gaacaatgag 720

aacggcttct ggcagttcac cgcaggcggc tactccatcg gtggcggcaa cggcacgtca 780

ggcagcaacg cgaccacagg cagcattggc acctccatcg cggacaccgg caccaccctc 840

ctcttgttgc ccagcaacgt agtcacggct tactacaagc aagtctcggg cgcttcttat 900

aactcggcgc aaggcggtta cacttacccg tgcggtgcca ctctgcccga cttcaacgtg 960

gccattggcg gcaagacttt cgtcgtcccc ggcaccgatc tcaattacgc gcctatcaac 1020

agcgcgggca ccacgtgctt cggcgggatt caagctaaca cgggcatcgg attcaacatc 1080

ttcggcgaca ttttcctaaa gagcgtctac gccgtcttcg accagactca gagctcgccg 1140

cgcctcggct ttgccgagca atcgtaa 1167

Claims (9)

1. the acid protease Bs2688 mutant that thermal stability improves, which is characterized in that the acid protease Bs2688 is prominent The amino acid sequence of variant is as shown in SEQ ID No.3.

2. the acid protease Bs2688 mutant gene that thermal stability improves, which is characterized in that coding is described in claim 1 Acid protease Bs2688 mutant.

3. the acid protease Bs2688 mutant gene that thermal stability according to claim 2 improves, which is characterized in that The nucleotide sequence of the acid protease Bs2688 mutant gene is as shown in SEQ ID No.4.

4. including the recombinant expression carrier of acid protease Bs2688 mutant gene as claimed in claim 2.

5. including the recombinant bacterial strain of acid protease Bs2688 mutant gene as claimed in claim 2.

6. the method for preparing the acid protease Bs2688 mutant of thermal stability raising, which is characterized in that the method includes Following steps:

(1) to convert host cell comprising the recombinant vector of encoding acidic Cathepsin B s2688 mutant gene, recombinant bacterium is obtained Strain；

(2) recombinant bacterial strain, inducing expression acid protease Bs2688 mutant are cultivated；

(3) the acid protease Bs2688 mutant of acquisition is isolated and purified.

7. the application for the acid protease Bs2688 mutant that thermal stability described in claim 1 improves.

8. the amino acid sequence application of albumen in terms of caseinhydrolysate as shown in SEQ ID No.3.

9. the amino acid sequence application of albumen in feed, food or field of medicaments as shown in SEQ ID No.3.