CN102021125A - Organic solvent resistant protease producing strain, gene of organic solvent resistant protease produced thereby and application of organic solvent resistant protease - Google Patents
Organic solvent resistant protease producing strain, gene of organic solvent resistant protease produced thereby and application of organic solvent resistant protease Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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Abstract
The invention relates to an organic solvent resistant protease producing strain, a gene of organic solvent resistant protease and an application of the organic solvent resistant protease, in particular relating to the organic solvent resistant protease producing strain as well as the gene of the organic solvent resistant protease thereby and the application of the organic solvent resistance protease in catalytic synthesis of small peptides in nonaqueous phases, belonging to the fields of microbiology and enzymology. Bacterial strains are named as Bacilluscereus WQ9-2 by classification, are gram-positive strains and can resist a plurality of organic solvents with a certain concentration. The protease-coding gene which is separated and cloned on organic solvent resistant protease produced by the producing strain is provided with a nucleotide sequence shown as SEQ ID NO:1, and an amino acid sequence shown as SEQ ID NO:2. The protease has the characteristics of high yield, high specific activity, wide and strong tolerance and the like. The protease has industrial application value for the catalytic synthesis of the small peptides in the nonaqueous phases.
Description
Technical field
The present invention relates to a kind of organic solvent tolerant protease and produce bacterium, and the application of the gene of the organic solvent tolerant protease that is produced and its synthetic little peptide of catalysis in nonaqueous phase, microbiology and zymetology field belonged to.
Background technology
Proteolytic enzyme is meant the class of enzymes of energy catalysis peptide bond hydrolysis, because its important commercial is worth and also become one of three big industrial enzymes gradually by extensive concern.The output of current proteolytic enzyme has occupied more than 40% of commercial enzyme market, is widely used in fields such as washing composition, food, medicine, leather, organic synthesis, waste treatment.Though proteolytic enzyme is present in nearly all biology, but because the proteolytic enzyme of microorganisms producing mainly is extracellular enzyme, compare with the proteolytic enzyme of animal, plant origin and to be more suitable for industrialization, so the output of microbe-derived proteolytic enzyme accounts for more than 2/3 of present proteolytic enzyme ultimate production.
Generally, the reaction of proteolytic enzyme catalytic hydrolysis peptide bond is to carry out in the buffered soln of specific pH, and proteolytic enzyme has the specificity requirement to the amino-acid residue of its catalysis peptide bond, and the regioselectivity and the stereoselectivity of height promptly arranged.In recent years, it is found that the reaction that proteolytic enzyme can not carry out in can some aqueous solution of catalysis in organic solvent, such as: under non-water condition, the formation that proteolytic enzyme can the catalysis peptide bond.A large amount of studies show that carrying out enzymatic reaction in the nonaqueous phase has many advantages: the solubleness that 1) increases various organic substrates; 2) have stereoselectivity and regioselectivity highly, organic medium can change selectivity; 3) the control molecular balance moves to required direction, as lytic enzyme energy catalytic dehydration condensation reaction in organic medium; 4) effectively prevent microbial contamination, product is easy to separation and purification etc.Organic phase biological catalysis has been showed the big potentiality of tool that enzyme is used in organic solvent.The advantage of proteolytic enzyme in nonaqueous phase is can the catalysis peptide bond and the formation of ester bond, thereby in the synthetic fractionation that reaches chiral drug that is applied to some important intermediate.
The contradiction of enzymatic tempting prospect and the easy inactivation of enzyme has promoted the transformation of enzyme and the development of non-water zymetology in the organic solvent system.In past more than 20 year, the investigator is devoted to improve the stability of enzyme in organic solvent.Natural proteolytic enzyme with organic solvent stability is a class novel protein enzyme of discovered in recent years, and having can the natural characteristic of stable existence in organic solvent.This proteinoid enzyme is produced by the organic solvent-resistant extreme microorganism usually.Although organic solvent has great murder by poisoning to microorganism cells, the extreme microorganism of relevant organic solvent-resistant becomes a research focus in recent years.Because the organic solvent-resistant extreme microorganism can be survived in being rich in the environment of organic solvent, therefore be secreted into the outer enzyme of born of the same parents and also have certain organic solvent tolerance by this quasi-microorganism.
It is false single Pseudomonas (Pseudomonas) that the organic solvent tolerant protease of having reported at present produces most the concentrating of bacterium, yet the related solvents of the proteolytic enzyme of report mensuration tolerance concentration is lower by (about 25% mostly, v/v), and exist production of enzyme low, be difficult for purifying, substrate and select shortcomings such as specificity is strong, thereby influence its application in Industrial Catalysis.
Summary of the invention
The technical problem to be solved in the present invention provides the bacterial strain that organic solvent tolerant protease is produced in a strain, and it is synthetic to make the proteolytic enzyme of the resisting high-concentration organic solvent that it produced can be successfully applied to the little peptide of industrialization nonaqueous phase.
In order to realize technical purpose of the present invention, technical scheme of the present invention is:
The present invention screens from the greasy dirt soil sample and obtains strain organic solvent tolerant protease generation bacterium, identify that through BIOLOG automatic bacterial assessing instrument sequential analysis shows that this bacterium of this bacterial strain is a bacillus cereus with 16SrDNA, classification called after bacillus cereus Bacillus cereus WQ9-2, its preserving number registration number is CCTCC NO:M 2010010.
The present invention has carried out the biological characteristic evaluation to Bacillus cereus WQ9-2, and the result shows that this bacterium is a gram-positive microorganism, has gemma to generate; Under aerobic conditions, grow; Behind the growth 24h, the colony diameter size is 3mm～5mm in broth culture, and growth scope is 20 ℃～40 ℃, and optimum growth temperature is 35 ℃, and growth pH is 6.0～11.0, and the suitableeest growth pH is 8.0.The physio-biochemical characteristics of Bacillus cereus WQ9-2 show: the oxydase reaction result is negative, and the result that glucose, maltose, sucrose and D-fructose utilize is positive, D-wood sugar and lactose utilize the result negative.
The present invention has carried out condition of enzyme production optimization to Bacillus cereus WQ9-2, optimizes the back yield of enzyme up to 3528U/mL, is 2.9 times of the preceding 1230U/mL of optimization.
The present invention has carried out purifying to the extracellular enzyme that this bacterium produces, and obtains electrophoretically pure proteolytic enzyme through two step separation and purification, called after organic solvent-resistant WQ9-2 proteolytic enzyme, behind the purifying its alives than enzyme be 78509U/mg.
The present invention has carried out the research of zymologic property to organic solvent-resistant WQ9-2 proteolytic enzyme, experimental results show that this proteolytic enzyme all has good tolerability to multiple organic solvent, and tolerance concentration is up to 50% (v/v), the organic solvent tolerant proteases tolerance concentration of having reported more than being better than greatly (25%, v/v); The optimal pH of organic solvent tolerant protease WQ9-2 is 8.0, and the pH value scope of useful effect is 7～10, is neutral protease, and its optimal reaction temperature is 45 ℃, the Ca of 5mM
2+The effect of increasing significantly of the optimum temperuture of this enzyme of ion pair and thermostability.
The present invention is isolated and cloned into the encoding gene of organic solvent tolerant protease that this bacterial strain produces, by PCR method separating clone this organic solvent tolerant protease gene, it has the nucleotide sequence shown in the SEQ ID NO:1, the DNA complete sequence analysis is the result show, 1701 Nucleotide of organic solvent tolerant protease gene WQ9-2 total length, 567 amino acid of encoding, wherein mature peptide is 317 amino acid.The dna homolog of this gene and Bacillus cereus ATCC 14579 coding bacillomyxins is 92%, and wherein mature peptide amino acid moiety homology is 98%.Its aminoacid sequence is shown in SEQ ID NO:2.
The invention provides the application of the little peptide of organic solvent tolerant protease WQ9-2 catalysis in organic phase in synthetic.The WQ9-2 proteolytic enzyme of purifying is applied in the different little peptide building-up reactions of organic synthesis, the result shows the Tris-HCl (pH8.5 that contains 50% (v/v) DMSO, 0.05M) solution is a kind of good building-up reactions system, WQ9-2 proteolytic enzyme can be in this system the multiple little peptide of catalysis synthetic and successfully realized many group reactions and isolating coupling.
Beneficial effect of the present invention is:
The organic solvent tolerance of bacillus cereus Bacillus cereus WQ9-2 excretory organic solvent tolerant protease is strong, the productive rate height, has advantages such as substrate category that height ratio lives, can discern is wide; The successful Application of the little peptide precursor of organic solvent-resistant WQ9-2 catalysis in organic solvent proves that further this enzyme has good performance in the organic solvent catalyzed reaction, has huge application potential at little peptide in the industry such as synthetic and pharmacy.
Description of drawings
Fig. 1 is the SDS-PAGE electrophorogram of organic solvent-resistant WQ9-2 proteolytic enzyme,
Wherein, swimming lane 1-Marker; Swimming lane 2-crude enzyme liquid; The pure enzyme liquid of swimming lane 3-;
Fig. 2 is the optimal reaction pH of organic solvent tolerant protease WQ9-2;
Fig. 3 is the pH stability of organic solvent tolerant protease WQ9-2;
Fig. 4 is the optimal reactive temperature of organic solvent tolerant protease WQ9-2;
Fig. 5 is the temperature stability of organic solvent tolerant protease WQ9-2.
Microorganism classification called after bacillus cereus Bacillus cereus WQ9-2 of the present invention, preservation date is on January 15th, 2010, depositary institution's full name is Chinese typical culture collection center, be called for short CCTCC, the depositary institution address is a China. Wuhan. and Wuhan University, deposit number: CCTCC NO:M 2010010.
Embodiment
Embodiment one
The present embodiment explanation produces the screening method of the natural bacterial strain of organic solvent tolerant protease.
Organic solvents such as the hexanaphthene of employing different concns, toluene screen from the greasy dirt soil sample as screening pressure and obtain the organic solvent-resistant extreme microorganism: adopt the milk Agar Plating, concrete prescription is (g/L): Tryptones 5, yeast powder 3, skim-milk 25, agar 12.With the organic solvent-resistant microbial inoculant that screened in the milk agar plate, according to the ratio of bacterium colony and transparent circle size, the bacterial strain that preliminary screening proteolytic enzyme output is high.This method screens has high organic solvent-resistant extreme microorganism 13 strains of proteolytic enzyme output.
In order further to detect the solvent tolerance of the proteolytic enzyme that primary dcreening operation obtains, the protease-producing ability of 13 strain bacterium and the organic solvent-resistant character of the proteolytic enzyme that produces are comprehensively detected.The protease-producing inoculation that primary dcreening operation is obtained arrives product enzymic fermentation substratum, and concrete prescription is: pancreas egg peptone 10g/L, (NH
4)
2SO
41.0g/L, KH
2PO
40.5g/L, MgSO
40.3g/L, CaCl
21.0g/L, NaCl 1.0g/L, glycerine 6.3g/L, pH7.0.Culture temperature is 37 ℃, and incubation time is 72h, and shaking speed is 180rmp.After the fermentation ends, 10000rmp is in 4 ℃ of centrifugal 15min, and getting supernatant is crude enzyme liquid.The methyl alcohol, toluene, hexanol, primary isoamyl alcohol, butanols, Virahol, acetone, ethanol, dimethyl formamide (DMF) and the dimethyl sulfoxide (DMSO) (DMSO) that in the 1mL crude enzyme liquid, add equal-volume (1mL) respectively, oscillation treatment 24h under 30 ℃, 150rpm condition is that substrate detects the proteolytic enzyme residual enzyme and lives with the casein; Choose have the highest vigor bacterial strain as the strain of purpose organic solvent protease production strain.
Wherein, the proteinase activity detection method is: the preparation casein content is that the Tris-HCl damping fluid of 2% pH 8.0 is as reaction substrate.Get the enzyme liquid that 200 μ L dilute suitable multiple and add 200 μ L reaction substrates, 40 ℃ are accurately reacted 10min, add 400 μ L TCA reaction terminating liquid (0.11M trichoroacetic acid(TCA)s, 0.22M sodium-acetate, 0.33M termination reaction acetic acid), place 15min for 4 ℃, the centrifugal 15min of 15000rpm detects the ultraviolet light absorption value down in 280nm.
Protease activity unit of force (U) is defined as: at 40 ℃, in the pH8.0 damping fluid, per minute catalysis produces the needed enzyme amount of 1 μ g tyrosine (U=μ g/mLmin).Detect and the organic solvent Detection of Stability by enzymic activity, wherein the protease production strain strain of a strain with good tolerance laid eggs white enzyme activity up to 1230U/mL, called after WQ9-2.
Embodiment two
Present embodiment explanation organic solvent tolerant protease produces biological property, evaluation and the condition of enzyme production research thereof of bacterium WQ9-2.
The biological property of bacterial strain WQ9-2: gramstaining shows that this bacterial strain is a gram positive bacterial strain, and gemma is arranged, and grows under aerobic conditions.In broth culture the growth 24h after, the bacterium colony size diameter is 3mm～5mm, growth temperature range is 20 ℃～40 ℃, optimum growth temperature is 35 ℃, growth pH scope is 6.0～11.0, and the suitableeest growth pH is 8.0, and its physio-biochemical characteristics show the oxydase reaction feminine gender, the result that glucose, maltose, sucrose and D-fructose utilize is positive, D-wood sugar and lactose utilize the result negative.
The strain identification of bacterial strain WQ9-2: identify and the 16SrDNA sequential analysis through BIOLOG automatic bacteria assessing instrument, show that this bacterial strain is that bacillus cereus belongs to, and called after Bacillus cereus WQ9-2.
The Bacillus cereus WQ9-2 white enzymic fermentation condition research of laying eggs: the substratum after by experiment of single factor and response surface method fermention medium being optimized is (g/L): glucose 17.0, yeast powder 8.0, KH
2PO
40.5, MgSO
40.3, CaCl
20.5 NaCl 1.Culture condition after the optimization is: the initial pH 7.5 that ferments, and shaking speed 180rpm, inoculum size 2% (v/v), inoculation 12h in age, leavening temperature is 25 ℃, liquid amount is 50mL/250mL.The proteolytic enzyme enzyme activity is 3528U/mL after cultivating 48 hours under this fermentation condition.
Embodiment three
The purifying procedure of present embodiment explanation organic solvent-resistant WQ9-2 proteolytic enzyme.
Bacillus cereus WQ9-2 cultivated 48h in producing the enzyme substratum after, fermented liquid is 10,000rpm, and 4 ℃ of centrifugal 10min get supernatant liquor as crude enzyme liquid; Crude enzyme liquid is placed ice bath, adopt ethanol sedimentation to collect the Tris-HCl damping fluid dissolving that 45%～75% (v/v) partly precipitated albumen is used the pH8.5 of 0.05M then, 10,000rpm, 4 ℃ of centrifugal 10min remove undissolvable foreign protein.The enzyme liquid that above processing is obtained adopts DEAE-Sepharose FF ion exchange column to carry out purifying, carries out wash-out with Tris-HCl (pH 8.5) damping fluid of 0.05M, collects the proteinase activity peak.By SDS-PAGE electrophorogram (Fig. 1), it is pure that the organic solvent tolerant protease (called after organic solvent-resistant WQ9-2 proteolytic enzyme) behind the discovery two-step purifying has reached electrophoresis, and this proteolytic enzyme molecular weight subunit is about 37kDa.The purifying multiple is 3.8 times, and the rate of recovery is 57%, and final proteolytic enzyme reaches 78509U/mg than living, and gathers to see Table 1.
Purification step and the result of table 1. organic solvent tolerant protease WQ9-2
Annotate: protein concn adopts the Coomassie brilliant blue method to measure
Embodiment four
The measuring method of the zymologic property of present embodiment explanation organic solvent tolerant protease WQ9-2.
The solvent tolerance of organic solvent tolerant protease detects: the 1mL proteolytic enzyme diluent behind the purifying is added 17 kinds of isopyknic organic solvents respectively place the test tube of sealing, to add equal-volume 0.05M Tris-HCl damping fluid group (pH8.0) is contrast, 30 ℃, 150rpm vibration.Respectively at 0h, 12h, 24h and 48h sampling are that substrate detects proteinase activity with the casein, the result is as shown in table 2.
Table 2.WQ9-2 organic solvent tolerance
Organic solvent tolerant protease WQ9-2 has good organic solvent tolerance, and DMF, dodecane, toluene are to the reduction effect maximum of enzyme activity in 17 kinds of organic solvents after deliberation; Acetonitrile group proteinase activity after temperature is bathed 24h is zero substantially; Octanol, DMSO, hydrophilic organic solvent group enzyme activity 24h such as glycerol are higher than initial vigor.The enzyme activity of all the other organic solvent groups is greater than the enzyme activity of synchronous control group.Experiment shows that there is good tolerance in organic solvent tolerant protease WQ9-2 to multiple organic solvent.
The mensuration of organic solvent tolerant protease optimal reaction pH value and pH stability: 2% casein damping fluid with different pH values is a substrate, is reference with the reaction vigor under the pH8.0 condition, measures enzyme activity, and the result as shown in Figure 2.WQ9-2 proteolytic enzyme that bacterial strain produces has best vigor in the pH8.0 reaction system, as can be seen from Figure 2 the pH value scope of proteolytic enzyme WQ9-2 useful effect is 7～9, is neutral protease.With the work of protoenzyme liquid enzyme serves as with reference to the pH stability that detects proteolytic enzyme WQ9-2, to measure enzyme activity behind 40 ℃ of insulation 60min in the buffered soln of proteolytic enzyme WQ9-2 adding pH 6～11, experiment shows that this proteolytic enzyme can stable existence (shown in Figure 3) in the damping fluid of pH7～10.
The mensuration of organic solvent tolerant protease optimal reactive temperature and thermostability: being determined as in the 0.05M Tris-HCl buffer system (pH 8.0) of the optimum temperuture of organic solvent tolerant protease is that substrate carries out enzymatic reaction with the casein under different temperature condition.The Ca that behind purifying, adds 5mM simultaneously in the enzyme liquid
2+, group check calcium ion is to the influence of WQ9-2 proteolytic enzyme optimal reactive temperature in contrast.Measurement result shows (shown in Figure 4), is not adding under the calcium ion situation, and this proteolytic enzyme optimal reactive temperature is 45 ℃, and optimal reactive temperature is 65 ℃ under adding 5mM calcium ion situation, illustrates that calcium ion increases significantly to the optimum temperuture of this enzyme.
Proteolytic enzyme is handled down 150min at 60 ℃, and wherein 30min sampling is at interval lived and is determined the thermostability of WQ9-2 under this temperature thereby measure residual enzyme down at 40 ℃.The Ca that adds 5mM simultaneously in the enzyme liquid behind purifying
2+, group check calcium ion is to the influence of the thermostability of WQ9-2 proteolytic enzyme in contrast.Measuring result shows (shown in Figure 5), and the calcium ion of 5mM can significantly improve this protease heat stability.
Embodiment five
The separating clone program of present embodiment explanation organic solvent tolerant protease WQ9-2 encoding gene.
Adopt the total DNA of phenol-chloroform method extracting thalline.The organic solvent-resistant WQ9-2 proteolytic enzyme of purifying is carried out sequential analysis by LC-MS/MS measure its amino acid fragment, the result shows that the sequence of the bacillomyxin among this enzyme and the bacillus cereus Bacilluscereus ATCC 14579 is the most close, therefore outside this bacillomyxin CDS two ends, design primer, the CDS encoding sequence of amplification organic solvent-resistant WQ9-2 proteolytic enzyme.The PCR fragment electrophoresis that will contain the CDS encoding sequence reclaims rear clone to the pMD18-T carrier, carries out sequential analysis.The primer of design is:
EUF：ATTTTTATTAGGGGGAAGGTTC
EUR：CATAGGCTCCTTCTTTATTTTTGA
The PCR reaction parameter is: 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 30sec; 55 ℃ of annealing 30sec, 72 ℃ are extended 1min 30sec; After circulation 30 is taken turns, 72 ℃ of insulation 10min.According to this reaction conditions, the PCR fragment of the 1.732kb that increased.This fragment is connected to the pMD18-T carrier, carries out sequencing.The result shows that it has the nucleotide sequence shown in the SEQ IDNO:1, and the DNA complete sequence analysis is the result show, 1701 Nucleotide of organic solvent tolerant protease gene WQ9-2 total length, and 567 amino acid of encoding, wherein mature peptide is 317 amino acid.The dna homolog of this gene and Bacilluscereus ATCC 14579 coding bacillomyxins is 92%, and wherein mature peptide amino acid moiety homology is 98%.Its aminoacid sequence is shown in SEQ ID NO:2.
Embodiment six
The application of present embodiment explanation organic solvent tolerant protease WQ9-2 in the little peptide of organic catalysis is synthetic.
With each seed amino acid (Cbz-AA-OH) of the amido protecting of 100mM and the L-Phe-NH of 200mM
2As substrate, after containing purifying, add equal-volume DMSO, DMF, methyl alcohol respectively in the 50mM Tris-HCl (pH8.5) of the WQ9-2 proteolytic enzyme reaction buffering, the reaction cumulative volume is 1mL, add, oscillatory reaction under 37 ℃ of conditions, sampling in 12 hours is diluted to 1/20 with distilled water (0.05% trifluoroacetic acid)/acetonitrile=(60/40), product with chemosynthesis is that standard substance adopt reverse hplc detecting product, the result shows, DMSO is an optimum solvent, under this condition, multiple little peptide is successfully synthesized (as table 3).
The synthetic different little peptide productive rates of table 3.WQ9-2 proteolytic enzyme catalysis
Claims (4)
1. an organic solvent tolerant protease produces bacterium, the new bacterial strain of its classification called after bacillus cereus, and called after Bacillus cereus WQ9-2, its preserving number registration number is CCTCC NO:M 2010010.
2. organic solvent tolerant protease according to claim 1 produces bacterium, it is characterized in that it produces organic solvent-resistant WQ9-2 proteolytic enzyme and has the aminoacid sequence shown in the SEQ ID NO:2.
3. organic solvent tolerant protease according to claim 2 produces bacterium, it is characterized in that the encoding gene of organic solvent-resistant WQ9-2 proteolytic enzyme has the nucleotide sequence shown in the SEQ ID NO:1.
4. organic solvent tolerant protease according to claim 1 produces the application of organic solvent tolerant protease in the little peptide synthetic system of nonaqueous phase that bacterium produces.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774891A (en) * | 2015-01-15 | 2015-07-15 | 南京工业大学 | Technology for efficiently synthesizing benzyloxycarbonyl aspartame through using enzyme method |
| CN107083353A (en) * | 2017-05-27 | 2017-08-22 | 江南大学 | A kind of method that use resting cell produces Aspartame |
| CN107227267A (en) * | 2017-05-12 | 2017-10-03 | 上海海洋大学 | A kind of soybean small peptide and preparation method and application |
| CN109161539A (en) * | 2018-09-18 | 2019-01-08 | 安徽大学 | A kind of organic solvent tolerance aminopeptidase LapA and its preparation method and application |
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2010
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774891A (en) * | 2015-01-15 | 2015-07-15 | 南京工业大学 | Technology for efficiently synthesizing benzyloxycarbonyl aspartame through using enzyme method |
| CN104774891B (en) * | 2015-01-15 | 2018-12-25 | 南京工业大学 | A kind of technique that enzyme process efficiently synthesizes benzyloxycarbonyl group Aspartame |
| CN107227267A (en) * | 2017-05-12 | 2017-10-03 | 上海海洋大学 | A kind of soybean small peptide and preparation method and application |
| CN107083353A (en) * | 2017-05-27 | 2017-08-22 | 江南大学 | A kind of method that use resting cell produces Aspartame |
| CN109161539A (en) * | 2018-09-18 | 2019-01-08 | 安徽大学 | A kind of organic solvent tolerance aminopeptidase LapA and its preparation method and application |
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