CN101215534A - Organic solvent-resistant alkaline protease producing strain, gene of organic solvent-resistant alkaline protease and application of organic solvent-resistant alkaline protease - Google Patents
Organic solvent-resistant alkaline protease producing strain, gene of organic solvent-resistant alkaline protease and application of organic solvent-resistant alkaline protease Download PDFInfo
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- CN101215534A CN101215534A CNA2007101919691A CN200710191969A CN101215534A CN 101215534 A CN101215534 A CN 101215534A CN A2007101919691 A CNA2007101919691 A CN A2007101919691A CN 200710191969 A CN200710191969 A CN 200710191969A CN 101215534 A CN101215534 A CN 101215534A
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- organic solvent
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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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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses an organic solvent resistant alkaline protease producing strain, a gene thereof and application thereof in catalyzing peptide synthesis and resolving racemic amine and amino acid in an organic phase. The strain is classified and named as Bacillus licheniformis YP1A, and the preservation registration number is as follows: CCTCC No: m207021, a gram-positive bacillus, is able to tolerate a range of organic solvents at certain concentrations. The invention separates and clones the coding gene of protease produced by the strain, which has the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. the organic solvent resistant alkaline protease has the properties of high specific activity, strong solvent tolerance, wide action pH range, high temperature resistance, strong alkali resistance and the like. The protease can be applied to the purposes of catalyzing peptide synthesis, resolving racemic amine and amino acid in an organic phase and the like.
Description
Technical field
The present invention relates to a kind of organic solvent-resistant Sumizyme MP produce bacterium with and the organic solvent-resistant alkaline protease gene, with and synthetic, resolution of racemic amine of catalysis peptide and amino acid whose application in organic phase, belong to microbiology and zymetology field.
Background technology
Proteolytic enzyme is since quilt test in 1914 is used for the added ingredients of washing composition, owing to having important commercial use and by extensive concern.The output of current proteolytic enzyme occupies more than 40% of enzyme market, is widely used in fields such as washing composition, food, pharmacy, process hides, diagnostic reagent, sewage disposal.And after 2005, the consumption of proteolytic enzyme is unprecedented soaring according to statistics.Proteolytic enzyme extensively is present in all biologies such as animal, plant, fungi and prokaryotic organism, and wherein microbe-derived proteolytic enzyme occupies more than 2/3 of present proteolytic enzyme production ultimate production.Microbe-derived proteolytic enzyme can be divided into metalloprotease, aspartate protease and serine protease etc. according to the reaction pH of enzyme, the feature of active group again.
Sumizyme MP (EC.3.4.21-24,99) be defined as in neutrality to alkaline pH scope and have active proteolytic enzyme, this fermentoid has Serine active centre (serine protease) or metal active center (metalloprotease), is the enzyme with important commercial use with alkaline serine protease again wherein.Serine protease has vigor in neutrality usually to the alkaline pH condition, pH is 7～11 during best vigor, and this enzyme molecular weight is less usually, and scope is 18～35kDa.To the effect specificity of oxidized form Regular Insulin β chain and further classification, serine protease can be divided into chymotrypsin protein enzyme, subtilisin class, wheat serine carboxypeptidase II type proteolytic enzyme and SP etc. usually according to this enzyme.
Along with the development of solvent and enzyme engineering technology, it is found that enzyme can catalysis some reaction that can not carry out in water in organic solvent.Under normal water condition, proteolytic enzyme catalysis peptide bond hydrolysis, but can reversal reaction in the medium of water restriction and promote the formation of peptide bond.Hydrolysis reaction is reversible in theory, and studies show that of relevant peptide bond hydrolysis, when certain mechanism of employing makes the thermodynamics obstacle that can overcome the hydrolysis reversed reaction when production concentration is lower than equilibrium concentration in the reaction system, reaction is carried out to the peptide compound direction.Chemical method catalysis peptide is synthetic to occupy critical role for a long time in peptide is synthetic, but chemical method catalysis peptide is synthetic significant disadvantages arranged, as: 1. the racemization in the peptide bond forming process; 2. protection and the deprotection to amino acid side chain causes cost increase and product to reduce; 3. the recycling of acry radical donor etc.; 4. prepare the potential risk that there is poisonous organic solvent in the process that is used for the food grade peptide.
Utilizing proteolytic enzyme organic phase catalysis peptide to synthesize has following 3 kinds of mode: a. to contain the water-soluble system of water dissolvable organic solvent; B. organic-the water diphasic system; C. the organic solvent system of micro-water content.In mode a, the content of organic solvent in reaction system has material impact to output; Because the pH value of aqueous phase is easy to adjust and can the synthetic of peptide can be carried out continuously by separating step, therefore, it is synthetic that mode b is successfully applied to peptide at first, as Aspartame in mode b
Precursor synthetic.This reaction is a catalyzer with thermolysin (thermolysin), synthetic precursor dipeptides in two-phase, the organic phase that contains substrate (Z-L-Asp and L-PheOMe) is added continuously in the reactor, substrate is extracted to the aqueous phase that contains enzyme, and at the synthetic precursor dipeptides of aqueous phase, dipeptides is extracted in the organic phase more then, and reaction is carried out continuously.Because the existence of water has promoted hydrolysis reaction, become the greatest weakness of enzymatic peptide in synthetic, so system C becomes optimal system.
Enzyme process resolving chiral compound is emphasis and the focus of studying both at home and abroad at present, enzymatic chiral separation technology, have other method for splitting the numerous advantages that can not compare, as the catalytic activity height, the few environmentally safe of power consumption, the by product of reaction is few, high specificity and selectivity, and it is high to split efficient.
The amino acid that non-natural exists mostly is racemic modification greatly, must just can obtain optically pure enantiomorph by splitting.Different with synthetic peptide, the not easy-to-use chemical method of most of amino acid whose racemic modifications splits, and splits simpler effective with enzyme process.Although at present solvent is to enzymic activity with stereoselectively influence rule and mechanism is still not fully aware of, but a large amount of experimental results show, by changing activity and the selectivity that solvent can regulatory enzyme, change zymologic properties such as the dynamics of enzyme and stability, can significantly improve the catalytic activity and the stereoselectivity of enzyme by the change of solvent.In the catalytic chiral separation reaction of proteolytic enzyme, especially in dissimilar organic solvents, the molecular structure degree of rigidity difference of organic solvent tolerant protease, the stereoselectivity degree all has evident difference.
Proteolytic enzyme in many weak points, is competed strong in the catalytic Chirality Reaction of aqueous phase as hydrolytic side reactions; Enzyme dosage is more relatively, and product easily covers around the enzyme, causes diffusional limitation, therefore is restricted on using.The selectivity of enzyme and the specific inductivity of solvent and moment of dipole have better dependency, and enzyme is compared its intramolecularly and had stronger noncovalent interaction power in non-aqueous media with in the water, thereby the structure rigidity of height is arranged.The catalytic chiral separation reaction of proteolytic enzyme in organic solvent, advantages such as it is simple that especially the enzyme process in the two-phase system splits product specific rotation height, by product is few, product separation is purified, and control the hard and soft degree of the reactive site of enzyme molecule by changing organic solvent, improve enantio-selectivity.
In research to proteolytic enzyme, people trend towards using protein engineering proteolytic enzyme are transformed, as improve the thermostability, oxidation-resistance etc. of proteolytic enzyme, or improve in the correlative studys such as stability of proteolytic enzyme to organic solvent (DMF, DMSO) by the orthogenesis technology of enzyme.Chen (1993) and You (1996) adopt the fallibility round pcr respectively subtilisin E to be improved 170 times and 500 times to the stability of DMF; Zhao (1999) adopts fallibility PCR and DNA shuffling technology to improve the vigor of proteolytic enzyme E under differing temps, and the Tm value is improved 17 ℃; Miyazaki (1999) employing fallibility PCR and cassette mutagenesis technology improve 100 times (Appl.Microbiol Biotechnol, 59:15～32) with the thermostability of subtilisin S41.
Even if protein engineering can generally use current, also have many problems still unresolved, as adopt the SDM technology that subtilisin BPN2 has been carried out 5 kinds of sudden changes, attempt by producing intramolecular disulfide linkage (26-232,29-119,36-210,41-80 and 148-243) improve the stability of enzyme; These 5 kinds of mutant all can produce disulfide linkage at specific site behind the B.subtilis secreting, expressing, and these mutant that produce disulfide linkage fail to improve the stability of enzyme; On the contrary, there is not the wild-type subtilisin BPN2 of disulfide linkage but to show higher stability (Biochem28:4807～4815).
Solvent stability proteolytic enzyme is a class novel protein enzyme of discovered in recent years, is produced by the organic solvent-resistant extreme microorganism usually.Although 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 extreme microorganism of organic solvent-resistant can be survived in being rich in the environment of solvent, therefore, the enzyme (as proteolytic enzyme, lipase, amylase etc.) that is produced by this quasi-microorganism also may have certain solvent tolerance.The relevant enzyme that screens some specific functions from extreme microorganism also appears in the newspapers repeatly.H.Ogino (Appl.Environ.Microbiol.61:4258～4262), L.P.Geok (Biochem.Eng.Journal, 13:73～77), A.Gupta (Jour.ofChromatography A, 1069:155～161) successively screened product solvent tolerance protease strain, bacterial classification all is accredited as Rhodopseudomonas (Pseudomonas) bacterial strain; And B.Ghorbel (Enzy.and Microb.Tech.32:513～518) screens strain product solvent stability protease strain, is accredited as bacillus cereus (Bacilluscereus).The optimum response pH of the solvent tolerance proteolytic enzyme of having delivered at present is 8.0～8.5, reaction system pH〉10 o'clock, proteolytic enzyme loses vigor; And this proteinoid enzyme shows that to multiple solvent (hydrophilic solvent and hydrophobic solvent) tolerance studies about about 25% (v/v) of its relevant tolerance concentration, its stability is relatively low.Present not insight clothing bacillus licheniformis produces the report of organic solvent tolerant protease.
Summary of the invention
The purpose of this invention is to provide a kind of organic solvent-resistant Sumizyme MP produce bacterium with and the organic solvent-resistant alkaline protease gene, with and the synthetic and resolution of racemic amine of catalysis peptide, amino acid whose application in organic phase, by engineered method, utilize the reorganization bio-reactor to come the cheap organic solvent-resistant Sumizyme MP of producing of industrialization that genetic resources is provided for further.
The present invention screens from samples such as greasy dirt soil sample and obtains the bacterial strain that the organic solvent-resistant Sumizyme MP is produced in a strain, classification called after Bacillus licheniformis Bacillus licheniformis YP1A, and its preserving number registration number is CCTCC No:M207021.
The present invention identifies the biological characteristic of lichens bacillus licheniformis YP1A, gramstaining observation to this bacterium shows that this bacterial strain is the G+ bacillus, gemma is arranged, adopt transmission electron microscope observing to show that this bacterial strain has peritrichous, size is 0.5 μ m * 2～3 μ m.In broth culture the growth 14h after, the bacterium colony size is 1.5～2mm, growth temperature range is 25～42 ℃, and optimum temperuture is 35 ℃, and growth pH is 7～12, optimal pH is 8.5, its physio-biochemical characteristics show that to the catalase reaction, citric acid, D-wood sugar and N.F,USP MANNITOL utilize the result positive, D-pectinose, α-D-lactose and D-galactose utilization result are negative, grow under aerobic conditions.
The present invention identifies through BIOLOG automatic bacterial assessing instrument Bacillus licheniformis YPlA, show that this bacterial strain and Bacillus licheniformis Bacillus licheniformis similarity (SIM) are 0.62, showing that through the 16SrDNA sequential analysis being higher than 98% bacterial strain with this sequence similarity degree is Bacillus and belongs to bacterial strain, is 99% with bacterial strain BacilluslicheniformisATCC 14580 similarities wherein.
The present invention is directed to this bacterial strain and carried out the solvent tolerance experiment, invention bacterial strain YPlA can grow in the substratum that contains the different concns organic solvent.
The present invention has carried out purifying to this organic solvent tolerant protease YPlA, and its specific activity is up to 117826.2U/mg.
The present invention has carried out the mensuration of zymologic property to this organic solvent tolerant protease YP1A, prove that this organic solvent tolerant protease YP1A has good tolerability to multiple solvent (hydrophilic solvent and hydrophobic solvent), tolerance concentration reaches 50% (v/v), and in the high density solvent, have satisfactory stability, be better than the relevant tolerance concentration 25% (v/v) of existing organic solvent-resistant Sumizyme MP; And the optimal pH of this organic solvent tolerant protease is 9.5, useful effect pH value broad, be 7～12, compare with the document of having reported and to have tangible alkaline-resisting speciality, and its optimal reaction temperature is 60 ℃, has good thermostability, handles 1 hour for 60 ℃, its enzymic activity also maintains about 90%, and is very stable.
The present invention is isolated and cloned into the encoding gene that this bacterial strain produces proteolytic enzyme, and it has the nucleotide sequence shown in the SEQ ID NO:1, for this reason the transformation of gene and efficiently express the genetic material that provides good in various heterologous gene expression systems.By PCR method separating clone this organic solvent tolerant protease gene OSTBpr, the DNA complete sequence analysis is the result show, these organic solvent tolerant protease full length gene 1140 Nucleotide, 379 amino acid of encoding, with Bacillus licheniformisATCC14580 basic protein enzyme coding gene homology be 96.3%, the prlmary structure of protein homology is 98.4%.Its aminoacid sequence is shown in SEQ ID NO:2.
The invention provides the application of organic solvent tolerant protease YP1A in organic phase catalysis peptide is synthetic.The YP1A proteolytic enzyme successful Application that purifying is obtained is in the reaction of organic synthesis peptide, and the result shows that under the solvent strength condition of 50% (v/v), DMSO is an optimum solvent, under this condition, and Cbz-Arg-Leu-NH
2The production rate of Cbz-Arg is up to 78% relatively.
The present invention also provides organic solvent tolerant protease YP1A resolution of racemic amine, amino acid whose application in organic phase.
In the chiral separation of the YP1A proteolytic enzyme successful Application that purifying is obtained racemic amines in the organic phase, the result shows that YP1A proteolytic enzyme has higher activity and stereoselectivity during the catalysis resolution reaction in organic solvent, and the e.e value of the amine that splits can reach about 98%.
The YP1A proteolytic enzyme successful Application that purifying is obtained is in the organic phase in the amino acid whose chiral separation, particularly the fractionation test-results of methionine(Met), phenylalanine, L-Ala shows that YP1A proteolytic enzyme has higher activity and stereoselectivity during the catalysis resolution reaction in organic solvent, and the e.e value of the chiral amino acid that splits can reach 95～98.9%.
Beneficial effect of the present invention is that the organic solvent-resistant Sumizyme MP that provided produces advantages such as bacterium Bacilluslicheniformis YP1A and organic solvent-resistant alkaline protease gene thereof have proved that this organic solvent-resistant Sumizyme MP has the specific activity height, solvent tolerance is strong, action pH haves a wide reach, high temperature resistant, strong basicity resisting condition.This organic solvent-resistant alkaline protease gene is the successful Application in synthetic and resolution of racemic amine, the amino acid at organic phase catalysis peptide, proved that further Bacillus licheniformis YP1A can tolerate certain density multiple organic solvent, its this proteolytic enzyme is huge at the application potential that organic phase catalysis peptide synthesizes industries such as reaching pharmacy, wool spinning, washing, leather processing future.
Description of drawings
The transmission electron microscope photo (5000x) of Fig. 1 YP1 thalline
The solvent tolerance of Fig. 2 bacterial strain YP1A
The SDS-PAGE of the organic solvent tolerant protease behind Fig. 3 purifying analyzes
The solvent tolerance of Fig. 4 organic solvent tolerant protease
The optimal pH of Fig. 5 organic solvent tolerant protease
The optimal reactive temperature of Fig. 6 organic solvent tolerant protease
The microbial preservation date of the present invention is on March 6th, 2007, and depositary institution's full name is Chinese typical culture collection center, is called for short CCTCC, deposit number: CCTCC No:M 207021.
Embodiment
Embodiment one
This description of test produces the screening procedure of the natural bacterial strain of organic solvent tolerant protease.
Adopting different concns hexanaphthene, toluene, acetone and other organic solvent is that selective pressure is screened acquisition organic solvent-resistant extreme microorganism from samples such as greasy dirt soil sample.Adopt the SMA substratum, concrete prescription is: skim-milk 12g/L, and nutrient agar medium 13.8g/L, inoculation organic solvent-resistant extreme microorganism according to the ratio of bacterium colony and transparent circle size, obtains to produce the bacterial strain of high vigor proteolytic enzyme at the SMA flat board.This method screening obtains having organic solvent-resistant extreme microorganism 8 strains of higher proteinase activity.
In order further to detect the solvent tolerance of secreted proteolytic enzyme, the organic solvent-resistant character of the white enzyme activity of laying eggs of 8 strain bacterium and the proteolytic enzyme that produces has been carried out complete detection.The generation bacterium that will have high vigor proteolytic enzyme is inoculated into and produces the enzymic fermentation substratum, and concrete prescription is: glucose 5g/L, corn steep liquor 10mL/L, peptone 5g/L, KH
2PO
41g/L, MgSO
40.5g/L pH 7.5.Culture temperature is 30 ℃, and incubation time is 36h, shaking speed 180rpm.Fermentation ends, the centrifugal 10min of 8000rpm gets supernatant; The 1mL supernatant is added isopyknic dimethyl formamide (DMF), dimethyl sulfoxide (DMSO) (DMSO), hexanaphthene, octane, toluene, acetone, butanols respectively, and in 30 ℃, 180rpm oscillation treatment 30min is a substrate with the casein, detects proteinase activity; Getting the bacterial strain with high vigor is solvent stability protease-producing bacterium.
The proteinase activity detection method is: be substrate with the casein.Get fermented liquid supernatant 1mL and join 3mL substrate (casein O.6%, 0.1M Tris-HCl damping fluid, pH9.0), place 40 ℃ of reaction 10min down, add 3.2mLTCA mixed solution (0.11M trichoroacetic acid(TCA), 0.22M sodium-acetate, 0.33M termination reaction acetic acid), room temperature is placed 30min, adopts 10, the centrifugal 5min of 000rpm detects the filtrate light absorption value in the 280nm place.Per 1 unit (U) protease activity is defined as under corresponding conditions, and every milliliter of fermented liquid supernatant per minute catalysis produces 1ug tyrosine [U, (ug/ml.min)].Detect and the solvent stability detection by enzymic activity, wherein a strain has the protease-producing bacterial strain vigor of tolerance up to 528 U, identify and the 16SrDNA sequential analysis through BIOLOG, show that this bacterial strain is Bacillus licheniformis YP1A, and as the material of further research.
Embodiment two
The biological property of this description of test organic solvent-resistant extreme microorganism Bacillus licheniformis YP1A.
Physiology and biochemistry character
Gramstaining observation to this bacterium shows that this bacterial strain is G
+Bacillus has gemma, adopts transmission electron microscope observing to show that this bacterial strain has peritrichous, and size is 0.5 μ m * 2～3 μ m.In broth culture the growth 14h after, the bacterium colony size is 1.5～2mm, growth temperature range is 25～42 ℃, and optimum temperuture is 35 ℃, and growth pH is 7～12, optimal pH is 8.5, its physio-biochemical characteristics show that to the catalase reaction, citric acid, D-wood sugar and N.F,USP MANNITOL utilize the result positive, D-pectinose, α-D-lactose and D-galactose utilization result are negative, grow under aerobic conditions.Part Physiology and biochemistry identification experiment feature such as the table 1 of this bacterium.
The part physio-biochemical characteristics of table 1 bacterial strain YP1
| Feature | The result | Feature | The result |
| The catalase reaction | + | The D-pectinose | - |
| Gemma | + | N.F,USP MANNITOL | + |
| Citric acid | + | α-D-lactose | - |
| The D-wood sugar | + | The D-semi-lactosi | - |
The solvent tolerance experiment of bacterial strain
With the overnight culture is seed liquor, inoculum size inoculating strain with 2% is to the MLB substratum, and 12 kinds of organic solvents (Fig. 2) of adding different concns, every 500mL triangular flask liquid amount is 30mL, seals with rubber plug, place 30 ℃, rotating speed 180rpm cultivates 24h, and the optical density value (OD660) of detection culture as shown in Figure 2, bacterial strain YP1A can grow in the substratum that contains the different concns organic solvent, wherein logP
O/WValue is higher than 3.0 organic solvent such as hexanaphthene (3.2), heptane (4.0), decane alkane solvents such as (5.6) is less relatively to the growth effect of YP1A; And logP
O/WSolvent such as the octanol (2.9), benzene (2.0), toluene (2.5) equal solvent of value between 3.0～2.0 has remarkable restraining effect to the growth of YP1A; LogP
O/WValue is lower than the growth that 2.0 solvent such as acetone (0.23), butanols (0.8) (2%) under low concentration just can suppress YP1A; This bacterial strain can also better growth in containing 10% hydrophilic solvent DMSO and DMF.
Embodiment three
The purifying procedure of this description of test organic solvent tolerant protease.
At first carry out the ammonium sulfate precipitation of organic solvent tolerant protease, bacterial strain is after shaking table is cultivated 48h in producing the enzyme substratum, and clear enzyme solution on the centrifuging and taking places ice bath with crude enzyme liquid, and it is saturated slowly to add ammonium sulfate to 80% while stirring, 4 ℃ of following standing over night.Then 13, the centrifugal 30min of 000rpm is added to the enzyme liquid of 80% ammonium sulfate precipitation gained with containing 1mol (NH
4)
2SO
4, the Phenyl sepharose CL-4B chromatography column that 50mmol/L Glycine-NaOH buffer A (pH9.0) balance is crossed adopts A, and the B buffer mixture carries out stepwise elution, and (buffer B is 50mmol/L Glycine-NaOH, pH9.0).Proteinase activity appears in the component of collecting at the 50%B place.Adopt SP Sepharose
TmFF strong cation exchange chromatography, selecting column-loading buffer pH is 8.6, the enzyme liquid that obtains after the dialysis of hydrophobic chromatography active ingredient is added on the SP strong cat ion exchange column of crossing with the 50mmol/LGlycine-NaOH damping fluid balance of pH8.6, carries out gradient elution with the same buffer that contains 0 → 1MNaCl.Fermented liquid supernatant crude protein enzyme is through above three steps, and it is pure to have reached electrophoresis by SDS-PAGE (Fig. 3) analysis revealed purifying protein.The molecular weight of this protein protomer is about 32kDa.Each rate of recovery and purifying multiple that goes on foot purifying sees Table 2, and total purifying multiple is 37.2 after three steps separated, and the rate of recovery is 20.8%, and final proteolytic enzyme reaches 117826.2 U/mg than living.
Purification step and the result of table 2 organic solvent tolerant protease YP1A
| Total activity (U) | Total protein (mg) | Than vigor (U/mg) | Yield (%) | The purifying multiple | |
| Crude enzyme liquid | 167713.0 | 41.5 | 3165.8 | 100 | 1 |
| Ammonium sulfate precipitation | 132943.0 | 31.3 | 4243.9 | 79.3 | 1.3 |
| Phenly sepharose FF | 63831.3 | 1.1 | 58830.6 | 38.1 | 18.6 |
| SP Sepharose Tm FF | 34930.5 | 0.3 | 117826.2 | 20.8 | 37.2 |
Annotate: protein concn adopts the Coomassie brilliant blue method to measure
Embodiment four
The measuring method of the zymologic property of this description of test organic solvent tolerant protease YP1A.
The solvent tolerance of organic solvent tolerant protease detects:
Proteolytic enzyme enzyme liquid or the trypsinase diluent of getting 0.5mL YP1A purifying and dilution add 11 kinds of equal-volume organic solvents (as Fig. 4) respectively and place the sealing test tube, to add equal-volume 0.1M Tris-HCl damping fluid (pH9.0) is contrast, in 40 ℃, the 180rpm 1h that vibrates; Detect proteinase activity, the result as shown in Figure 3.The relative trypsinase of organic solvent tolerant protease YP1A has good solvent tolerance, in 11 kinds of organic solvents having studied, only have isopyknic acetone, butanols after handling this proteinase activity to be had certain influence, proteinase activity keeps 78% and 86% respectively; And decane and dodecanol can promote proteinase activity, reach 108% and 121% respectively; Vigor kept 99% and 97% respectively after DMF and DMSO handled proteolytic enzyme; All the other organic solvents are handled the back proteinase activity and are changed not remarkable.And trypsinase vigor after the acetone under the same concentrations, butanols are handled only keeps 31% and 3%; Vigor keeps 51% and 77% respectively after DMF and DMSO processing.The YP1A crude enzyme liquid is mixed with isopyknic DMF, in 40 ℃, detect enzyme behind the 180rpm oscillation treatment different time and live, the result as shown in Figure 4.The YP1A crude enzyme liquid is after adding isopyknic solvent DMF, and stability shows that far above contrast solvent DMF can significantly improve the stability of this proteolytic enzyme.
The optimal pH of organic solvent tolerant protease is measured:
Casein with different pH values is a substrate, is reference with pH9.5 reaction vigor, surveys enzyme slip-knot fruit as shown in Figure 5.Proteolytic enzyme that YP1A produces has best vigor in the pH9.5 reaction system, keep 80% of best vigor in the reaction system of pH12.0, and in the scope of pH7～12, enzymic activity all maintains more than 60% of maximum enzyme activity.
The optimal reactive temperature of organic solvent tolerant protease and the mensuration of thermostability:
Enzymatic reaction is carried out in being determined as under 0.05 M glycine-NaOH buffer system (pH9.5) and differing temps of the optimum temperuture of organic solvent tolerant protease.Temperature tolerance is determined as proteolytic enzyme and carries out enzyme activity determination again behind the processing different time under 40 ℃ down at 50 ℃, 60 ℃, 70 ℃.The optimum temperuture measurement result (Fig. 6) of enzyme shows that the proteolytic enzyme behind the purifying has higher proteinase activity when 40～60 ℃ of reactions.When temperature of reaction was 40 ℃ and 50 ℃, proteinase activity was respectively 81% and 90% of high enzymatic activity, and its optimal reactive temperature is 60 ℃.The heat stability test of enzyme shows (Fig. 6), and behind 60 ℃ of processing 60min, enzymic activity still maintains more than 90%.
Embodiment five
The separating clone program of this description of test organic solvent-resistant basic protein enzyme coding gene OSTBpr
Adopt the total DNA of phenol-chloroform method extracting thalline.According to lichens bacillus licheniformisATCC14580 and other alkaline protease gene sequence, design degenerated primer, the encoding sequence of amplification organic solvent-resistant Sumizyme MP.The PCR fragment electrophoresis that contains the maturing enzyme encoding sequence reclaims rear clone to the pMD18-T carrier, carries out sequential analysis.The degenerated primer of design is:
PF：AAAGAGTTTTTGGYTTGGGATG
PR：TTGATCAGACCTTTYCCATA
The PCR reaction parameter is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec; 55 ℃ of annealing 30sec; 72 ℃ are extended 1min; After circulation 30 is taken turns, 72 ℃ of insulation 10min.According to this reaction conditions, the PCR fragment of about 1.1kb that increased.According to measuring this proteic molecular weight, infer that the encoding gene length of this zymoprotein is no more than 0.9kb.This fragment is connected to the pMD18-T carrier, carries out sequencing.The result shows that this fragment has the reading frame of a total length 1140, contains 104 amino acid of signal peptide sequence, and the amino acid number of encoding mature proteolytic enzyme is 275.
Embodiment six
The application of this description of test organic solvent tolerant protease YP1A in organic phase catalysis peptide is synthetic.
With the Cbz-Arg of 10mM and the Leu-NH of 500mM
2Be substrate, be the organic phase solvent with DMSO, DMF, methyl alcohol, ethanol, glycerine respectively, in 50mM Tris-HCl (pH9.0) reaction buffer, add isopyknic above-mentioned solvent respectively, the reaction cumulative volume is 10mL, the YP1A proteolytic enzyme 1mg that adds purifying, under 40 ℃, 150rpm oscillatory reaction 180min, reaction solution is diluted to 1/20 with acetonitrile/50mM sodium phosphate buffer (pH3.0), adopts reverse hplc to detect product C bz-Arg-Leu-NH
2Content.The result shows that under the solvent strength condition of 50% (v/v), DMSO is an optimum solvent, under this condition, and Cbz-Arg-Leu-NH
2The production rate of Cbz-Arg reaches 78% relatively.
Embodiment 7
The application of this description of test organic solvent tolerant protease in organic phase catalysis resolution of racemic amine.
With the racemic 1-of 2mmol (1-naphthyl)-ethamine is substrate; 1.8mmol penta-obtusilic acid cyanomethyl ester is as acylating agent; be reaction solvent with methyl alcohol, ethanol, 3-methyl-3-amylalcohol respectively; add solvent volume and be 15mL; the YP1A proteolytic enzyme 60mg that adds purifying; 30 ℃ of shaking table reactions, rotating speed is controlled at 200rpm.Timing sampling dilutes and makes HPLC after centrifugal and detect penta-4-alkene acyl 1-(1-naphthyl)-ethamine.Amidated transformation efficiency is to stop in 40% o'clock.Filter; the filtrate solvent evaporated; resistates gets S type acid amides with silica gel column chromatography separating purification; elutriant is an ethyl acetate: normal hexane (V: V)=1: 10; be catalyzer again with the tetrahydrofuran (THF); add excess iodine, go acidylate to get optically active amine, promptly optically active penta-4-alkene acyl 1-(1-naphthyl)-ethamine.Automatically polarimeter is surveyed its specific rotatory power, calculates the e.e value.The result shows under the equal solvent volume, in methyl alcohol, the ethanol, penta-4-alkene acyl 1-(1-naphthyl)-ethamine transformation efficiency can't reach 40%, penta-obtusilic acid cyanomethyl ester as itself just can with amino group generation ammonolysis reaction.And in 3-methyl-3-amylalcohol, reaching at 40% o'clock at transformation efficiency, the 1-of S type (1-naphthyl)-ethamine e.e can reach 98%, is the most suitable solvent.
Embodiment 8
The application of this description of test organic solvent tolerant protease in organic phase catalysis resolution of racemic amine.
(S)-pipecoline acid is important chiral synthon, so that the racemic piperidines of 3mmol-the 2-carboxylic acid amide is a substrate, contains 5.4%H with 10mL respectively
2The acetonitrile of O and 10mL toluene are to react in the organic solvent, add lyophozyme 50mg, 30 ℃ of shaking table reactions, rotating speed 150rpm.The 20h stopped reaction, reaction solution end of a period thing is washed 3 times with isopyknic 0.5NaOH solution, separatory.Water in the reaction solution with 0.1moL/L hydrochloric acid adjust pH to 3, is obtained the acid of product pipecoline after the filtration.Sampling detects the acid of (S)-pipecoline with HPLC, calculates hydrolysis conversion.Pipecoline acid is measured (S)-pipecoline acid specific rotatory power with automatic polarimeter.Contain 5.4% H under the experimental result demonstration equivalent responses condition
2Transformation efficiency can reach 43% in the acetonitrile of O, and e.e is 87%.And when toluene was reaction solvent, transformation efficiency was 40%, and e.e can reach 98%.When a certain amount of toluene was solvent, stereoselectivity was higher.Used proteolytic enzyme and the common freeze-drying of methyl-beta-cyclodextrin of this experiment in addition compared with the lyophozyme powder of methylate-beta-cyclodextrin not, has higher activity and stereoselectivity in organic solvent during the catalysis resolution reaction.
Embodiment 9
This description of test organic solvent tolerant protease is in the application of organic phase catalysis chiral separation methionine(Met).
Methionine(Met) Met, especially L-methionine(Met) are one of eight kinds of indispensable amino acids of human body, have important physical function and medical value.N-acetyl-DL-methionine methyl esters is self-control.In the toluene and excessive 100% sodium bicarbonate at room temperature vigorous stirring is to pH=7, separation of methylbenzene concentrates mutually; With the sodium bicarbonate neutralization, toluene layer washes with water in toluene and water two-phase system, and anhydrous sodium sulfate dehydration, vacuum concentration must contain the toluene solution of N-acetyl-DL-methionine methyl esters 200g/L.With V (toluene): V (water)=2: 1,4: 1,5: 1,6: 1,8: 1, be reaction solvent at 10: 1 respectively, and water is that pH is 6.8 NaH
2PO
4-Na
2HPO
4Damping fluid.The enzyme-to-substrate mass ratio is 3: 20.37 ℃ of oscillatory reaction 10h of water-bath.Water phase separated, liquid is transferred iso-electric point, adds the dehydrated alcohol stand at low temperature, separates out crystal, and suction filtration is used the dehydrated alcohol repetitive scrubbing, the crystal oven dry, recrystallization, suction filtration, oven dry obtains L-Met.With dissolve with methanol, measure calculating e.e value down in automatic polarimeter.Methionine solution with 3mmol/L is a standard amino acid solution, and the 570nm place measures solution absorbency A, calculates L-Met content in the solution, transformation efficiency.Experimental result shows that toluene: water=transformation efficiency reaches 40%, and e.e is 95% at 6: 1 o'clock.
This description of test organic solvent tolerant protease is in the application of organic phase catalysis chiral separation phenylalanine.
The L-phenylalanine is a kind of humans and animals essential amino acid, and this experiment is adopted in the enzyme process fractionation and produced the L-phenylalanine.First with D, acetylize of L-phenylalanine and esterification, N-acetyl-D, L-phenylalanine ethyl ester are this laboratory self-control.With N-acetyl-D of 2g, the L-phenylalanine ethyl ester is a substrate.In following solvent, react respectively:
(1) mixing solutions of the cobalt chloride solution of the 1.5mmoL of the Sodium phosphate dibasic-citric acid solution of 100mLpH7.0 (Mcllvaine) and 50mL, adding adds the proteolytic enzyme behind the 0.25g purifying again, 37 ℃ of following stirring reactions, 20h stops.HPLC measure N-acetyl-D, the transformation efficiency of L-phenylalanine ethyl ester is 50%, reaction solution heating back is centrifugal, the 30mL xylene extraction surplusly is rotated evaporation or underpressure distillation mutually and gets the aqueous solution about 15mL.This aqueous solution is carried out crystallization, get 0.4g L-phenylalanine, the e.e value is 99%.
(2) proteolytic enzyme behind the 0.25g purifying is dissolved in the mixing solutions that the cobalt chloride solution of the Mcllvaine damping fluid of 100mLpH7.0 and 50mL 1.5mmoL is formed, add the 2g N-acetyl-D that is dissolved in the 50mL n-Octanol, the L-phenylalanine ethyl ester, proteolytic enzyme behind the adding 0.025g purifying, temperature of reaction is 37 ℃, oscillatory reaction 20h, standing demix, water layer reaction mixture are cooled to room temperature promptly has the L-phenylalanine to separate out.After L-phenylalanine crystal leached, the L-phenylalanine transformation efficiency that makes was 50%, and e.e reaches 98.9%.Enzyme solid property selected and activity improve a lot in n-Octanol and buffering two-phase system.
Embodiment 11
The catalysis in organic phase of this description of test organic solvent tolerant protease splits the DL-L-Ala and produces the D-L-Ala.
With 2.5gN-acetyl-DL-alanine methyl ester (self-control) is substrate, is dissolved in respectively in 10mL hexanaphthene, methyl alcohol, DMSO, the toluene.Be mixed with reaction solvent-two-phase system with the phosphoric acid buffer of 0.01mol/LpH 7.0 according to 2: 1 ratios.Add the organic solvent tolerant protease 0.2g of purifying,, then solution is taken out heating 10min, make enzyme flocculation sex change, remove by filter the enzyme of sex change in 37 ℃ of constant temperature oscillatory reaction 10h.Filtrate decompression distillation is concentrated, transfer pH to 6.0, drip the 100mL dehydrated alcohol, refrigeration is spent the night, filter, small amount of solid, get the pure product of D-Ala through vacuum-drying.Wherein toluene is the organic solvent phase, aqueous phase separation be refiltered.Experimental result shows that productive rate was minimum when methyl alcohol was organic phase, and when toluene was organic solvent, productive rate and enantioselectivity are the highest to be 98% at automatic polarimeter mensuration D-L-Ala e.e, gets D-L-Ala 0.55g.
Sequence table
＜110〉Nanjing University of Technology
＜120〉a kind of organic solvent-resistant Sumizyme MP produces the gene and the application of bacterium and this organic solvent-resistant Sumizyme MP
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<213>Artificial
<220>
<223>PR
<400>4
ttgatcagac ctttyccata 20
Claims (6)
1. an organic solvent-resistant Sumizyme MP produces bacterium, the new bacterial strain that its classification called after Bacillus licheniformis belongs to, and called after Bacillus licheniformis YPlA, its preserving number registration number is CCTCC No:M 207021.
2. organic solvent-resistant Sumizyme MP according to claim 1 produces bacterium, it is characterized in that it produces the organic solvent-resistant Sumizyme MP and has the aminoacid sequence shown in the SEQ ID NO:2.
3. the encoding gene of organic solvent-resistant Sumizyme MP according to claim 2, it has the nucleotide sequence shown in the SEQID NO:1.
4. organic solvent-resistant Sumizyme MP according to claim 1 produces the application of organic solvent-resistant Sumizyme MP in organic phase peptide synthetic system that bacterium produces.
5. organic solvent-resistant Sumizyme MP according to claim 1 produces the application of organic solvent-resistant Sumizyme MP resolution of racemic amine in organic phase of bacterium generation.
6. the organic solvent-resistant Sumizyme MP that organic solvent-resistant Sumizyme MP according to claim 1 produces the bacterium generation splits amino acid whose application in organic phase.
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| CNA2007101919691A CN101215534A (en) | 2007-12-28 | 2007-12-28 | Organic solvent-resistant alkaline protease producing strain, gene of organic solvent-resistant alkaline protease and application of organic solvent-resistant alkaline protease |
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| CNA2007101919691A CN101215534A (en) | 2007-12-28 | 2007-12-28 | Organic solvent-resistant alkaline protease producing strain, gene of organic solvent-resistant alkaline protease and application of organic solvent-resistant alkaline protease |
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| CN101215534A true CN101215534A (en) | 2008-07-09 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703482A (en) * | 2011-08-01 | 2012-10-03 | 南京工业大学 | Organic solvent-resistant alkaline protease |
| CN103275958A (en) * | 2011-08-01 | 2013-09-04 | 南京工业大学 | Organic solvent-resistant alkaline protease |
| CN107384897A (en) * | 2017-08-02 | 2017-11-24 | 北京科为博生物科技有限公司 | A kind of alkali protease and its gene and application |
| CN108384771A (en) * | 2018-02-06 | 2018-08-10 | 珠海市双指环投资有限公司 | A kind of alkali protease mutation body and its encoding gene improving Rate activity |
| CN114540330A (en) * | 2022-04-21 | 2022-05-27 | 深圳润康生态环境股份有限公司 | Alkaline protease mutant AprBpM and application thereof |
-
2007
- 2007-12-28 CN CNA2007101919691A patent/CN101215534A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703482A (en) * | 2011-08-01 | 2012-10-03 | 南京工业大学 | Organic solvent-resistant alkaline protease |
| CN103275958A (en) * | 2011-08-01 | 2013-09-04 | 南京工业大学 | Organic solvent-resistant alkaline protease |
| CN103275958B (en) * | 2011-08-01 | 2014-07-16 | 南京工业大学 | Organic solvent-resistant alkaline protease |
| CN102703482B (en) * | 2011-08-01 | 2014-08-06 | 南京工业大学 | Organic solvent-resistant alkaline protease |
| CN107384897A (en) * | 2017-08-02 | 2017-11-24 | 北京科为博生物科技有限公司 | A kind of alkali protease and its gene and application |
| CN107384897B (en) * | 2017-08-02 | 2020-10-23 | 北京科为博生物科技有限公司 | Alkaline protease, gene and application thereof |
| CN108384771A (en) * | 2018-02-06 | 2018-08-10 | 珠海市双指环投资有限公司 | A kind of alkali protease mutation body and its encoding gene improving Rate activity |
| CN108384771B (en) * | 2018-02-06 | 2020-06-09 | 珠海市双指环投资有限公司 | Alkaline protease mutant for improving specific activity and coding gene thereof |
| CN114540330A (en) * | 2022-04-21 | 2022-05-27 | 深圳润康生态环境股份有限公司 | Alkaline protease mutant AprBpM and application thereof |
| CN114540330B (en) * | 2022-04-21 | 2022-07-12 | 深圳润康生态环境股份有限公司 | Alkaline protease mutant AprBpM and application thereof |
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Open date: 20080709 |