CN103275958A - Organic solvent-resistant alkaline protease - Google Patents
Organic solvent-resistant alkaline protease Download PDFInfo
- Publication number
- CN103275958A CN103275958A CN2013102438001A CN201310243800A CN103275958A CN 103275958 A CN103275958 A CN 103275958A CN 2013102438001 A CN2013102438001 A CN 2013102438001A CN 201310243800 A CN201310243800 A CN 201310243800A CN 103275958 A CN103275958 A CN 103275958A
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- CN
- China
- Prior art keywords
- ala
- val
- gly
- ser
- lys
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- 239000003960 organic solvent Substances 0.000 title claims abstract description 66
- 108091005658 Basic proteases Proteins 0.000 title abstract description 12
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 27
- 108091005804 Peptidases Proteins 0.000 abstract description 20
- 150000001413 amino acids Chemical class 0.000 abstract description 14
- 235000001014 amino acid Nutrition 0.000 abstract description 13
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 abstract description 5
- 235000018102 proteins Nutrition 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 239000004365 Protease Substances 0.000 abstract description 4
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 abstract description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 abstract description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 abstract description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract 1
- 239000004472 Lysine Substances 0.000 abstract 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 235000004279 alanine Nutrition 0.000 abstract 1
- 235000013922 glutamic acid Nutrition 0.000 abstract 1
- 239000004220 glutamic acid Substances 0.000 abstract 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 abstract 1
- 238000002741 site-directed mutagenesis Methods 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 32
- 108090000790 Enzymes Proteins 0.000 description 32
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 24
- 230000008859 change Effects 0.000 description 24
- 102000035195 Peptidases Human genes 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 16
- 241000282326 Felis catus Species 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 229940024606 amino acid Drugs 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 230000003321 amplification Effects 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- KFQDSSNYWKZFOO-LSJOCFKGSA-N His-Val-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O KFQDSSNYWKZFOO-LSJOCFKGSA-N 0.000 description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 8
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 8
- 108010047495 alanylglycine Proteins 0.000 description 8
- 108010050848 glycylleucine Proteins 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 6
- 108010079364 N-glycylalanine Proteins 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 6
- 239000003471 mutagenic agent Substances 0.000 description 6
- 238000000137 annealing Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
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- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 4
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the technical field of protease gene engineering, particularly relates to an organic solvent resistant protease, and particularly relates to a mutant gene and a protein coded by the mutant gene. From SEQ ID NO: 1 or SEQ ID NO: 3, the organic solvent-resistant alkaline protease of the invention has site-directed mutagenesis at 385 th or 436 th of the prokaryotic nucleotide sequence, namely, the 24 th mutated amino acid of the mature peptide segment is glutamic acid or glutamine; the amino acid at position 41 is alanine or lysine. The tolerance of the mutated four encoded proteins to organic solvents, particularly hydrophilic organic solvents, is obviously greatly enhanced.
Description
The application is to be on 08 01st, 2011 the applying date, and application number is 201110217989.8, and invention and created name is divided an application for " a kind of organic solvent-resistant Sumizyme MP ".
Technical field
The invention belongs to the proteinase gene field of engineering technology, be specifically related to a kind of organic solvent tolerant protease, be specifically related to its mutator gene and encoded protein matter thereof.
Technical background
Proteolytic enzyme refers to the class of enzymes of energy catalysis peptide bond hydrolysis, has been tested for since the added ingredients of washing composition from 1914, owing to having the important commercial purposes and by extensive concern.The output of current proteolytic enzyme occupies more than 40% of enzyme market, fields such as widespread use and washing composition, food, pharmacy, process hides, diagnostic reagent, sewage disposal.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.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.
Albumen the most general enzymatic reaction is the peptide bond in the protein hydrolysate.Generally, the reaction of proteolytic enzyme catalytic hydrolysis peptide bond is to carry out in the damping fluid of specific pH, and proteolytic enzyme has the specificity requirement to the amino-acid residue of its catalysis peptide bond, and regioselectivity and the stereoselectivity of height namely arranged.Along with the development of enzyme engineering and solvent engineering, it is found that the reaction that proteolytic enzyme can not carry out in some aqueous solution of energy catalysis in organic solvent in organic solvent.As, 1984, people such as Klibanov found that proteolytic enzyme preserves the long period and still have activity in some organic solvents, and can catalyze and synthesize the reaction of peptide bond.Studies show that in a large number carrying out enzymatic reaction in the organic solvent has many advantages: 1, increase various organic substrates in solvent, particularly the solubleness in hydrophilic organic solvent improves reaction efficiency; 2, have stereoselectivity and the regioselectivity of height, 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.This discovery has promoted the rise of non-water zymetology.Utilize the organic solvent tolerant protease that screens voluntarily in hydrophilic organic solvent dimethyl sulfoxide (DMSO) (DMSO), to realize the precursor of sweeting agent Aspartame and synthesizing of interior morphine dai 1 as this research department, productive rate reaches more than 90%, and realizing the Reaction Separation coupling of substrate and product, improve productive rate greatly, simplified the separation and purification of product.Enzymic catalytic reaction is carried out in explanation in the hydrophilic organic solvent system market outlook are very huge.
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, common method: 1, use hydrophobic organic solvent as the water content in reaction medium and the control agent, to guarantee " necessary water " of enzyme; 2, in reaction medium, add freezing drying protective agents such as protective material glycerine, ethylene glycol, polyhydric alcohol polymer etc., or adding cyclodextrin, to improve the stability of enzyme molecule in organic medium.3, utilize physics and chemistry modifying method such as immobilization, entrapping method, macromole modification to improve enzymes active and stable in organic medium; 4, utilize genetic modification to improve the organic solvent stability of enzyme.For example, Arnold group utilizes fallibility PCR directional transformation subtilisin (Subtilisin), improve its vigor in organic phase, dna fragmentation to this proteolytic enzyme mature peptide of encoding suddenlys change, and screening obtains the obviously mutant strain of raising of in high density dimethyl formamide (DMF) enzymic activity, and wherein the enzyme activity of mutant PC3 in 60% and 85% DMF is respectively 256 and 131 times of natural enzyme.After this suddenly change taller 3 times than PC3 of the mutant 13M enzyme activities that obtains again on the basis of PC3.
Though obtaining certain progress aspect the organic solvent stability of utilizing genetic modification raising enzyme in recent years, but the organic solvent tolerance of the enzyme that often sets out is relatively low, organic solvent patience, particularly the amplitude that improves in hydrophilic organic solvent is little, and industrial application is also had a certain distance.
Summary of the invention
Technical purpose of the present invention is to provide a kind of mutator gene of high organic solvent-resistant Sumizyme MP, make this proteolytic enzyme of the present invention for the similar proteolytic enzyme of prior art, the tolerance of its organic solvent, particularly hydrophilic organic solvent further improves greatly.
In order to realize technical purpose of the present invention, technical program of the present invention lies in:
One, a kind of organic solvent-resistant Sumizyme MP is characterized in that it has the nucleotide sequence shown in the SEQ ID NO:1, and its aminoacid sequence is shown in SEQ ID NO:2; Perhaps it has the nucleotide sequence shown in the SEQ ID NO:3, and its aminoacid sequence is shown in SEQ ID NO:4.
The original protein enzyme gene that sets out of organic solvent-resistant alkaline protease gene of the present invention comes from the described organic solvent-resistant alkaline protease gene of Chinese patent application (patent application publication number CN101215534A), and its nucleotide sequence is shown in sequence table SEQ ID NO:5 of the present invention.From the SEQ ID NO:1 of sequence table or SEQ ID NO:3 as seen, rite-directed mutagenesis has taken place at the 385th or the 436th of former nucleotide sequence in organic solvent-resistant Sumizyme MP of the present invention, and namely the 24th of its mature peptide section the mutating acid is L-glutamic acid or glutamine; The 41st amino acids is L-Ala or Methionin.The tolerance of the organic solvent, particularly hydrophilic organic solvent of the protein behind four kinds of codings after the sudden change is obviously strengthened greatly.
Two, the clone of organic solvent-resistant alkaline protease gene of the present invention and preparation method comprise the steps.
(1) selects for use described in the patent application publication number CN101215534A
Bacillus licheniformiYP1A(CCTCC NO:M207021) the cloning process clones coding organic solvent-resistant alkaline protease gene of organic solvent-resistant basic protein enzyme coding gene, basis simultaneously
Bacillus subtilisThe gene order of p43 promotor in 168 (GenBank number: K02714) design primer amplification p43 promotor, method with overlapping PCR is connected promotor p43 with above-mentioned organic solvent-resistant alkaline protease gene, be so kind as to give by professor Li Shunpeng of Agricultural University Of Nanjing with expression vector pHY300(at last) be connected, transforming subtilis host WB800(is so kind as to give by professor Li Shunpeng of Agricultural University Of Nanjing), build reorganization bacterium WB800-pHY300-p43-YP1A and express.
(2) select for use the genetic engineering bacterium WB800-pHY300-p43-YP1A that builds in 37 ℃ of shaking table overnight incubation, extract plasmid as the template of orthogenesis.
(3) according to the principle of fallibility rolling-circle replication, design six aggressiveness primer: 5 '-NpNpNpNpsNpsN-3 ' of 3 ' terminal thio-modification.
(4) be template with the plasmid DNA that obtains, carry out the amplification of fallibility rolling-circle replication with Φ 29 DNA polysaccharases (Fermentas #EP0097), program is as follows: 95 ℃ of sex change 3min; Put cooled on ice rapidly to room temperature; 30 ℃ of reaction 24 h.
(5) reaction product is carried out glue and is reclaimed concentrated with Dpn I enzyme (Fermentas #ER1701) digestion 1 h.
(6) glue reclaims product and transforms Bacillus subtilus WB800, and coating milk flat board carries out primary dcreening operation.
(7) select have the mutant strain of transparent circle to carry out shake-flask culture 7 days, the centrifuging and taking supernatant liquor is crude enzyme liquid, carry out organic solvent tolerance and sieve again, find that the mutant strain of called after K8 and P8 is at 50%(v/v) DMF in enzyme activity be respectively 2.29 and 1.88 times of protoenzyme; And the mutant strain of called after Z8 and Z25 is at 50%(v/v) DMF in enzyme activity be respectively 1.76 and 1.8 times of protoenzyme, as table two.
(8) main character of the described mutant strain of step (7) is studied, found four kinds of mutant optimal reaction pH values, considerable change does not all take place in the pH value stabilization; The optimal reactive temperature of three kinds of mutant P8, Z8, Z25 and temperature stability do not have significantly yet and change; And the optimal reactive temperature of K8 and temperature stability have all increased; The stability of mutant strain in various organic solvents all increases significantly.Nucleotide and amino acid analysis find that mutator gene is compared with the YP1A gene, respectively 385 bp(K8/P8 after the initiator codon) and 436 bp(Z8/Z25) sudden change has taken place, sport CAG and GAG from GCG respectively at the 385th; Amino acid sports Gln and Glu from Ala respectively; Sport GCA and AAG from GAC; Amino acid sports Ala and Lys from Asp.
Three, organic solvent-resistant Sumizyme MP of the present invention catalyzes and synthesizes the application in the little peptide in organic solvent.
Beneficial effect of the present invention is:
(1) the present invention is from natural organic solvent-resistant Sumizyme MP, means by orthogenesis are to its genetic modification, four various organic solvents have been obtained, the stronger mutator gene of hydrophilic organic solvent tolerance particularly, mutant strain is at 50%(v/v) enzyme work in the hydrophilic organic solvent is alive higher more than 1.5 times than protoenzyme, the enzyme work in acetone and acetonitrile solvent be protoenzyme live more than 30 times.Not seeing at present has relevant report.
(2) open reading frame of YP1A gene of the present invention contains 1140bp, 379 amino acid of encoding.Nucleotide and amino acid analysis find that mutator gene is compared with the YP1A gene, respectively 385 bp(K8/P8 after the initiator codon) and 436 bp(Z8/Z25) sudden change taken place; From the SEQ ID NO:1 of sequence table or SEQ ID NO:3 as seen, rite-directed mutagenesis has taken place at the 385th or the 436th of former nucleotide sequence in organic solvent-resistant Sumizyme MP of the present invention, and namely the 24th of its mature peptide section the mutating acid is L-glutamic acid or glutamine; The 41st amino acids is L-Ala or Methionin.The organic solvent, particularly hydrophilic organic solvent tolerance of the protein behind four kinds of codings after the sudden change obviously strengthened greatly.
(3) organic solvent-resistant Sumizyme MP of the present invention can be applied to catalyze and synthesize in organic solvent in the little peptide, the organic solvent of its superelevation, particularly hydrophilic organic solvent tolerance are applicable to the synthetic field of little peptide more for the organic solvent tolerant protease of prior art.
Description of drawings
Fig. 1 shows that fallibility rolling-circle replication electrophorogram and mutant strain do the preliminary screening on the milk flat board.
Fig. 2 shows mutant strain optimal reaction pH value.
Fig. 3 shows mutant strain pH value stabilization.
Fig. 4 shows the mutant strain optimal reactive temperature.
Fig. 5 shows the mutant strain temperature stability.
Fig. 6 and 7 shows the stability of mutant strain in DMF/DMSO.
Embodiment
The present invention is described in further detail below in conjunction with specific examples.
Embodiment 1
Present embodiment illustrates the orthogenesis of organic solvent-resistant alkaline protease gene of the present invention.
Select for use described in the patent application publication number CN101215534A
Bacillus licheniformiYP1A(CCTCC NO:M207021) the cloning process clones coding organic solvent-resistant alkaline protease gene of organic solvent-resistant basic protein enzyme coding gene, simultaneously according to the gene order of p43 promotor among the Bacillus subtilis 168 (GenBank number: K02714) design primer amplification p43 promotor, method with overlapping PCR is connected promotor p43 with the organic solvent-resistant alkaline protease gene, be so kind as to give by professor Li Shunpeng of Agricultural University Of Nanjing with expression vector pHY300(at last) be connected, transforming subtilis host WB800(is so kind as to give by professor Li Shunpeng of Agricultural University Of Nanjing), build reorganization bacterium WB800-pHY300-p43-YP1A and express, specific implementation method is as follows:
(1) design amplification YP1A gene and promotor p43 primer.
The YP1A primer:
YP1AF:5-GAGAGGAATGTACACATGATGAGGAAAAAG-3；
YP1AR:5-CGGATCCTTATTGAGCGGCAGCTTC-3。
Promoter primer:
p43F:5-GCAGATCTTGATAGGTGGTATGTTTTCGCT-3；
p43R:5-CTCTTTTTCCTCATCATGTGTACATTCCTC-3。
(2) respectively by following program amplification YP1A gene and promotor p43.
1. the amplification of YP1A gene: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30 sec; 55 ℃ of annealing 30 sec; 72 ℃
Extend 1 min; After 30 circulations, 72 ℃ of insulation 10 min, according to this reaction conditions, the PCR fragment of about 1.1 kb has been arrived in amplification.
2. the amplification of promotor p43: 94 ℃ of pre-sex change 5 min; 94 ℃ of sex change 30 sec; 50 ℃ of annealing 30 sec; 72 ℃
Extend 30 sec; After 30 circulations, 72 ℃ of insulation 10 min, according to this reaction conditions, the PCR fragment of about 300 bp has been arrived in amplification.
3. YP1A-p43 amplification: the method by overlapping PCR connects YP1A gene and promotor p43,94 ℃ of pre-sex change 5 min; 94 ℃ of sex change 30 sec; 65 ℃ of annealing 30 sec(annealing temperatures design gradients descend 1 ℃); 72 ℃ are extended 2 min; After 19 circulations; 94 ℃ of sex change 30 sec; 45 ℃ of annealing 30 sec; 72 ℃ are extended 2 min; After 16 circulations, 72 ℃ of insulation 10 min.
4. overlapping PCR product being carried out carrying out enzyme with restriction enzyme Bgl II (the precious biotech firm in Dalian) with BamH I (the precious biotech firm in Dalian) behind the purifying cuts, with same enzyme carrier pHY300 being carried out enzyme simultaneously cuts, purifying enzyme is cut product with the precious biotech firm in T4(Dalian) ligase enzyme connects down at 16 ℃ and spends the night, transform subtilis WB800 and express.
Select for use the genetic engineering bacterium WB800-pHY300-p43-YP1A that builds in 37 ℃ of shaking table overnight incubation, extract plasmid as the sudden change template; According to the principle of fallibility rolling-circle replication, six aggressiveness primer: 5 '-NpNpNpNpsNpsN-3 ' of design 3 ' terminal thio-modification; Suddenly change according to following program:
(1) 50 μ L reaction system: Tris-HCl(pH7.5) final concentration 50 mM; Ammonium sulfate final concentration 10mM, MgCl
2 Final concentration 10 mM, DTT final concentration 4 mM, BSA final concentration 200 ng/ μ L, dNTP0.2 mM, primer six aggressiveness 4 μ M, plasmid DNA 40 pM, MnCl
20.8 mM, Φ 29 DNA polysaccharase 5U.
(2) response procedures: above-mentioned reactive component is removed polysaccharase and MnCl
2, be added to mixing in the 500 μ L reaction tubess outward, in 95 ℃ of following sex change 3 min; Place reaction tubes cooled on ice to room temperature rapidly; Add polysaccharase and MnCl
2In 30 ℃ of reaction 24 h.
(3) reaction product adds Dpn I enzymic digestion 1 h according to certain system, carries out glue and reclaims.
(4) reclaim product and directly be transformed into Bacillus subtilus WB800 expression system, selecting on the milk flat board has the transparent circle mutant strain further to screen (as Fig. 1).
(5) select have the mutant strain of transparent circle to carry out shake-flask culture 7 days, the centrifuging and taking supernatant liquor is crude enzyme liquid, carries out the organic solvent tolerance screening according to follow procedure.
Mutant strain is inoculated in the fermention medium cultivated 7 days, centrifugal recovery supernatant liquor is the crude enzyme liquid of mutant strain, get 45%(V/V at ice bath) crude enzyme liquid of volume, adding 55%(V/V) volume hydrophilic organic solvent (DMF) is in 3 mL sealed vial, in 37 ℃, 200 rpm vibrate and measure the residual protein enzyme activity behind 1.5 h.Control group is for adding 55% volume (V/V) damping fluid.Carry out the organic solvent tolerance screening, select tolerance to change tangible mutant strain and extract the plasmid order-checking, determine the mutational site.Find that mutant strain K8 and the tolerance of P8 in 55% DMF have improved 55% and 30% respectively; And Z8 and Z25 have improved 29% and 51% respectively, as table one:
Table one screen mutation result
The present invention also studies the main character of mutant strain, finds four kinds of mutant optimal reaction pH values, and considerable change (as Fig. 2,3) does not all take place the pH value stabilization; The optimal reactive temperature of three kinds of mutant P8, Z8, Z25 and temperature stability yet and significantly do not change (as Fig. 4,5); And the optimal reactive temperature of K8 and temperature stability have all increased (as Fig. 4,5); The stability of mutant strain in various organic solvents, particularly hydrophilic organic solvent all increase significantly (as table two, Fig. 6,7).The open reading frame of YP1A gene of the present invention contains 1140bp, 379 amino acid of encoding.Nucleotide and amino acid analysis find that mutator gene is compared with the YP1A gene, respectively 385 bp(K8/P8 after the initiator codon) and 436 bp(Z8/Z25) sudden change has taken place, sport CAG and GAG from GCG respectively at the 385th; Amino acid sports Gln and Glu from Ala respectively; Sport GCA and AAG from GAC; Amino acid sports Ala and Lys from Asp.
Table two mutant strain solvent stability
Annotate: DMSO concentration is 65%(v/v), other solvent strength is 50%(v/v).
Sequence table
＜110〉Nanjing University of Technology
＜120〉a kind of organic solvent-resistant Sumizyme MP
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Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
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Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
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Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
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Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
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Xaa Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
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Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
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Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
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Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
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Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
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Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
120 125 130 135
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
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Ala Gly Asn Ser Gly Pro Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
155 160 165
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200 205 210 215
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
220 225 230
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
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Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
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tcc ttg tac gcg gtt aaa gta ctg aat tca agc gga agc gga tca tac 624
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90 95 100
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105 110 115
gtt atc aat atg agc ctt ggg gga gca tca ggc tcg aca gcg atg aaa 720
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
120 125 130 135
cag gca gtc gac aat gca tat gca aga ggg gtt gtc gtt gta gct gca 768
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
140 145 150
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Ala Gly Asn Ser Gly Pro Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
155 160 165
gcg aaa tac gat tct gtc atc gct gtt ggc gcg gta gac tct aac agc 864
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
170 175 180
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185 190 195
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Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
200 205 210 215
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Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
220 225 230
ttg atc ttg tca aaa cat ccg aac ctt tca gct tca caa gtc cgc aac 1056
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
235 240 245
cgt ctc tcc agc acg gcg act tat ttg gga agc tcc ttc tac tat ggg 1104
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
250 255 260
aaa ggt ctg atc aat gtc gaa gct gcc gct caa taa 1140
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
265 270
<210> 4
<211> 379
<212> PRT
<213> Artificial
<220>
<221> misc_feature
<222> (41)..(41)
＜223〉Xaa=Ala or Lys.
<220>
<223> Synthetic Construct
<400> 4
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Leu Met
-105 -100 -95 -90
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Leu
-85 -80 -75
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
-70 -65 -60
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
-55 -50 -45
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
-40 -35 -30
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
-25 -20 -15 -10
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
-5 -1 1 5
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
10 15 20
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
25 30 35
Pro Xaa Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
40 45 50 55
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
60 65 70
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
75 80 85
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
90 95 100
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
105 110 115
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
120 125 130 135
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
140 145 150
Ala Gly Asn Ser Gly Pro Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
155 160 165
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
170 175 180
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
185 190 195
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
200 205 210 215
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
220 225 230
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
235 240 245
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
250 255 260
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
265 270
<210> 5
<211> 1140
<212> DNA
＜213〉Bacillus licheniformis YP1A proteinase gene
<400> 5
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttaatgct cgtgttcacg 60
atggcattca gcgattccgc ttctgctgct caactggcga aaaatgttga aaaggattat 120
atcgtcggat ttaagtcagg agtgaaaacc gcatccgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcaca catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtatcct tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggaccttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggcgcgg tagactctaa cagcaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
Claims (1)
1. an organic solvent-resistant Sumizyme MP is characterized in that it has the nucleotide sequence shown in the SEQ ID NO:3, and its aminoacid sequence is shown in SEQ ID NO:4.
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| CN201110217989.8A Division CN102703482B (en) | 2011-08-01 | 2011-08-01 | Organic solvent-resistant alkaline protease |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111979215A (en) * | 2020-07-28 | 2020-11-24 | 江苏海洋大学 | Bacillus sphaericus organic solvent-resistant protease mutant |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215534A (en) * | 2007-12-28 | 2008-07-09 | 南京工业大学 | Organic solvent-resistant alkaline protease producing strain, gene of organic solvent-resistant alkaline protease and application of organic solvent-resistant alkaline protease |
| WO2010126156A2 (en) * | 2009-04-30 | 2010-11-04 | Kao Corporation | Alkaline protease variants |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215534A (en) * | 2007-12-28 | 2008-07-09 | 南京工业大学 | Organic solvent-resistant alkaline protease producing strain, gene of organic solvent-resistant alkaline protease and application of organic solvent-resistant alkaline protease |
| WO2010126156A2 (en) * | 2009-04-30 | 2010-11-04 | Kao Corporation | Alkaline protease variants |
Non-Patent Citations (1)
| Title |
|---|
| 何小丹等: "地衣芽孢杆菌YP1A耐有机溶剂蛋白酶基因的克隆与功能表达", 《生物加工过程》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111979215A (en) * | 2020-07-28 | 2020-11-24 | 江苏海洋大学 | Bacillus sphaericus organic solvent-resistant protease mutant |
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| Publication number | Publication date |
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| CN103275958B (en) | 2014-07-16 |
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