CN103275958B - Organic-solvent-resistant alkaline protease - Google Patents
Organic-solvent-resistant alkaline protease Download PDFInfo
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- CN103275958B CN103275958B CN201310243800.1A CN201310243800A CN103275958B CN 103275958 B CN103275958 B CN 103275958B CN 201310243800 A CN201310243800 A CN 201310243800A CN 103275958 B CN103275958 B CN 103275958B
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- Prior art keywords
- ala
- val
- gly
- ser
- lys
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- 239000003960 organic solvent Substances 0.000 title claims abstract description 69
- 108091005658 Basic proteases Proteins 0.000 title abstract description 16
- 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 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 26
- 108091005804 Peptidases Proteins 0.000 abstract description 19
- 150000001413 amino acids Chemical class 0.000 abstract description 14
- 235000001014 amino acid Nutrition 0.000 abstract description 12
- 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
- 238000010353 genetic engineering Methods 0.000 abstract description 3
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- 239000004472 Lysine Substances 0.000 abstract 1
- 108010033276 Peptide Fragments Proteins 0.000 abstract 1
- 102000007079 Peptide Fragments Human genes 0.000 abstract 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 235000004279 alanine Nutrition 0.000 abstract 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 abstract 1
- 238000002741 site-directed mutagenesis Methods 0.000 abstract 1
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Abstract
The invention belongs to the technical field of protease genetic engineering and particularly relates to an organic-solvent-resistant alkaline protease, a mutant gene of the organic-solvent-resistant alkaline protease and proteins encoded by the organic solvent resistance alkaline protease. As shown from SEQ ID NO:1 or SEQ ID NO:3 in a sequence table, site-specific mutagenesis occurs to the organic solvent resistance alkaline protease disclosed by the invention at a 385th or 436th site of an original nucleotide sequence, namely 24th bit mutated amino acid of the mature peptide fragment of the organic solvent resistance alkaline protease is alanine or lysine. Tolerance of an organic solvent comprising four mutated and encoded proteins, especially the tolerance of a hydrophilic organic solvent is obviously and greatly reinforced.
Description
The application is to be on 08 01st, 2011 the applying date, and application number is 201110217989.8, and what invention and created name was " a kind of organic solvent-resistant Sumizyme MP " divides an application.
Technical field
The invention belongs to proteinase gene field of engineering technology, be specifically related to a kind of organic solvent tolerant protease, be specifically related to the protein of its mutator gene and coding thereof.
Technical background
Proteolytic enzyme refer to can catalysis the class of enzymes of peptide bond hydrolysis, from 1914 by test for since the added ingredients of washing composition, owing to thering is important commercial use by extensive concern.The output of current proteolytic enzyme occupies the more than 40% of enzyme market, the fields such as widespread use and washing composition, food, pharmacy, process hides, diagnostic reagent, sewage disposal.Proteolytic enzyme is extensively present in all biologies such as animal, plant, fungi and prokaryotic organism, and wherein microbe-derived proteolytic enzyme occupies the more than 2/3 of current proteolytic enzyme production.Microbe-derived proteolytic enzyme can be divided into metalloprotease, aspartate protease and serine protease etc. according to the feature of the reaction pH of enzyme, active group again.
Albumen the most general enzymatic reaction is the peptide bond in 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 specificity requirement to the amino-acid residue of its catalysis peptide bond, has regioselectivity and the stereoselectivity of height.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, the 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.Quantity research shows greatly, and in organic solvent, carrying out enzymatic reaction has many advantages: 1, increase various organic substrates in solvent, particularly the solubleness in hydrophilic organic solvent, improves reaction efficiency; Stereoselectivity and the regioselectivity 2, with height, organic medium can change selectivity; 3, control molecular balance and move 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.Ru Zhe research department utilizes the organic solvent tolerant protease screening voluntarily in hydrophilic organic solvent dimethyl sulfoxide (DMSO) (DMSO), to realize the precursor of sweeting agent Aspartame and synthesizing of interior morphine dai 1, productive rate reaches more than 90%, and realizing the Reaction Separation coupling of substrate and product, greatly improve productive rate, simplified the separation and purification of product.The market outlook that enzymic catalytic reaction is carried out in explanation in hydrophilic organic solvent system are very huge.
In organic solvent system, 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 past more than 20 year, 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 control agent, to guarantee " necessary water " of enzyme; 2, in reaction medium, add protective material glycerine, ethylene glycol, polyhydric alcohol polymer etc., or add the freezing drying protective agents such as cyclodextrin, to improve the stability of enzyme molecule in organic medium.3, utilize the physics and chemistry modifying method such as immobilization, entrapping method, macromole modification to improve activity and the stability of enzyme 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, wherein the enzyme activity of mutant PC3 in 60% and 85% DMF is respectively 256 and 131 times of natural enzyme.After this on the basis of PC3, suddenly change again, taller 3 times than PC3 of the mutant 13M enzyme activities obtaining.
Although make some progress utilizing genetic modification to improve aspect the organic solvent stability of enzyme in recent years, but the organic solvent tolerance of the enzyme that often sets out is relatively low, organic solvent patience, the amplitude particularly improving in hydrophilic organic solvent is little, and industrial application is also had to 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, an organic solvent-resistant Sumizyme MP, is characterized in that it has the nucleotide sequence shown in SEQ ID NO:1, and its aminoacid sequence is shown in SEQ ID NO:2; Or it has the nucleotide sequence shown in SEQ ID NO:3, 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 as shown in sequence table SEQ ID NO:5 of the present invention.SEQ ID NO:1 or SEQ ID NO:3 from sequence table, there is rite-directed mutagenesis at the 385th or the 436th of former nucleotide sequence in organic solvent-resistant Sumizyme MP of the present invention, 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 after four kinds of codings after 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) select described in 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 basis
bacillus subtilisthe gene order of p43 promotor in 168 (No. GenBank: K02714) design primer amplification p43 promotor, by the method for overlapping PCR, promotor p43 is connected with above-mentioned organic solvent-resistant alkaline protease gene, finally be so kind as to give by professor Li Shunpeng of Agricultural University Of Nanjing with expression vector pHY300() be connected, transforming subtilis host WB800(is so kind as to give by professor Li Shunpeng of Agricultural University Of Nanjing), build recombinant bacterium WB800-pHY300-p43-YP1A and express.
(2) select 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, six aggressiveness primer: 5 '-NpNpNpNpsNpsN-3 ' of design 3 ' end thio-modification.
(4) take the plasmid DNA obtaining is template, with Φ 29 DNA polysaccharases (Fermentas #EP0097), carries out the amplification of fallibility rolling-circle replication, and program is as follows: 95 ℃ of sex change 3min; Put rapidly cooled on ice to room temperature; 30 ℃ of reaction 24 h.
(5) Dpn I enzyme (Fermentas #ER1701) digestion 1 h for reaction product, carries out glue and reclaims concentrated.
(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, 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 mutant strain step (7) Suo Shu being studied, finding four kinds of mutant optimal reaction pH values, all there is not considerable change in pH value stabilization; The optimal reactive temperature of three kinds of mutant P8, Z8, Z25 and temperature stability significantly do not change yet; 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, mutator gene is compared with YP1A gene, respectively from 385 bp(K8/P8 after initiator codon) and 436 bp(Z8/Z25) there is sudden change, at the 385th, from GCG, sport CAG and GAG respectively; Amino acid sports Gln and Glu from Ala respectively; From GAC, sport GCA and AAG; Amino acid sports Ala and Lys from Asp.
Three, organic solvent-resistant Sumizyme MP of the present invention catalyzes and synthesizes the application in 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 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.At present there are no 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, mutator gene is compared with YP1A gene, respectively from 385 bp(K8/P8 after initiator codon) and 436 bp(Z8/Z25) sudden change there is; SEQ ID NO:1 or SEQ ID NO:3 from sequence table, there is rite-directed mutagenesis at the 385th or the 436th of former nucleotide sequence in organic solvent-resistant Sumizyme MP of the present invention, 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 after four kinds of codings after 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 little peptide, the organic solvent of its superelevation, particularly hydrophilic organic solvent tolerance are more applicable for the synthetic field of little peptide for the organic solvent tolerant protease of prior art.
Accompanying drawing explanation
Fig. 1 shows that fallibility rolling-circle replication electrophorogram and mutant strain do the preliminary screening on milk flat board.
Fig. 2 shows mutant strain optimal reaction pH value.
Fig. 3 shows mutant strain pH value stabilization.
Fig. 4 shows mutant strain optimal reactive temperature.
Fig. 5 shows mutant strain temperature stability.
Fig. 6 and 7 shows the stability of mutant strain in DMF/DMSO.
Embodiment
Below in conjunction with specific examples, the present invention is described in further detail.
embodiment 1
The present embodiment illustrates the orthogenesis of organic solvent-resistant alkaline protease gene of the present invention.
Select described in 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 in Bacillus subtilis 168 (No. GenBank: K02714) design primer amplification p43 promotor, by the method for overlapping PCR, promotor p43 is connected with organic solvent-resistant alkaline protease gene, finally be so kind as to give by professor Li Shunpeng of Agricultural University Of Nanjing with expression vector pHY300() be connected, transforming subtilis host WB800(is so kind as to give by professor Li Shunpeng of Agricultural University Of Nanjing), building recombinant bacterium WB800-pHY300-p43-YP1A expresses, specific implementation method is as follows:
(1) design amplification YP1A gene and promotor p43 primer.
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 denaturation 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 approximately 1.1 kb has been arrived in amplification.
2. the amplification of promotor p43: 94 ℃ of denaturation 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 approximately 300 bp has been arrived in amplification.
3. YP1A-p43 amplification: the method by overlapping PCR connects YP1A gene and promotor p43,94 ℃ of denaturation 5 min; 94 ℃ of sex change 30 sec; 65 ℃ of annealing 30 sec(annealing temperatures design 1 ℃ of Gradient Descent); 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 to by restriction enzyme Bgl II (the precious biotech firm in Dalian), carry out enzyme with BamH I (the precious biotech firm in Dalian) after purifying cuts, with same enzyme, carrier pHY300 is carried out to enzyme cuts simultaneously, purifying enzyme is cut the precious biotech firm in T4(Dalian for product) ligase enzyme connects and spends the night at 16 ℃, and conversion subtilis WB800 expresses.
Select the genetic engineering bacterium WB800-pHY300-p43-YP1A building in 37 ℃ of shaking table overnight incubation, extract plasmid as sudden change template; According to the principle of fallibility rolling-circle replication, six aggressiveness primer: 5 '-NpNpNpNpsNpsN-3 ' of design 3 ' end thio-modification; According to following program, suddenly change:
(1) 50 μ L reaction system: Tris-HCl(pH7.5) final concentration 50 mM; Ammonium sulfate final concentration 10mM, MgCl
2final 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 to polysaccharase and MnCl
2outward, be added in 500 μ L reaction tubess and mix, sex change 3 min at 95 ℃; Reaction tubes is placed in rapidly to cooled on ice to room temperature; Add polysaccharase and MnCl
2; In 30 ℃ of reaction 24 h.
(3) reaction product adds Dpn I enzymic digestion 1 h according to certain system, carries out glue recovery.
(4) reclaim product and be directly transformed into Bacillus subtilus WB800 expression system, on milk flat board, selecting has 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, centrifuging and taking supernatant liquor is crude enzyme liquid, according to follow procedure, carries out organic solvent tolerance screening.
Mutant strain is inoculated in fermention medium and is cultivated 7 days, centrifugal recovery supernatant liquor is the crude enzyme liquid of mutant strain, on ice bath, get 45%(V/V) 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 after 1.5 h and measure residual protein enzyme activity.Control group is for adding 55% volume (V/V) damping fluid.Carry out organic solvent tolerance screening, select tolerance to change obvious mutant strain and extract plasmid order-checking, determine mutational site.Find that mutant strain K8 and the tolerance of P8 in 55% DMF have improved respectively 55% and 30%; And Z8 and Z25 have improved respectively 29% and 51%, as table one:
Table one screen mutation result
The present invention is also studied 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 occur pH value stabilization; The optimal reactive temperature of three kinds of mutant P8, Z8, Z25 and temperature stability significantly do not change (as Fig. 4,5) yet; 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, mutator gene is compared with YP1A gene, respectively from 385 bp(K8/P8 after initiator codon) and 436 bp(Z8/Z25) there is sudden change, at the 385th, from GCG, sport CAG and GAG respectively; Amino acid sports Gln and Glu from Ala respectively; From GAC, sport GCA and AAG; Amino acid sports Ala and Lys from Asp.
Table two mutant strain solvent stability
Note: DMSO concentration is 65%(v/v), other solvent strength is 50%(v/v).
Sequence table
<110> Nanjing University of Technology
<120> organic solvent-resistant Sumizyme MP
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<170> PatentIn version 3.3
<210> 1
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<212> DNA
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<222> (1)..(1137)
<220>
<221> mat_peptide
<222> (316)..(1137)
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<221> misc_feature
<222> (385)..(387)
<223> is nnn=gaa or gag or caa or cag herein; Xaa=Gln or Glu.
<400> 1
atg atg agg aaa aag agt ttt tgg ctt ggg atg ctg acg gcc tta atg 48
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Leu Met
-105 -100 -95 -90
ctc gtg ttc acg atg gca ttc agc gat tcc gct tct gct gct caa ctg 96
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Leu
-85 -80 -75
gcg aaa aat gtt gaa aag gat tat atc gtc gga ttt aag tca gga gtg 144
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
-70 -65 -60
aaa acc gca tcc gtc aaa aag gac atc atc aaa gag agc ggc gga aaa 192
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
-55 -50 -45
gtg gac aag cag ttt aga atc atc aac gcg gca aaa gcg aag cta gac 240
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
-40 -35 -30
aaa gaa gcg ctt aag gaa gtc aaa aat gat ccg gat gtc gct tat gtg 288
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
-25 -20 -15 -10
gaa gag gat cat gtg gcc cat gcc ttg gcg caa acc gtt cct tac ggc 336
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
-5 -1 1 5
att cct ctc att aaa gcg gac aaa gtg cag gct caa ggc ttt aag gga 384
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
10 15 20
nnn aat gta aaa gta gcc gtc ctg gat aca gga atc caa gct tct cat 432
Xaa Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
25 30 35
ccg gac ttg aac gta gtc ggc gga gca agc ttt gtg gct ggc gaa gct 480
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
40 45 50 55
tat aac acc gac ggc aac gga cac ggc aca cat gtt gcc ggt aca gta 528
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
60 65 70
gct gcg ctt gac aat aca acg ggt gta tta ggc gtt gcg cca agc gta 576
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
75 80 85
tcc ttg tac gcg gtt aaa gta ctg aat tca agc gga agc gga tca tac 624
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
90 95 100
agc ggc att gta agc gga atc gag tgg gcg aca aca aac ggc atg gat 672
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
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
gca ggg aac agc gga cct tca gga aac acg aat aca att ggc tat cct 816
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
aac aga gct tca ttt tcc agt gtg gga gca gag ctt gaa gtc atg gct 912
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
185 190 195
cct ggc gca ggc gta tac agc act tac cca acg aac act tat gca aca 960
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
200 205 210 215
ttg aac gga acg tca atg gct tct cct cat gta gcg gga gca gca gct 1008
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
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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
Xaa Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
25 30 35
Pro Asp 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
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<223> organic solvent-resistant alkaline protease gene 2
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Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Leu Met
-105 -100 -95 -90
ctc gtg ttc acg atg gca ttc agc gat tcc gct tct gct gct caa ctg 96
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Leu
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gcg aaa aat gtt gaa aag gat tat atc gtc gga ttt aag tca gga gtg 144
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
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aaa acc gca tcc gtc aaa aag gac atc atc aaa gag agc ggc gga aaa 192
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
-55 -50 -45
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Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
-40 -35 -30
aaa gaa gcg ctt aag gaa gtc aaa aat gat ccg gat gtc gct tat gtg 288
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
-25 -20 -15 -10
gaa gag gat cat gtg gcc cat gcc ttg gcg caa acc gtt cct tac ggc 336
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
-5 -1 1 5
att cct ctc att aaa gcg gac aaa gtg cag gct caa ggc ttt aag gga 384
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
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Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
25 30 35
ccg nnn ttg aac gta gtc ggc gga gca agc ttt gtg gct ggc gaa gct 480
Pro Xaa Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
40 45 50 55
tat aac acc gac ggc aac gga cac ggc aca cat gtt gcc ggt aca gta 528
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
60 65 70
gct gcg ctt gac aat aca acg ggt gta tta ggc gtt gcg cca agc gta 576
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
75 80 85
tcc ttg tac gcg gtt aaa gta ctg aat tca agc gga agc gga tca tac 624
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
90 95 100
agc ggc att gta agc gga atc gag tgg gcg aca aca aac ggc atg gat 672
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
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
gca ggg aac agc gga cct tca gga aac acg aat aca att ggc tat cct 816
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
aac aga gct tca ttt tcc agt gtg gga gca gag ctt gaa gtc atg gct 912
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
185 190 195
cct ggc gca ggc gta tac agc act tac cca acg aac act tat gca aca 960
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
200 205 210 215
ttg aac gga acg tca atg gct tct cct cat gta gcg gga gca gca gct 1008
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 is the nucleotide sequence shown in 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-tolerant alkali protease |
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| 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 resisting basified protease producing strain, gene and application thereof |
| WO2010126156A2 (en) * | 2009-04-30 | 2010-11-04 | Kao Corporation | Alkaline protease variants |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215534A (en) * | 2007-12-28 | 2008-07-09 | 南京工业大学 | Organic solvent resisting basified protease producing strain, gene and application thereof |
| WO2010126156A2 (en) * | 2009-04-30 | 2010-11-04 | Kao Corporation | Alkaline protease variants |
Non-Patent Citations (2)
| 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》.2009,第7卷(第6期), |
| 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;何小丹等;《生物加工过程》;20091130;第7卷(第6期);67-73 * |
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