CN101045933A - Cryophilous proteinase gene mcp01 and its prepn process - Google Patents

Abstract

The present invention is one kind of cryophilous protease gene mcp01 and its preparation process, and belongs to the field of biotechnology. The cryophilous proteinase gene mcp01 has one genome DNA segment of 2676 bp containing one opened reading frame with 2508 nucleotides. The opened reading frame is the gene encoding protease MCP-01, and the protease MCP-01 gene mcp01 expressed cryophilous protease has optimal enzyme activity temperature of about 35 deg.c and optimal enzyme pH of 9.0. The present invention may be applied in low temperature preservation of meat, dairy product processing, detergent production, etc.

Description

Gene mcp01 of a kind of cold-adapted protease and preparation method thereof

Technical field

The present invention relates to gene of a kind of novel deep sea cold-adapted protease and preparation method thereof, belong to the marine biotechnology field.

Background technology

(proteases proteinases) also claims peptase (peptidases) to proteolytic enzyme, is the hydrolase of a class catalysis peptide bond rupture.Proteolytic enzyme extensively is present in pluck, plant stem-leaf and fruit, and multiple microorganism can secrete extracellular protease, is the main source of industrial production proteolytic enzyme.According to mode of action difference, proteolytic enzyme is divided into exopeptidase (exopeptidases) and endopeptidase (endopeptidases).One or several amino acid of polypeptide chain N end of exopeptidase cutting or C end.Therefore, exopeptidase has aminopeptidase (cutting from the N end-grain cutting) and carboxypeptidase (cutting from the C end-grain cutting) two big classes.The peptide bond of endopeptidase fracture polypeptide chain inside.According to the catalyst mechanism difference, endopeptidase is divided into arginine proteolytic enzyme, serine protease, L-Cysteine HCL Anhydrous, metalloprotease and serine/threonine protein enzyme five big classes.Proteolytic enzyme commodity production started from for 20 beginnings of the century, because its purposes widely becomes topmost zymin already, sales volume is huge in countries in the world.The main application of proteolytic enzyme has following 5 aspects:

(1) makes protein hydrolystate: peptone for example, kilnitamin, protolysate transfusion, elements diet, amino acid sauce, beef extract, proheparin, placenta hydrolyzate etc.

(2) as deproteinizing agent: remove protein when making Oils,glyceridic,cod-liver, spices, useless film comes unstuck and reclaims concise, the clarifications such as wool is concise, beer and grape wine of only coming unstuck of sheet base and silver granuel, silk fabric, lactose Deproteinization and be used for washing composition, tooth powder etc. to strengthen washing effect.

(3) being used to reform technology improves the quality of products: the depilation of leather material skin, and fur softening, low temperature dyeing of wool is made collegen filament, the meat tenderization, the bread manufacturing, soy sauce brewing is produced medicines such as chondroitin sulfate, arklemin.

(4) as medicine and reagent: as digestion, anti-inflammatory, preventing phlegm from forming and stopping coughing medicine and treatment fall injure, oedema hemotoncus and eliminate medicine such as necrotic tissue.

(5) as fodder additives, promote cub to grow up, save feed, increase the poultry spawning rate.

Because the extensive use of proteolytic enzyme is all paid attention to by the countries in the world scientist the research and development of proteolytic enzyme always.The novel protein enzyme that exploitation has new purposes has very important theory significance and using value.Since the seventies in 20th century, along with gene recombination technology and development of molecular biology, the encoding gene of many proteolytic enzyme and primary structure are studied in great detail.Serine protease is research and a most widely used class of enzymes in the proteolytic enzyme, is divided into 14 clan.Subtilin proteinoid enzyme (subtilase) belongs to the S8A subfamily (subfamily) among the clan SB.Such proteolytic enzyme major part is by microorganism secretions such as bacterium and fungies, its precursor generally comprises signal peptide (signal peptide), N end structure territory and catalyst structure domain, the enzyme of fungus secretion also has a structural domain that is called P-domain at the C end usually, but their maturing enzyme only contains a catalyst structure domain, and molecular weight is generally about 3kDa.Such proteolytic enzyme is of many uses, for example, the subtilin of producing bacillus subtilis (subtilisin) is widely used in articless for washing such as washing powder, agent is widely used in molecular biology and biological technical field to Proteinase K as Deproteinization, and subtilicin and Proteinase K be also widespread use in the field of protein production peptide.

Studies show that in recent years, the deep-sea exists a large amount of microorganisms, these microorganism long term growth are under near the extreme environment of high salt, high pressure, oligotrophic, low temperature or high temperature (the submarine hydrothermal solution mouth), the special construction and the special mechanism that adapt to these envrionment conditionss have been formed, therefore, Deep-Sea Microorganisms is to find the good material of novel enzyme.But because the difficulty of sampling and cultivation is at present less relatively to the research of Deep-Sea Microorganisms.

The present inventor is separated to a strain cold-adaptive microbe bacterium strain in 1855 meters dark marine bottom sediments, belong to Rhodopseudomonas through evaluation, called after Pseudomonas sp.SM9913.Referring to Chen Xiulan etc., " low-temperature protease that deep sea cold-adaptive microbe bacterium strain SM9913 produces ", " ocean science " calendar year 2001, the 5th volume, the 1st phase, 4-8 page or leaf.The applicant's ZL01127405.0 patent provides the method (Granted publication CN1141388C) of utilizing this deep sea cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913 to prepare properly cooled proteinase with special flavor.

Summary of the invention:

The invention provides gene mcp01 of a kind of novel deep sea cold-adapted protease MCP-01 and preparation method thereof.The cold-adapted protease of expressing by the gene mcp01 of proteolytic enzyme MCP-01 can be used for low temperature meat fresh-keeping, increase aquatic foods, also can be used for industries such as washing composition.

The cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913 preparation method who the present invention relates to is referring to Chen Xiulan etc., " low-temperature protease that deep sea cold-adaptive microbe bacterium strain SM9913 produces ", " ocean science " calendar year 2001, the 5th volume, the 1st phase, 4-8 page or leaf.

With a kind of proteolytic enzyme called after of bacterial strain cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913 excretory MCP-01.Being extracted among the embodiment 2 of this proteolytic enzyme will elaborate.

Extract the genomic dna of Pseudomonas sp.SM9913.N end and its self-dissolving fragment N end to proteolytic enzyme MCP-01 check order, according to sequencing result design primer.Then, utilize the part of the gene of pcr clone proteins encoded enzyme MCP-01.According to the portion gene design primer of preparation method, utilize the Tail pcr clone to go out the complete genome of proteolytic enzyme MCP-01 again.Sequencing result shows in the dna fragmentation of 2676bp of acquisition and contains an open reading frame with 2508 Nucleotide, this open reading frame promptly is the gene of proteins encoded enzyme MCP-01, initiator codon is positioned at 143bp, and terminator codon is positioned at 2648bp, and coded amino acid is 835 altogether.

The structure of the precursor of proteolytic enzyme MCP-01 comprises signal peptide, N end structure territory, catalyst structure domain, connecting zone, P-domain structural domain, PKD structural domain and C end regions seven parts, its maturing enzyme comprises catalyst structure domain, connecting zone, P-domain structural domain and PKD structural domain four parts, it is the subtilin proteinoid enzyme of a Multidomain, this type proteolytic enzyme is not reported at present as yet, proteolytic enzyme MCP-01 is present unique the type proteolytic enzyme that is purified, and we are with this novel protein enzyme called after deseasin.The suitableeest enzyme of this enzyme temperature alive is about 35 ℃, and optimal pH is 9.0.

The present invention clone's the genomic DNA fragment that contains deep-sea cold-adapted protease gene mcp01 has following sequence, SEQ IDNO.1:

gttttgttcc tgattttgct ttttttactt gagttagtga cggattaccc gttttcttaa 60

cgcttgccac gtgattactt aaattcataa acgtaaatat ttcgcttagt aaagctaagt 120

taacttaatt acggatagaa caatgaaaac aaaattatct attattacac ttgcactttt 180

accttgttta gctgcggcta aattgcctga tgttaacagt acaactcaaa aagtaactgc 240

gagcagtgac tcgattattg ttaagtataa aaagaatgct tcgccgaaaa tgcgtaagca 300

agctcgttcg ttagtaaaag ccaaaatatc tgatctaaat aacgatgaaa ttgatgataa 360

cttcacctcg ttattttctg gccgcctcgc aaaatttaaa atatcaggaa tgagcgctaa 420

agaagcaatt gagcgcttaa aatcgcatca agctattgag tatgttgagc ctgattaccg 480

agtgagcata gcaagcgcaa cgaacgaccc gcgatttgac gatttatggg gcttaaacaa 540

cgaaggccaa actggtggca cggctgatgc cgatattgat gcaccagaag catggtcaat 600

ctctacaggt agtcgtgatg ttgtagtagg tgttattgat acgggtgttg attatagtca 660

ccctgattta gctgcgaacg cgtgggtaaa cagtggtgaa attgccggtg atggtatcga 720

taatgacgga aacggttaca ttgatgacgt tcacggtatt aatgcaatta ctgatgttgg 780

tgatccgatg gatgacgaag gccatggtac acacgtatcg ggtacaatcg gtgccagtgg 840

taacaatggt gtcggtgttg ttggtgtaaa ccatgatgtt tctattgttg gttgtaagtt 900

tttagccgca gatggtacag gttctacctc tggtgctatt aagtgtatcg attacatggt 960

tggccttaaa aatgcgggtg ttaatctgcg tgtgttaaat aatagctggg gcggcggtgg 1020

ttttagccaa gcgttagctg atgcgattac ggctagtgag caagctgata ttttatttgt 1080

tgctgctgcg ggtaacgatg cggttgataa cgatcaaaat ccacactacc catctaatta 1140

tgaaaacgac aacgtattat cgatagctag tactgatagc agagataata tgtctagctt 1200

ttctcaatgg ggcctaacaa gtgttgatat gggcgcacct ggctcaggta ttttgtcgac 1260

cgttcctggt aatagctatg ctacttattc aggtacatct atggcaacac cgcatgtagc 1320

tggtgctgct gcacttgtac tttcagttaa tcctgattta acaacactag agcttaaaga 1380

attacttatg tctagtggtg atgctaatgc ggcattaaat ggtaaaacgg ttgcaggtac 1440

acgcctaaat gttaaccaag cgctaattga tgctgatcca actccaggct ttaaattatc 1500

tgtatcgccg gtttctcagc aagcgactgt aggtgatacg gttacttata cctttacgat 1560

tggctcaatt gcacaatggg aaggtgacgt ttcactggct ttagcttcag atcttaccga 1620

tgcatcacta agcgcaacta ccgctcgtcc gggcgatgaa gttgttctaa cggttgcaac 1680

taacagcgat actcagtggg gaaattacga ctttactgtt accgcaacaa ctgaagagca 1740

agttaaagaa caaactgtta gcttaatgct tcaacctgct ggtttaaatg actttactta 1800

cagcagtgat gaaaacatcg ctattccaga taattctcca gagggcgcaa gctcagttat 1860

tacggttgct gatgatttaa ctatttttgg caccactgct gatgtagata ttacgcatac 1920

ttggtctggt gacttagtac ttacgcttat ttcagcgcaa ggtacagaag taactttaca 1980

aagcaatgaa ggtggcagtg cagacgatat tgttaagtca tttacatcaa gcgtatttaa 2040

ttctgaagtg gcaaccggag attggacact tcatgttgaa gatacagctg gtgcagatac 2100

gggtaatatc aatggttggt cacttacatt tagcgctatt ggtgaggtaa gcccacaacc 2160

accacaagct ggctttagtg tttcagcaca aggtttaact gctacatttg taggtacaag 2220

tcgtgatgta aatgatgaca tttcacagtg gagctggaac tttggtgatg gttcaacatc 2280

aacaaaccca aatcctacac atgtttacgc tcagtcagga agttatgagg ttgaattaac 2340

cgtaacggat agtgaaaata actcagatac attcactcaa acagtggttg ttagtgatgt 2400

tgaaattgaa ttgagcttaa aacgcgctaa caagtcacgt cttgatacta tgcgtgttga 2460

gttaaactgg gcagaagtag gggctgagtc acttagccta taccgaaatg gcgaactagt 2520

agataccgta tcagataatg gccgttaccg tgattatgtg cgtaacgcaa cattaccggc 2580

atacgactat cagttatgcg tatctgaaaa cgtatgttcg aatattatta cggtttcatt 2640

ttctgaataa aacgtttacc ccttagacat tcaaag 2676

Nucleotide and coded amino acid corresponding relation are as follows in the said gene:

1 ATG AAA ACA AAA TTA TCT ATT ATT ACA CTT GCA CTT TTA CCT TGT

1 M K T K L S I I T L A L L P C

46 TTA GCT GCG GCT AAA TTG CCT GAT GTT AAC AGT ACA ACT CAA AAA

16 L A A A K L P D V N S T T Q K

91 GTA ACT GCG AGC AGT GAC TCG ATT ATT GTT AAG TAT AAA AAG AAT

31 V T A S S D S I I V K Y K K N

136 GCT TCG CCG AAA ATG CGT AAG CAA GCT CGT TCG TTA GTA AAA GCC

46 A S P K M R K Q A R S L V K A

181 AAA ATA TCT GAT CTA AAT AAC GAT GAA ATT GAT GAT AAC TTC ACC

61 K I S D L N N D E I D D N F T

226 TCG TTA TTT TCT GGC CGC CTC GCA AAA TTT AAA ATA TCA GGA ATG

76 S L F S G R L A K F K I S G M

271 AGC GCT AAA GAA GCA ATT GAG CGC TTA AAA TCG CAT CAA GCT ATT

91 S A K E A I E R L K S H Q A I

316 GAG TAT GTT GAG CCT GAT TAC CGA GTG AGC ATA GCA AGC GCA ACG

106 E Y V E P D Y R V S I A S A T

361 AAC GAC CCG CGA TTT GAC GAT TTA TGG GGC TTA AAC AAC GAA GGC

121 N D P R F D D L W G L N N E G

406 CAA ACT GGT GGC ACG GCT GAT GCC GAT ATT GAT GCA CCA GAA GCA

136 Q T G G T A D A D I D A P E A

451 TGG TCA ATC TCT ACA GGT AGT CGT GAT GTT GTA GTA GGT GTT ATT

151 W S I S T G S R D V V V G V I

496 GAT ACG GGT GTT GAT TAT AGT CAC CCT GAT TTA GCT GCG AAC GCG

166 D T G V D Y S H P D L A A N A

541 TGG GTA AAC AGT GGT GAA ATT GCC GGT GAT GGT ATC GAT AAT GAC

181 W V N S G E I A G D G I D N D

586 GGA AAC GGT TAC ATT GAT GAC GTT CAC GGT ATT AAT GCA ATT ACT

196 G N G Y I D D V H G I N A I T

631 GAT GTT GGT GAT CCG ATG GAT GAC GAA GGC CAT GGT ACA CAC GTA

211 D V G D P M D D E G H G T H V

676 TCG GGT ACA ATC GGT GCC AGT GGT AAC AAT GGT GTC GGT GTT GTT

226 S G T I G A S G N N G V G V V

721 GGT GTA AAC CAT GAT GTT TCT ATT GTT GGT TGT AAG TTT TTA GCC

241 G V N H D V S I V G C K F L A

766 GCA GAT GGT ACA GGT TCT ACC TCT GGT GCT ATT AAG TGT ATC GAT

256 A D G T G S T S G A I K C I D

811 TAC ATG GTT GGC CTT AAA AAT GCG GGT GTT AAT CTG CGT GTG TTA

271 Y M V G L K N A G V N L R V L

856 AAT AAT AGC TGG GGC GGC GGT GGT TTT AGC CAA GCG TTA GCT GAT

286 N N S W G G G G F S Q A L A D

901 GCG ATT ACG GCT AGT GAG CAA GCT GAT ATT TTA TTT GTT GCT GCT

301 A I T A S E Q A D I L F V A A

946 GCG GGT AAC GAT GCG GTT GAT AAC GAT CAA AAT CCA CAC TAC CCA

316 A G N D A V D N D Q N P H Y P

991 TCT AAT TAT GAA AAC GAC AAC GTA TTA TCG ATA GCT AGT ACT GAT

331 S N Y E N D N V L S I A S T D

1036 AGC AGA GAT AAT ATG TCT AGC TTT TCT CAA TGG GGC CTA ACA AGT

346 S R D N M S S F S Q W G L T S

1081 GTT GAT ATG GGC GCA CCT GGC TCA GGT ATT TTG TCG ACC GTT CCT

361 V D M G A P G S G I L S T V P

1126 GGT AAT AGC TAT GCT ACT TAT TCA GGT ACA TCT ATG GCA ACA CCG

376 G N S Y A T Y S G T S M A T P

1171 CAT GTA GCT GGT GCT GCT GCA CTT GTA CTT TCA GTT AAT CCT GAT

391 H V A G A A A L V L S V N P D

1216 TTA ACA ACA CTA GAG CTT AAA GAA TTA CTT ATG TCT AGT GGT GAT

406 L T T L E L K E L L M S S G D

1261 GCT AAT GCG GCA TTA AAT GGT AAA ACG GTT GCA GGT ACA CGC CTA

421 A N A A L N G K T V A G T R L

1306 AAT GTT AAC CAA GCG CTA ATT GAT GCT GAT CCA ACT CCA GGC TTT

436 N V N Q A L I D A D P T P G F

1351 AAA TTA TCT GTA TCG CCG GTT TCT CAG CAA GCG ACT GTA GGT GAT

451 K L S V S P V S Q Q A T V G D

1396 ACG GTT ACT TAT ACC TTT ACG ATT GGC TCA ATT GCA CAA TGG GAA

466 T V T Y T F T I G S I A Q W E

1441 GGT GAC GTT TCA CTG GCT TTA GCT TCA GAT CTT ACC GAT GCA TCA

481 G D V S L A L A S D L T D A S

1486 CTA AGC GCA ACT ACC GCT CGT CCG GGC GAT GAA GTT GTT CTA ACG

496 L S A T T A R P G D E V V L T

1531 GTT GCA ACT AAC AGC GAT ACT CAG TGG GGA AAT TAC GAC TTT ACT

511 V A T N S D T Q W G N Y D F T

1576 GTT ACC GCA ACA ACT GAA GAG CAA GTT AAA GAA CAA ACT GTT AGC

526 V T A T T E E Q V K E Q T V S

1621 TTA ATG CTT CAA CCT GCT GGT TTA AAT GAC TTT ACT TAC AGC AGT

541 L M L Q P A G L N D F T Y S S

1666 GAT GAA AAC ATC GCT ATT CCA GAT AAT TCT CCA GAG GGC GCA AGC

556 D E N I A I P D N S P E G A S

1711 TCA GTT ATT ACG GTT GCT GAT GAT TTA ACT ATT TTT GGC ACC ACT

571 S V I T V A D D L T I F G T T

1756 GCT GAT GTA GAT ATT ACG CAT ACT TGG TCT GGT GAC TTA GTA CTT

586 A D V D I T H T W S G D L V L

1801 ACG CTT ATT TCA GCG CAA GGT ACA GAA GTA ACT TTA CAA AGC AAT

601 T L I S A Q G T E V T L Q S N

1846 GAA GGT GGC AGT GCA GAC GAT ATT GTT AAG TCA TTT ACA TCA AGC

616 E G G S A D D I V K S F T S S

1891 GTA TTT AAT TCT GAA GTG GCA ACC GGA GAT TGG ACA CTT CAT GTT

631 V F N S E V A T G D W T L H V

1936 GAA GAT ACA GCT GGT GCA GAT ACG GGT AAT ATC AAT GGT TGG TCA

646 E D T A G A D T G N I N G W S

1981 CTT ACA TTT AGC GCT ATT GGT GAG GTA AGC CCA CAA CCA CCA CAA

661 L T F S A I G E V S P Q P P Q

2026 GCT GGC TTT AGT GTT TCA GCA CAA GGT TTA ACT GCT ACA TTT GTA

676 A G F S V S A Q G L T A T F V

2071 GGT ACA AGT CGT GAT GTA AAT GAT GAC ATT TCA CAG TGG AGC TGG

691 G T S R D V N D D I S Q W S W

2116 AAC TTT GGT GAT GGT TCA ACA TCA ACA AAC CCA AAT CCT ACA CAT

706 N F G D G S T S T N P N P T H

2161 GTT TAC GCT CAG TCA GGA AGT TAT GAG GTT GAA TTA ACC GTA ACG

721 V Y A Q S G S Y E V E L T V T

2206 GAT AGT GAA AAT AAC TCA GAT ACA TTC ACT CAA ACA GTG GTT GTT

736 D S E N N S D T F T Q T V V V

2251 AGT GAT GTT GAA ATT GAA TTG AGC TTA AAA CGC GCT AAC AAG TCA

751 S D V E I E L S L K R A N K S

2296 CGT CTT GAT ACT ATG CGT GTT GAG TTA AAC TGG GCA GAA GTA GGG

766 R L D T M R V E L N W A E V G

2341 GCT GAG TCA CTT AGC CTA TAC CGA AAT GGC GAA CTA GTA GAT ACC

781 A E S L S L Y R N G E L V D T

2386 GTA TCA GAT AAT GGC CGT TAC CGT GAT TAT GTG CGT AAC GCA ACA

796 V S D N G R Y R D Y V R N A T

2431 TTA CCG GCA TAC GAC TAT CAG TTA TGC GTA TCT GAA AAC GTA TGT

811 L P A Y D Y Q L C V S E N V C

2476 TCG AAT ATT ATT ACG GTT TCA TTT TCT GAA TAA

826 S N I I T V S F S E *

The gene mcp01 cloning process of cold-adapted protease of the present invention is as follows, and step is as follows:

1, utilize the CTAB/NaCl method to extract the genomic dna of Pseudoomonas sp.SM9913,

2, N end and its self-dissolving fragment N end to proteolytic enzyme MCP-01 checks order, according to two degenerate primers of sequencing result design,

3, then, utilize the part of the gene of pcr clone proteins encoded enzyme MCP-01, again according to the portion gene design primer that obtains,

4, utilize the upstream and downstream sequence of Tail pcr clone known portions, splice after the order-checking, obtain the dna fragmentation of a 2676bp at last, the open reading frame that wherein contains the 2508bp of a coding MCP-01, initiator codon is positioned at 143bp, terminator codon is positioned at 2648bp, 835 amino acid of encoding altogether.

According to the two ends of this open reading frame design primer, utilize PCR from genomic dna, clone this gene and carry out sequence verification, the gene mcp01 that result's proof is cloned into is entirely true with the gene order of utilizing PCR and Tail pcr clone to arrive.

Utilize the CDD analysis software to carrying out analysis revealed according to the gene order deduced amino acid, the structure of the precursor of proteolytic enzyme MCP-01 comprises signal peptide, N end structure territory, catalyst structure domain, connecting zone, P-domain structural domain, PKD structural domain and C end regions seven parts.

The pre-enzyme of proteolytic enzyme MCP-01 and the structure of maturing enzyme are as shown in Figure 6.The maturing enzyme of deriving MCP-01 according to the N terminal sequence and the molecular weight (65.38kDa) of maturing enzyme comprises catalyst structure domain, connecting zone, P-domain structural domain, PKD structural domain, is a Multidomain subtilin proteinoid enzyme.The type proteolytic enzyme does not still have report at present.We are with this novel protein enzyme called after deseasin.

Excellent results of the present invention:

The cloned genes mcp01 of the present invention plain proteinoid enzyme of a kind of novel bacillus subtilis deseasin MCP-01 that encodes.The maturing enzyme of deseasin MCP-01 comprises a plurality of structural domains such as catalyst structure domain, connecting zone, P-domain structural domain, PKD structural domain, and the maturing enzyme of the subtilin proteinoid enzyme of report all had only a catalyst structure domain in the past.Compare with other subtilin proteinoid enzyme, deseasin MCP-01 can not only cut off and split the peptide bond that the P1 position is a hydrophobic amino acid, and is that the peptide bond of basic aminoacids has very high catalytic efficiency to the P1 position.Because the PKD structural domain can be in conjunction with the insolubility substrate, MCP-01 may have good degradation effect to insolubility albumen, and this enzyme is typical cold-adapted protease, low about 20 ℃ of the suitableeest enzyme temperature (35 ℃) alive middle temperature proteolytic enzyme the suitableeest enzyme temperature alive (55 ℃).Therefore, cloned genes mcp01 encoded protein enzyme of the present invention can have excellent usage in fields such as food-processing, milk-product processing and washing composition.

Description of drawings:

Fig. 1. the segmental electrophorogram of portion gene of the MCP-01 by pcr amplification clone.1: the dna fragmentation of amplification; 2:DNA molecular weight marker..

Fig. 2. the electrophorogram of dna fragmentation is closed in the segmental upstream of portion gene of the MCP-01 by the TAIL pcr amplification.1: the dna fragmentation of amplification; 2:DNA molecular weight marker.

Fig. 3 closes on the electrophorogram of dna fragmentation by the segmental downstream of the portion gene of the MCP-01 of TAIL pcr amplification.1:DNA molecular weight marker; 2: the dna fragmentation of amplification.

The aminoacid sequence of the gene nucleotide series of Fig. 4 .. clone's MCP-01 and the MCP-01 of coding thereof.Signal peptide cutting site and maturing enzyme N end and C end-grain cutting are cut the site and are indicated with arrow.Terminator is represented with asterisk.Underscore partly is the maturing enzyme sequence.First amino acid label of maturing enzyme is+1.Participate in catalytic three amino acid ((D ₄₉, H ₁₀₄And S ₂₆₉) indicate with black matrix.

The pre-enzyme of Fig. 5 .MCP-01 and maturing enzyme structural representation.

Fig. 6 .MCP-01 and other subtilin proteinoid enzyme sequence be evolutionary tree and structural representation thereof relatively.A: compare evolutionary tree, to indicate, except that MCP-01, all predict from genome sequence by all the other 12 deseasin proteinase genes in evolutionary tree for MCP-01; B: pre-enzyme structural representation.

Embodiment

Embodiment 1:

The foregoing SEQ ID of the encoding gene fragment sequence NO.1 of novel deep sea cold-adapted protease deseasin MCP-01.Nucleotide and coded amino acid corresponding relation are also as previously mentioned in the gene.This genomic DNA fragment is 2676bp altogether, wherein contains the open reading frame proteins encoded enzyme deseasin MCP-01 of a 2508bp, and initiator codon is positioned at 143bp, and terminator codon is positioned at 2648bp, 835 amino acid of encoding altogether.

Embodiment 2: gene fragment clone method of the present invention

Bacterial classification source: pseudomonas Pseudomonas sp.SM9913 separates in 1855 meters dark marine bottom sediments and obtains.Referring to Chen Xiulan etc., " low-temperature protease that deep sea cold-adaptive microbe bacterium strain SM9913 produces ", " ocean science " calendar year 2001, the 5th volume, the 1st phase, 4-8 page or leaf.

Concrete grammar is as follows:

1.MCP-01 purifying

(1) pseudomonas Pseudomonas sp.SM9913 bacteria suspension is inoculated in fermention medium (Semen Maydis powder 2%, wheat bran 1%, dregs of beans 2%, Na by 2% inoculum size ₂HPO ₄0.4%, K ₂PO ₄0.03%, CaCl ₂0.1%, Chen Haishui pH7.5), 50ml/500ml, 180 rev/mins, cultivated 72 hours by 12 ℃.

(2) cultivation finishes, collect nutrient solution, 4 ℃ following 11000 rev/mins centrifugal 15 minutes, get supernatant liquor, add ammonium sulfate powder to 55% saturation ratio while stirring in the ice bath kind, then 4 ℃ following 10000 rev/mins centrifugal 1 minute, remove supernatant, the precipitation dissolve in the 50mM Tris-HCl damping fluid (pH8.5).

(3) 4 ℃ following 11000 rev/mins centrifugal 15 minutes, with the supernatant liquor dialysis tubing of packing into, with 50mM Tris-HCl damping fluid (pH8.5) dialysis 20 hours, the polyoxyethylene glycol with molecular weight 20000 concentrated then.

(4) on the enzyme liquid after concentrating sephardex G100 post (1.6 * 100cm) carry out separation and purification, with 50mM Tris-HCl damping fluid (pH8.5) wash-out, flow velocity 8ml/h detects albumen with the nucleic acid-protein detector under 280nm, collect with automatic Fraction Collector, collected one and manage in 20 minutes.

(5) detect content and the purity that contains MCP-01 in proteic each pipe collection liquid with capillary electrophoresis.Pure MCP-01 solution is collected, be stored in-20 ℃ standby.

2.MCP-01 the mensuration of molecular weight

The MCP-01 solution of purifying is considered centrifuge tube and is concentrated into 1mg/ml. and serves sea base health biotech company with its molecular weight of MALDI-TOF mass spectroscopy with interception 3kDa is super.

3.MCP-01 and the segmental N end of self-dissolving order-checking.

(1) pure MCP-01 solution made its self-dissolving in 1 hour 35 ℃ of insulations, then will pure MCP-01 and the fragment of its self-dissolving electrophoresis together.Electrophoresis adopts the SDS-PAGE electrophoresis, adopts Mini-PROTEIN 3 electrophoresis apparatuss of Bio-Rad company, concentrates glue 4%, separation gel 12.5%, constant current 15mA electrophoresis 1 hour.

(2) MCP-01 in the running gel and self-dissolving fragment electricity thereof are gone on the pvdf membrane.Change the electrotransfer device that film adopts Bio-Rad company, operation is undertaken by its specification sheets.

(3) measure MCP-01 and the segmental N terminal sequence of its self-dissolving that goes on the film with the protein sequence automatic analyser.

4.MCP-01 the clone of encoding gene

4.1 the extraction of pseudomonas Pseudomonas sp.SM9913 genomic dna

With reference to " fine works molecular biology experiment guide ".

(1) cultivates the 50ml bacterial cultures to state of saturation, get the centrifugal 5min of culture 12000g of 15ml;

(2) throw out adds 5670 μ l TE, blows and beats repeatedly with suction pipe and makes it resuspended.Add 300 μ l 10%SDS and 30 μ l20mg/mL Proteinase Ks, mixing, 37 ℃ of incubation 2.5-3h;

(3) add 1000 μ l 5M NaCl, fully mixing adds 800 μ l CTAB/NaCl solution again, and mixing is in 65 ℃ of incubation 30-60min (beginning to remove the macromole of polysaccharide and other pollution) from this step;

(4) add isopyknic chloroform/primary isoamyl alcohol (24: 1), mixing, 4 ℃, the centrifugal 20min of 12000g changes supernatant liquor in the new pipe over to, shifts out supernatant as difficulty, removes boundary material with toothpick earlier;

(5) add isopyknic chloroform/primary isoamyl alcohol (24: 1), mixing, 4 ℃, the centrifugal 30min of 14000g forwards supernatant in the new pipe to;

(6) add isopyknic chloroform/primary isoamyl alcohol (24: 1), mixing, 4 ℃, the centrifugal 30min of 16000g forwards supernatant in the new pipe to;

(7) add 0.6 times of volume Virahol, mix (slowly turning upside down 3 minutes) gently, place 4 ℃ of refrigerators to keep 10min, the flocks ditch is gone out, change in the centrifuge tube of a 5ml with a thin curved glass rod;

(8) with washing in 70% ethanol of 3ml 2 times, use absolute ethanol washing again 2 times, the centrifugal 2min of each 12000g abandons supernatant, air-dry on the Bechtop (do not make the precipitation complete drying, otherwise extremely indissoluble);

(9) add 100-200 μ l TE damping fluid, 4 ℃ of dissolving DNAs that spend the night;

(10) adding 10mg/ml RNase is 50ug/mL to final concentration, 37 ℃ of incubation 1h;

(11) add equal-volume phenol/chloroform/primary isoamyl alcohol extracting 1 time, supernatant liquor is used chloroform/primary isoamyl alcohol extracting 1 time again;

(12) dehydrated alcohol of adding 1/10 volume 3M NaAc (pH5.2) and 2 times of volume precoolings in supernatant is inverted and is mixed;

(13) supernatant discarded gently after centrifugal, the DNA precipitation is respectively washed 1 time with 70% ethanol and dehydrated alcohol, is dissolved among an amount of TE buffer after dried slightly, measures DNA concentration and purity with ultraviolet spectrophotometer and is placed on 4 ℃ of refrigerators preservations (can in-20 ℃ of prolonged preservation).

4.2 utilize the portion gene of PCR clone gene MCP-01

(1) according to N terminal sequence SATNDP and the segmental N terminal sequence of the one bar self-dissolving AVDNDQ of MCP-01, design two degenerate primer M1:TCNGCNACNAA (TC) GA (TC) CC, M2:TG (GA) TC (GA) TT (GA) TCNACNGC, synthetic by Bo Ya biotech company.

(2) being primer with M1 and M2, is template with the genomic dna, does pcr amplification.The PCR reaction conditions is: 95 ℃, and 5 minutes; 95 ℃ then, 1 minute; 55 ℃, 1 minute; 72 ℃, 2 minutes, after 30 circulations, 72 ℃, extended 10 minutes.

(3) pcr amplification product is carried out 1% agarose gel electrophoresis, the DNA with Omega company reclaims test kit according to its explanation recovery amplification of DNA fragments then.

Be connected on the pGEMT carrier of Promega company reclaiming dna fragmentation.The ligation system:

Ligase enzyme Buffer 1 μ l

Carrier pGEMT 1 μ l

Exogenous dna fragment 3 μ l

T ₄Ligase enzyme 1 μ l

Add ddH ₂O to 10 μ l

Cover tight lid, finger flicks centrifuge tube, and the mixing sample changes 2sec on whizzer, and sample is concentrated on the pipe end, connects in 16 ℃ of water-baths and spends the night.

(4) go up the competent method of preparation intestinal bacteria by " molecular cloning experiment guide " and prepare the bacillus coli DH 5 alpha competence.

(5) the reorganization pGEM-T carrier that will connect by the heat shock method for transformation on " molecular cloning experiment guide " goes to the bacillus coli DH 5 alpha competence.

(6) bacillus coli DH 5 alpha of Zhuan Huaing is applied to the Mai Kangkai substratum that contains 50 μ g/L penbritins, 37 ℃ of incubated overnight, select white transformant as template, as primer, verify by bacterium colony PCR whether plasmid in the white transformant contains the dna fragmentation of pcr amplification with M1 and M2.

(7) transformant that plasmid is contained the dna fragmentation of pcr amplification is served the order-checking of sea living worker Bioisystech Co., Ltd.Therefore, obtain the portion gene sequence 620bp of MCP-01.

4.3 utilize the full gene of TAIL PCR clone gene MCP-01

(1) 5 ' and 3 ' terminal sequence according to the 620bp gene order of the MCP-01 of being cloned into designs 3 special primers and two universal primers respectively.Primer sequence is as follows:

N1：5’GCATCAATATCGGCATCAGCCG3’

N2：5’GCCACCAGTTTGGCCTTCGT3’

N3：5’AATCGCGGGTCGTTCGTGGC3’

C1：5’GCCAAGCGTTAGCTGATGCG3’

C2：5’TACGGCTAGTGAGCAAGCTG3’

C3：5’CGGGTAACGATGCGGTGGTC3’

AND：5’AAGCGTATG3’

ADC：5’GCAGCGTTA3’

Primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.

(2) adopt the two ends of the known portion gene of TAIL pcr amplification to close on sequence.The reaction conditions of TAIL PCR such as following table.

PCR number	Cycle number	The PCR reaction conditions
			PCR PCR PCR for the third time for the second time for the first time	1 5 1 10 12 1 12 1 25 1	94℃，5min 94℃，1min，58℃，1min，72℃，2.5min 94℃，1min，30℃，2min，rise to 72℃with speed of 0.3℃/s，72℃，2.5min 94℃，30s，40℃，1min，72℃，2.5min 94℃，30s，58℃，1min，72℃，2.5min 94℃，30s，58℃，1min，72℃，2.5min 94℃，30s，40℃，1min，72℃，2.5min 72℃，10min 94℃，30s，58℃，1min，72℃，2.5min 94℃，30s，58℃，1min，72℃，2.5min 94℃，30s，40℃，1min，72℃，2.5min 72℃，10min 94℃，30s，40℃，1min，72℃，1.5min 72℃，10min

(3) amplified production of TAIL PCR carries out the agarose gel electrophoresis analysis, increase from the known array two ends respectively dna fragmentation of about 500bp and 1500bp of result.

(4) these two dna fragmentations are served the sea and given birth to the order-checking of worker Bioisystech Co., Ltd.

(5) these two order-checking fragments and PCR clone's known fragments sequence is spliced, obtain the dna fragmentation of a 2676bp, wherein contain the complete open reading frame of the coding MCP-01 of a 2508bp.

4.4 the clone and the checking of MCP-01 gene to the clone

(1) 5 ' and 3 ' terminal sequence according to the open reading frame of coding MCP-01 designs two primers, and primer sequence is as follows:

MCPN：5’ATGAAAACAAAATTATCTATT3’

MCPC：5’TTATTCAGAAAATGAAACCGT3’

(2) with the genomic dna be template, with the complete genome of pcr amplification MCP-01.The PCR reaction conditions is: 95 ℃, and 5 minutes; 95 ℃ then, 1 minute; 50 ℃, 1 minute; 72 ℃, 3 minutes, after 30 circulations, 72 ℃, extended 10 minutes.

(3) the MCP-01 gene that pcr amplification is gone out inserts the pGEM-T carrier, and transformed into escherichia coli DH5 α serves the sea and gives birth to the order-checking of worker Bioisystech Co., Ltd.The MCP-01 gene order that sequencing result and front are cloned into by PCR and TAIL PCR compares, the checking gene order.

5. result

By being checked order, MCP-01 and self-dissolving fragment thereof obtain MCP-01 and the segmental N terminal sequence of one bar self-dissolving.The N terminal sequence of MCP-01 is SATNDP, and wherein the segmental N terminal sequence of self-dissolving is AVDNDQ.According to the nucleotide sequence design primer of these two sequences of coding, be template with the genomic dna, be cloned into the dna fragmentation of a 620bp, the portion gene (Fig. 1) of coding MCP-01 by pcr amplification.According to the sequence dna fragment that obtains, the design primer, with the genomic dna is template, the sequence of closing on by TAIL pcr amplification known dna fragment two ends, obtain dna fragmentation (Fig. 2 of 554bp and 1622bp respectively, Fig. 3), obtain the dna sequence dna of a 2676bp by splicing, sequence is shown in SEQ ID N0.1.The open reading frame that wherein contains the coding MCP-01 of a 2508bp by the blastX software analysis discovery of NCBI website.With two primers of 5 ' and 3 ' terminal sequence design of open reading frame, be template with the genomic dna, be cloned into the complete genome of coding MCP-01 by pcr amplification.This gene order is carried out sequencing result to be proved with the MCP-01 gene order of being cloned into by PCR and TAIL PCR in full accord.The complete genome sequence of MCP-01 and amino acid sequence coded thereof are as shown in Figure 4.

Utilize the structure of the CDD software analysis MCP-01 of NCBI website, the result as shown in Figure 5, the structure of the pre-enzyme of proteolytic enzyme MCP-01 comprises signal peptide, N end structure territory, catalyst structure domain, connecting zone, P-domain structural domain, PKD structural domain and C end regions seven parts.The N terminal sequence of MCP-01 maturing enzyme is SATNDP, and its molecular weight of MALDI-TOF mass spectroscopy is 65.38kDa.Therefore, the maturing enzyme of deriving MCP-01 according to the N terminal sequence and the molecular weight of MCP-01 maturing enzyme comprises catalyst structure domain, connecting zone, P-domain structural domain, PKD structural domain, is a Multidomain subtilin proteinoid enzyme.By homology comparison with make up evolutionary tree find that MCP-01 is identical with other 12 the proteolytic enzyme structures of predicting from genome, and with other the structural difference very big (Fig. 6) of subtilin proteinoid enzyme.The type proteolytic enzyme does not still have report at present, and MCP-01 is the type proteolytic enzyme of first separated purifying.

Sequence table

＜110〉Shandong University

＜120〉gene mcp01 of a kind of cold-adapted protease and preparation method thereof

<140>

<141>

<160>1

<170>Patent In3.1

<210>1

<211>330

<212>DNA

<213>Pseudomonas sp.SM9913

<400>1

gttttgttcc tgattttgct ttttttactt gagttagtga cggattaccc gttttcttaa 60

cgcttgccac gtgattactt aaattcataa acgtaaatat ttcgcttagt aaagctaagt 120

taacttaatt acggatagaa caatgaaaac aaaattatct attattacac ttgcactttt 180

accttgttta gctgcggcta aattgcctga tgttaacagt acaactcaaa aagtaactgc 240

gagcagtgac tcgattattg ttaagtataa aaagaatgct tcgccgaaaa tgcgtaagca 300

agctcgttcg ttagtaaaag ccaaaatatc tgatctaaat aacgatgaaa ttgatgataa 360

cttcacctcg ttattttctg gccgcctcgc aaaatttaaa atatcaggaa tgagcgctaa 420

agaagcaatt gagcgcttaa aatcgcatca agctattgag tatgttgagc ctgattaccg 480

agtgagcata gcaagcgcaa cgaacgaccc gcgatttgac gatttatggg gcttaaacaa 540

cgaaggccaa actggtggca cggctgatgc cgatattgat gcaccagaag catggtcaat 600

ctctacaggt agtcgtgatg ttgtagtagg tgttattgat acgggtgttg attatagtca 660

ccctgattta gctgcgaacg cgtgggtaaa cagtggtgaa attgccggtg atggtatcga 720

taatgacgga aacggttaca ttgatgacgt tcacggtatt aatgcaatta ctgatgttgg 780

tgatccgatg gatgacgaag gccatggtac acacgtatcg ggtacaatcg gtgccagtgg 840

taacaatggt gtcggtgttg ttggtgtaaa ccatgatgtt tctattgttg gttgtaagtt 900

tttagccgca gatggtacag gttctacctc tggtgctatt aagtgtatcg attacatggt 960

tggccttaaa aatgcgggtg ttaatctgcg tgtgttaaat aatagctggg gcggcggtgg 1020

ttttagccaa gcgttagctg atgcgattac ggctagtgag caagctgata ttttatttgt 1080

tgctgctgcg ggtaacgatg cggttgataa cgatcaaaat ccacactacc catctaatta 1140

tgaaaacgac aacgtattat cgatagctag tactgatagc agagataata tgtctagctt 1200

ttctcaatgg ggcctaacaa gtgttgatat gggcgcacct ggctcaggta ttttgtcgac 1260

cgttcctggt aatagctatg ctacttattc aggtacatct atggcaacac cgcatgtagc 1320

tggtgctgct gcacttgtac tttcagttaa tcctgattta acaacactag agcttaaaga 1380

attacttatg tctagtggtg atgctaatgc ggcattaaat ggtaaaacgg ttgcaggtac 1440

acgcctaaat gttaaccaag cgctaattga tgctgatcca actccaggct ttaaattatc 1500

tgtatcgccg gtttctcagc aagcgactgt aggtgatacg gttacttata cctttacgat 1560

tggctcaatt gcacaatggg aaggtgacgt ttcactggct ttagcttcag atcttaccga 1620

tgcatcacta agcgcaacta ccgctcgtcc gggcgatgaa gttgttctaa cggttgcaac 1680

taacagcgat actcagtggg gaaattacga ctttactgtt accgcaacaa ctgaagagca 1740

agttaaagaa caaactgtta gcttaatgct tcaacctgct ggtttaaatg actttactta 1800

cagcagtgat gaaaacatcg ctattccaga taattctcca gagggcgcaa gctcagttat 1860

tacggttgct gatgatttaa ctatttttgg caccactgct gatgtagata ttacgcatac 1920

ttggtctggt gacttagtac ttacgcttat ttcagcgcaa ggtacagaag taactttaca 1980

aagcaatgaa ggtggcagtg cagacgatat tgttaagtca tttacatcaa gcgtatttaa 2040

ttctgaagtg gcaaccggag attggacact tcatgttgaa gatacagctg gtgcagatac 2100

gggtaatatc aatggttggt cacttacatt tagcgctatt ggtgaggtaa gcccacaacc 2160

accacaagct ggctttagtg tttcagcaca aggtttaact gctacatttg taggtacaag 2220

tcgtgatgta aatgatgaca tttcacagtg gagctggaac tttggtgatg gttcaacatc 2280

aacaaaccca aatcctacac atgtttacgc tcagtcagga agttatgagg ttgaattaac 2340

cgtaacggat agtgaaaata actcagatac attcactcaa acagtggttg ttagtgatgt 2400

tgaaattgaa ttgagcttaa aacgcgctaa caagtcacgt cttgatacta tgcgtgttga 2460

gttaaactgg gcagaagtag gggctgagtc acttagccta taccgaaatg gcgaactagt 2520

agataccgta tcagataatg gccgttaccg tgattatgtg cgtaacgcaa cattaccggc 2580

atacgactat cagttatgcg tatctgaaaa cgtatgttcg aatattatta cggtttcatt 2640

ttctgaataa aacgtttacc ccttagacat tcaaag 2676

Claims (8)

1, contain the genomic DNA fragment of deep-sea cold-adapted protease gene mcp01, have following sequence:

gttttgttcc tgattttgct ttttttactt gagttagtga cggattaccc gttttcttaa 60

cgcttgccac gtgattactt aaattcataa acgtaaatat ttcgcttagt aaagctaagt 120

taacttaatt acggatagaa caatgaaaac aaaattatct attattacac ttgcactttt 180

accttgttta gctgcggcta aattgcctga tgttaacagt acaactcaaa aagtaactgc 240

gagcagtgac tcgattattg ttaagtataa aaagaatgct tcgccgaaaa tgcgtaagca 300

agctcgttcg ttagtaaaag ccaaaatatc tgatctaaat aacgatgaaa ttgatgataa 360

cttcacctcg ttattttctg gccgcctcgc aaaatttaaa atatcaggaa tgagcgctaa 420

agaagcaatt gagcgcttaa aatcgcatca agctattgag tatgttgagc ctgattaccg 480

agtgagcata gcaagcgcaa cgaacgaccc gcgatttgac gatttatggg gcttaaacaa 540

cgaaggccaa actggtggca cggctgatgc cgatattgat gcaccagaag catggtcaat 600

ctctacaggt agtcgtgatg ttgtagtagg tgttattgat acgggtgttg attatagtca 660

ccctgattta gctgcgaacg cgtgggtaaa cagtggtgaa attgccggtg atggtatcga 720

taatgacgga aacggttaca ttgatgacgt tcacggtatt aatgcaatta ctgatgttgg 780

tgatccgatg gatgacgaag gccatggtac acacgtatcg ggtacaatcg gtgccagtgg 840

taacaatggt gtcggtgttg ttggtgtaaa ccatgatgtt tctattgttg gttgtaagtt 900

tttagccgca gatggtacag gttctacctc tggtgctatt aagtgtatcg attacatggt 960

tggccttaaa aatgcgggtg ttaatctgcg tgtgttaaat aatagctggg gcggcggtgg 1020

ttttagccaa gcgttagctg atgcgattac ggctagtgag caagctgata ttttatttgt 1080

tgctgctgcg ggtaacgatg cggttgataa cgatcaaaat ccacactacc catctaatta 1140

tgaaaacgac aacgtattat cgatagctag tactgatagc agagataata tgtctagctt 1200

ttctcaatgg ggcctaacaa gtgttgatat gggcgcacct ggctcaggta ttttgtcgac 1260

cgttcctggt aatagctatg ctacttattc aggtacatct atggcaacac cgcatgtagc 1320

tggtgctgct gcacttgtac tttcagttaa tcctgattta acaacactag agcttaaaga 1380

attacttatg tctagtggtg atgctaatgc ggcattaaat ggtaaaacgg ttgcaggtac 1440

acgcctaaat gttaaccaag cgctaattga tgctgatcca actccaggct ttaaattatc 1500

tgtatcgccg gtttctcagc aagcgactgt aggtgatacg gttacttata cctttacgat 1560

tggctcaatt gcacaatggg aaggtgacgt ttcactggct ttagcttcag atcttaccga 1620

tgcatcacta agcgcaacta ccgctcgtcc gggcgatgaa gttgttctaa cggttgcaac 1680

taacagcgat actcagtggg gaaattacga ctttactgtt accgcaacaa ctgaagagca 1740

agttaaagaa caaactgtta gcttaatgct tcaacctgct ggtttaaatg actttactta 1800

cagcagtgat gaaaacatcg ctattccaga taattctcca gagggcgcaa gctcagttat 1860

tacggttgct gatgatttaa ctatttttgg caccactgct gatgtagata ttacgcatac 1920

ttggtctggt gacttagtac ttacgcttat ttcagcgcaa ggtacagaag taactttaca 1980

aagcaatgaa ggtggcagtg cagacgatat tgttaagtca tttacatcaa gcgtatttaa 2040

ttctgaagtg gcaaccggag attggacact tcatgttgaa gatacagctg gtgcagatac 2100

gggtaatatc aatggttggt cacttacatt tagcgctatt ggtgaggtaa gcccacaacc 2160

accacaagct ggctttagtg tttcagcaca aggtttaact gctacatttg taggtacaag 2220

tcgtgatgta aatgatgaca tttcacagtg gagctggaac tttggtgatg gttcaacatc 2280

aacaaaccca aatcctacac atgtttacgc tcagtcagga agttatgagg ttgaattaac 2340

cgtaacggat agtgaaaata actcagatac attcactcaa acagtggttg ttagtgatgt 2400

tgaaattgaa ttgagcttaa aacgcgctaa caagtcacgt cttgatacta tgcgtgttga 2460

gttaaactgg gcagaagtag gggctgagtc acttagccta taccgaaatg gcgaactagt 2520

agataccgta tcagataatg gccgttaccg tgattatgtg cgtaacgcaa cattaccggc 2580

atacgactat cagttatgcg tatctgaaaa cgtatgttcg aatattatta cggtttcatt 2640

ttctgaataa aacgtttacc ccttagacat tcaaag 2676。

2, the genomic DNA fragment of proteinase gene mcp01 as claimed in claim 1, it is characterized in that wherein containing a open reading frame with 2508 Nucleotide, this open reading frame is the gene of proteins encoded enzyme MCP-01, initiator codon is positioned at 143bp, terminator codon is positioned at 2648bp, and coded amino acid is 835 altogether.

3, the genomic DNA fragment of proteinase gene mcp01 as claimed in claim 2 is characterized in that the structure of the precursor of described proteolytic enzyme MCP-01 comprises signal peptide, N end structure territory, catalyst structure domain, connecting zone, P-domain structural domain, PKD structural domain and C end regions seven parts.

4, the genomic DNA fragment of proteinase gene mcp01 as claimed in claim 2, the maturing enzyme that it is characterized in that described proteolytic enzyme MCP-01 comprises catalyst structure domain, connecting zone, P-domain structural domain, PKD structural domain, is a Multidomain subtilin proteinoid enzyme.

5, the genomic DNA fragment of proteinase gene mcp01 as claimed in claim 2 is characterized in that the suitableeest enzyme temperature alive of described proteolytic enzyme MCP-01 is about 35 ℃, and optimal pH is 9.0.

6, the gene mcp01 cloning process of cold-adapted protease, step is as follows:

(1) utilize the CTAB/NaCl method to extract the genomic dna of Pseudoomonas sp.SM9913,

(2) N end and its self-dissolving fragment N end to proteolytic enzyme MCP-01 checks order, according to two degenerate primers of sequencing result design,

(3) then, utilize the part of the gene of pcr clone proteins encoded enzyme MCP-01, again according to the portion gene design primer that obtains,

(4) utilize the upstream and downstream sequence of Tail pcr clone known portions, splice after the order-checking, obtain the dna fragmentation of the 2676bp of a described sequence of claim 1 at last, the open reading frame that wherein contains the 2508bp of a coding MCP-01, initiator codon is positioned at 143bp, terminator codon is positioned at 2648bp, 835 amino acid of encoding altogether.

7, the gene mcp01 cloning process of cold-adapted protease as claimed in claim 6 is characterized in that, described two degenerate primers of step (2) are M1:TCNGCNACNAA (TC) GA (TC) CC, M2:TG (GA) TC (GA) TT (GA) TCNACNGC.

8, the gene mcp01 cloning process of cold-adapted protease as claimed in claim 6, it is characterized in that, step (3) is that 5 ' and 3 ' terminal sequence according to the 620bp gene order of the MCP-01 of being cloned into designs 3 special primers and two universal primers respectively, and primer sequence is as follows:

N1：5’GCATCAATATCGGCATCAGCCG 3’

N2：5’GCCACCAGTTTGGCCTTCGT 3’

N3：5’AATCGCGGGTCGTTCGTGGC 3’

C1：5’GCCAAGCGTTAGCTGATGCG 3’

C2：5’TACGGCTAGTGAGCAAGCTG 3’

C3：5’CGGGTAACGATGCGGTGGTC 3’

AND：5’AAGCGTATG 3’

ADC：5’GCAGCGTTA 3’。

Images