CN103215192A - Aspergillus bacterial strain and endo-chitosan enzyme CsnW2 encoding gene, preparation method and application thereof - Google Patents

Abstract

The invention discloses an endo-chitosan enzyme CsnW2 gene sequence and preparation method and application thereof. The invention relates to a soil new isolated bacterial strain Aspergillus clavatus from the endo-chitosan enzyme CsnW2. The invention also provides a method for preparing the novel chitosan enzyme, ie a gene engineering technique, which includes the following steps: cloning the novel chitosan enzyme gene to a pichia yeast expression vector, and obtaining a pichia yeast recombination bacterial strain for heterogenous express of the enzyme. The chitosan enzyme CsnW2 prepared by heterogenous expression of the bacterial strain has high activity at pH 3.5-5.0 and 40-60 DEG C. with Chitosan oligosaccharide preparation function by chitosan degradation. The chitosan enzyme CsnW2 provided by the invention can be widely applied to the chemical industry, the agriculture, the foodstuff, the forage adding, the medicine and other fields, and has large production potential and economic value.

Description

The encoding gene of Aspergillus bacterial strain and inscribe chitoanase, preparation and application

Technical field

The present invention relates to gene order of a kind of inscribe chitoanase CsnW2 and its production and application.The present invention also provides the recombinant plasmid and the recombination engineering strain of this inscribe chitoanase.Inscribe chitoanase CsnW2 of the present invention can be widely used in chemical industry, agricultural, food, feed interpolation and medicine and other fields.

Background technology

Chitosan (chitosan), have another name called poly-glucosamine, chitin, chitosan, chitosan, solubility chitin, Viartril-S, crust saccharan and poly-chitose etc., chemistry poly-β by name-(1 → 4)-2-amino-2-deoxy-D-glucose.Chitosan is the product that chitin obtains through deacetylation, and general chitinous deacetylation surpasses at 50% o'clock and is considered to chitosan.Chitosan extensively is present in the cell walls of shrimp, crab, insect shell and algae and mushroom.Because its molecular weight is bigger, poorly water-soluble makes its widespread use be subjected to considerable restraint.But its degraded product, a series of oligochitosans and glucosamine have unique physiologically active and functional property, as regulate the absorption of immunity, antitumor, antibiotic, reducing cholesterol and promotion calcium, and it is soluble in water, be a kind of emerging functional oligose, be widely used in fields such as food, medicine, agricultural and makeup.

At present, the method for preparing oligochitosan mainly contains chemical method, physics method and biological process.But the electrochemical conditions harshness, the stability of reaction and poor repeatability, the product polymerization degree is wayward, and is seriously polluted, is difficult to obtain the oligomeric oligochitosan of the relative homogeneous of the polymerization degree.The product polymerization degree that the physics method obtains is also wayward, and its repeatability is also relatively poor, so gentle, efficient, product is easy to control and stablize free of contamination biological process has caused people's attention gradually.It is to utilize the various chitoanases of the biology, particularly microorganisms of occurring in nature existence to come degrade chitosan that biological process prepares oligochitosan, how to obtain the microorganism strains of high yield chitoanase and the research of the chitoanase that high enzyme is lived and receives much concern.

Chitoanase distributes very extensive, detects the existence of chitoanase at present from the different tissues of bacterium (as: Bacillus, Myxobacter, Enterobacter), actinomycetes (as: Streptomyces, Nocardioides), mould (as: Aspergillus, Penicillium), virus (as chlorella virus PBCV-1, CVK-2) and plant.

The classification of chitoanase at present has dual mode.The one, according to the hydrolysis classification of enzyme to glycosidic link, substrate specificity according to enzyme is divided into 3 classes with chitoanase: the 1st class mainly is can hydrolysis GlcNAc-GlcN and the chitoanase of GlcN-GlcN glycosidic link, as the chitoanase from Bacillus pumilus BN-262 and Streptomyces sp.N174; The 2nd class can only hydrolysis GlcN-GlcN key, produces this zymoid bacterial strain and has only Bacillus sp.No.7-M at present; The 3rd class is hydrolyzable GlcN-GlcN key both, hydrolyzable GlcN-GlcNAc key again, and bacterium producing multi enzyme preparation comprises S.gmeus HUT6037 and B.circulans MH.K1 etc.Be chitoanase I, II, III class.The 2nd, according to the aminoacid sequence classification of enzyme, chitoanase mainly is distributed in 5,8,46,75 and 80 5 families of glycoside hydrolysis enzyme family.Wherein the chitoanase of bacterial origin mainly belongs to 46 families, and the chitoanase of originated from fungus mainly belongs to 75 families.Two family's chitoanase homologys are poor, and the chitoanase of bacterial origin research at present is more, and for fungi, it is less especially to derive from excellent aspergillar chitoanase research.The cloning and expression that derives from excellent aspergillar chitosanase gene does not at present appear in the newspapers.

Summary of the invention

First purpose of the present invention provides a kind of Aspergillus bacterial strain that produces chitoanase, and it is excellent aspergillus W-2, classification name: excellent aspergillus Aspergillus clavatus, deposit number: CGMCC NO.7018.

Second purpose of the present invention provides a kind of new and effective inscribe chitoanase CsnW2 and encoding gene thereof.

The 3rd purpose of the present invention provides a kind of method for preparing new and effective inscribe chitoanase CsnW2.

The 4th purpose of the present invention provides the gene engineering expression system that contains described inscribe chitoanase CsnW2 gene.

The 5th purpose of the present invention provides the application of new and effective inscribe chitoanase CsnW2 in degradation of chitosan.

Inscribe chitoanase CsnW2 provided by the present invention derives from the Aspergillus strains A spergillus clavatus w-2 of separation and purification in the soil, and its aminoacid sequence has one of following feature:

1) 1-242 or the 18-242 amino acids residue sequence that SEQ ID NO.3 begins from aminoterminal in the sequence table;

2) 1-242 that the SEQ ID NO.3 in the sequence table is begun from aminoterminal or 18-242 amino acids residue sequence have the active protein of degrade chitosan through the replacement and/or the disappearance of amino-acid residue and/or after adding;

3) with sequence table in the homology of the aminoacid sequence that limited of SEQ ID NO.3 reach 90% and above and have the active protein of degrade chitosan.

The present invention also provides the encoding gene (called after csnW2) of inscribe chitoanase CsnW2, has one of following nucleotide sequence feature:

1) has Yeast Nucleic Acid (RNA) sequence of SEQ ID NO.1 in the sequence table and thymus nucleic acid (DNA) sequence of SEQ ID NO.2;

2) thymus nucleic acid (DNA) sequence of coding SEQ ID NO.3 aminoacid sequence;

3) have with sequence table in the homology of thymus nucleic acid (DNA) sequence that limited of SEQ ID NO.2 reach 90% and more than, and proteinic thymus nucleic acid (DNA) sequence of the degrade chitosan of encoding.

The aminoacid sequence of chitoanase CsnW2 of the present invention and nucleotide coding sequence thereof also can obtain according to aminoacid sequence and the nucleotide coding sequence synthetic thereof of the CsnW2 that predicts.

The method for preparing recombinase CsnW2 is that encoding gene csnW2 is cloned into recombinant expression vector, imports host cell, obtains recombinant expressed inscribe chitoanase.

The expression vector of described recombinant expressed inscribe chitoanase CsnW2 can be coli expression carrier, Yeast expression carrier, Bacillus subtilus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungus expression vector, plant expression vector, insect expression vector or mammalian cell expression vector etc.

The reorganization bacterium or the transgenic cell line that are used for recombinant expressed inscribe chitoanase CsnW2, can be that e. coli host cell is (as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α etc.), the yeast host cell is (as Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis etc.), the Bacillus subtilus host cell is (as Bacillus subtilis R25, Bacillus subtilis9920 etc.), milk-acid bacteria host cell (as Lactic acid bacteria COCC101 etc.), actinomycetes host cell (as Streptomyces spp. etc.), filamentous fungal host cell is (as Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans etc.), insect cell is (as Bombyx mori, Antharaea eucalypti etc.) or mammalian cell (as Chinese hamster ovary cell CHO, immature hamster kidney cell BHK, Chinese hamster pneumonocyte CHL etc.).

CsnW2 of the present invention derives from the Aspergillus strains A spergillus clavatus w-2 of separation and purification in the soil.Its gene and aminoacid sequence obtain by the method clone of Race.Concrete grammar:

(1) clone of its 3 ' end of inscribe chitoanase CsnW2 mRNA sequence.Fungi 18S and morphologic observation preliminary evaluation bacterial strain are excellent aspergillus.By the sequence of chitoanase in the nearer bacterial strain of homology is carried out the homology comparison.Find out about 7 amino acid whose conserved sequences; MRNA to correspondence carries out the homology comparison again, designs a degenerated primer.Extracting total RNA of excellent aspergillus w-2, according to Takara RNA PCR Kit(Ver.3.0) specification sheets carries out RT-PCR amplification., reclaim the purpose band and is connected with 1% agarose gel electrophoresis analysis through the product of two-wheeled pcr amplification, transform Top10, after PCR identifies, serve Beijing order-checking portion of extra large Ying Jun Bioisystech Co., Ltd and check order, obtain 3 ' and hold the mRNA sequence with the pMD19-T carrier.

(2) clone of its 5 ' end of inscribe chitoanase CsnW2 mRNA sequence: 3 ' end mRNA based on aforesaid method obtains, design two reverse primers.Carry out 5 ' RACE according to Clontech SMARTerTM RACE cDNA Amplification Kit specification sheets., reclaim the purpose band and is connected with 1% agarose gel electrophoresis analysis through the product of two-wheeled pcr amplification, transform Top10, after PCR identifies, serve Beijing order-checking portion of extra large Ying Jun Bioisystech Co., Ltd and check order, obtain 5 ' and hold the mRNA sequence with the pMD19-T carrier.

(3) 3 ' and 5 ' Race result is spliced, and carry out bioinformatic analysis.On NCBI,, and then derive the aminoacid sequence of inscribe chitoanase CsnW2 to the open reading frame analysis.Design corresponding primer, with mRNA is that template is carried out reverse transcription and PCR, the PCR product is with 1% agarose gel electrophoresis analysis, reclaim the purpose band and be connected with the pMD19-T carrier, transform Top10, after PCR identifies, serve the order-checking of Beijing order-checking portion of extra large Ying Jun Bioisystech Co., Ltd, obtain full length sequence.

Above-mentioned inscribe chitosanase gene coding head of district 729bp, 262 amino acid of having encoded, the about 29kD of molecular weight belongs to chitoanase family 75.The CsnW2 of the recombinant expressed acquisition of pichia spp when being substrate with the chitosan, has greater activity under 40 ℃-60 ℃, the condition of pH3.5-5.0.

Chitoanase CsnW2 provided by the invention can be widely used in chemical industry, agricultural, food, feed interpolation and medicine and other fields, has bigger production potential and economic worth.

Sequence table

SEQ?ID?NO.1

AUGCGCUAUCUUGCAACUGCUGCCGUUUUGGCUGGAGCAG

GCCUGGCCUCGGCCUAUAGUGUCCCAGCCAACCUACAGCAA

AUCUAUAACAAACACAAGACCGGAACAUGCCAGAACAAGC

UGCAAGACGGUUUUUCCGAUGGCAUCAGCGGCCCCGGCAC

CUCCGCCUACUGCGGCGACAUCCAGGGGGCGAUCUUCCUUC

UAGCUCCGCCAAUGGCGGCCAGUAUGAUAACAUGGACAU

UGACUGCGAUGGAGCGAACAACAGCGGCGGCGACUGUGCG

AAUGAUCCCUCCGGCCAGAGCAUGACGGCGUUCAUGGAUA

CGGUGAAGCAAUACGGCAUUUCGGAUCUGGACGCCAACAU

CCACCCGUACGUGGUGUUUGGUAACUCGGGCAGCUCGCCG

ACCUUUGAUCCUCAGCAGUAUGGAAUGCAGCCGUUAAGCG

UGAUGGCUGUGGUGUGCAAUAAUCAGCUGUUCUAUGGUGU

CUGGGGUGACACCAACGGUGAUAUCGCUACUGGGGAGGCU

UCCAUCUCGCUGGCCAAAUUGUGUUUCCCCAAUGAUGGUA

UCACCGGUGACAAUGGCCAUGACCAGGACGAUGUACUCUA

CAUUGGGUUUACGGGGCAGGAUACUGUCCCCGGUGCUAGU

GCUGCCUGGACUGCUAGCGACACUUCAACCUUUGAGGAGA

GUAUCAAGGGUCUCGGUGAUCGCCUUGUUGCUAAGCUUAG

CGCUUAA

SEQ?ID?NO.2

ATGCGCTATCTTGCAACTGCTGCCGTTTTGGCTGGAGCAGGCCT

GGCCTCGGCCTATAGTGTCCCAGCCAACCTACAGCAAATCTATA

ACAAACACAAGACCGGAACATGCCAGAACAAGCTGCAAGACG

GTTTTTCCGATGGCATCAGCGGCCCCGGCACCTCCGCCTACTGC

GGCGACATCCAGGGGGCGATCTTCCTTCATAGCTCCGCCAATGG

CGGCCAGTATGATAACATGGACATTGACTGCGATGGAGCGAAC

AACAGCGGCGGCGACTGTGCGAATGATCCCTCCGGCCAGAGCA

TGACGGCGTTCATGGATACGGTGAAGCAATACGGCATTTCGGAT

CTGGACGCCAACATCCACCCGTACGTGGTGTTTGGTAACTCGG

GCAGCTCGCCGACCTTTGATCCTCAGCAGTATGGAATGCAGCCG

TTAAGCGTGATGGCTGTGGTGTGCAATAATCAGCTGTTCTATGG

TGTCTGGGGTGACACCAACGGTGATATCGCTACTGGGGAGGCT

TCCATCTCGCTGGCCAAATTGTGTTTCCCCAATGATGGTATCACC

GGTGACAATGGCCATGACCAGGACGATGTACTCTACATTGGGTT

TACGGGGCAGGATACTGTCCCCGGTGCTAGTGCTGCCTGGACTG

CTAGCGACACTTCAACCTTTGAGGAGAGTATCAAGGGTCTCGG

TGATCGCCTTGTTGCTAAGCTTAGCGCTTAA

SEQ?ID?NO.3

MRYLATAAVLAGAGLASAYSVPANLQQIYNKHKTGTCQNKLQDG

FSDGISGPGTSAYCGDIQGAIFLHSSANGGQYDNMDIDCDGANNS

GGDCANDPSGQSMTAFMDTVKQYGISDLDANIHPYVVFGNSGSS

PTFDPQQYGMQPLSVMAVVCNNQLFYGVWGDTNGDIATGEASIS

LAKLCFPNDGITGDNGHDQDDVLYIGFTGQDTVPGASAAWTASD

TSTFEESIKGLGDRLVAKLSA

Description of drawings

Fig. 1: the polyacrylamide gel electrophoresis figure (SDS-PAGE) of recombined endo chitoanase CsnW2 expression and purifying.The sample that each swimming lane adds is respectively: M: protein marker (marker), band size from top to bottom are 170kD, 130kD, 100kD, 70kD, 55kD, 40kD, 35kD, 25kD; Swimming lane 1: fermented liquid supernatant, applied sample amount 20 μ l, the imidazoles wash-out of swimming lane 2:100mmol is collected liquid, applied sample amount 20 μ l.

Fig. 2: the pH value is to the activity influence curve of inscribe chitoanase CsnW2.

Fig. 3: temperature is to the activity influence curve of inscribe chitoanase CsnW2.

Fig. 4: the HPLC figure of chitoanase CsnW2 degrade chitosan products therefrom

The bacterial strain of Aspergillus, it is excellent aspergillus W-2, classification name: excellent aspergillus Aspergillus clavatus, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number: CGMCC NO.7018, preservation date on December 18th, 2012.

Embodiment

The present invention prepares the method for this novel chitosan enzyme, promptly utilize engineered technological method, with the gene clone of this new chitoanase to yeast expression vector, but obtain the pichia spp recombinant bacterial strain of this enzyme of heterogenous expression, under pH3.5-5.0 and 40 ℃-60 ℃, greater activity is arranged with the chitoanase CsnW2 of this bacterial strain heterogenous expression preparation, have the function that degrade chitosan prepares oligochitosan.

The cultivation of embodiment 1 Aspergillus strains A spergillus clavatus W-2 and production chitoanase

The bacterial classification that is adopted is excellent aspergillus, the mono-clonal of picking Aspergillus clavatus W-2 bacterial strain (CGMCC NO.7018) is inoculated in the 30ml liquid nutrient medium, then put into temperature and be 30 ℃, revolution and be the shaking table of 150rpmin and cultivate after 4 days, medium centrifugal is collected the supernatant substratum.

The liquid culture based formulas of using is (g/L): chitosan 5.0g, yeast soak powder 0.5g, NaCl0.5g, MgSO47H2O1.44g, CaCl20.1g, KCl0.5g, K2HPO42.0g, KH2PO41.0g, and the pH value is 7.0, and surplus is a water.

Enzyme activity unit (U) definition: per minute catalysis chitosan produces the needed enzyme amount of 1 μ mol reducing sugar.Record in the liquid nutrient medium supernatant of Aspergillus clavatus W-2 bacterial strain enzyme lives and is 0.53U/ml according to the DNS method.

The extraction of the total RNA of embodiment 2Aspergillus clavatus W-2 bacterial strain

Bacterial strain is carried out cultivating in 4 days, use filtered through gauze, thalline is directly put into liquid nitrogen preserve.Therefrom get about 40mg thalline, under liquid nitrogen, grind three times.Add 1ml Trizol reagent afterwards, wait to melt the back and move into the 1.5ml centrifuge tube.Add the 200ul chloroform in system, concuss leaves standstill 5min, the centrifugal 15min of 12000rpm.Carefully get supernatant in new centrifuge tube, add isopyknic Virahol, leave standstill 5min, the centrifugal 10min of 12000rpm.Outwell supernatant, adding volumetric concentration in precipitation is 75% ethanol 1ml, the centrifugal 5min of 7500rpm.The water 20-50ul that will precipitate at last with no ribose ribozyme activity melts.It is stand-by to put-80 ℃ of preservations.

The clone of embodiment 3csnW2 gene 3 ' end mRNA sequence

By operation steps in precious biotechnology (Dalian) company limited 3 '-Full RACE Core Set Ver.2.0 (Takara Code:D314) specification sheets, with wherein 3 ' RACEAdaptor is primer, and the total RNA of Aspergillus clavatus W-2 bacterial strain is article one chain of the synthetic cDNA of template reverse transcription; With 3 ' RACE Outer Primer (TACCGTCGTTCCACTAGTGATTT) and SP Primer (ATGGAYATCGACTGYGAYGG) is that primer carries out first round PCR, its reaction conditions is: 94 ℃ of 3min, 30 circulations (94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 1.2min), 72 ℃ of 10min; With 3 ' RACE Inner Primer (CGCGGATCCTACCGTCGTTCCACTAGTGATTTCACTATAGG) and SP Primer (ATGGAYATCGACTGYGAYGG) is that primer carries out second and takes turns PCR, reaction conditions is: 94 ℃ of 3min, 30 circulation (94 ℃ of 30sec, 58 ℃ of 30sec, 72 ℃ of 1.2min), 72 ℃ of 10min.Two-wheeled PCR product with 1% agarose gel electrophoresis analysis, is reclaimed the purpose band and is connected with the pMD19-T carrier, be transformed into Top10, after bacterium colony PCR checking, serve extra large Ying Jun Bioisystech Co., Ltd and check order.

The clone of embodiment 4csnW2 gene 5 ' end mRNA sequence

According to the requirement of Clontech SMARTerTM RACE cDNA Amplification Kit specification sheets, to the pre-treatment of carrying out of the total RNA of Aspergillus clavatus W-2 bacterial strain; With 3 ' terminal sequence is reference, designs two primers, 5RACE Outer sp W1(GGACACGAACGTATCAAATCGACTA) and 5RACE Inner sp W1(TCCTGCCCCGTAAACCCAA); With 5RACE Outer sp W1 is primer, and the total RNA of pre-treatment is article one chain that template is carried out the synthetic cDNA of reverse transcription; With UPM10X primer in 5RACEOuter sp W1 and the test kit, be that template is carried out first round PCR with above-mentioned cDNA article one chain, reaction conditions is: 94 ℃ of 3min, 25 circulations (94 ℃ of 30sec, 68 ℃ of 30sec, 72 ℃ of 1.5min), 72 ℃ of 10min; With 5RACE Inner sp W1 and UPM10X is primer, and taking turns the PCR product with one is that template is carried out second and taken turns PCR, and reaction conditions is: 94 ℃ of 3min, 30 circulations (94 ℃ of 30sec, 58 ℃ of 30sec, 72 ℃ of 1.5min), 72 ℃ of 10min.With two-wheeled PCR product with 1% agarose gel electrophoresis analysis, reclaim purpose band and pMD19-T carrier (Takara:D102A) and connect and compose recombinant plasmid pMD19-T-csnW2, be transformed into intestinal bacteria (Top10), after bacterium colony PCR checking, serve the order-checking of extra large Ying Jun Bioisystech Co., Ltd.

Embodiment 5csnW2 gene recombinant expressed in intestinal bacteria

With the recombinant plasmid pMD19-T-csnW2 in the foregoing description 4 is template, with following primer to carrying out pcr amplification.Primer is to as follows: forward primer NdeI-SignalP-F(GCCCATATGTATAGTGTCCCAGCCAACC), reverse primer XhoI-SignalP+ATG-R(AATGAGCTCCACGAACGTATCAATCGACTA), what the forward primer underscore marked is restriction enzyme Ned I site, and what the reverse primer underscore marked is restriction enzyme Xho I site.Archaeal dna polymerase (DRR200A) is available from precious biotech firm, and the PCR reaction system is operated according to the description of product that company provides.The PCR reaction conditions: 94 ℃ of pre-sex change 5 minutes, 94 ℃ sex change 30sec-50 ℃ annealing is extended 1min for 30sec-72 ℃ then, 30 circulations, last 72 ℃ are extended 10min.With PCR product Ned I and Xho I double digestion, agarose gel electrophoresis reclaims the PCR product that enzyme is cut.To purchase in the product pET-23b of the U.S. Novagen company carrier Ned I and Xho I double digestion, agarose gel electrophoresis reclaims enzyme and cuts carrier.Ned I and Xho I all purchase in precious biotech firm, the description of product operation that the system of enzyme-to-substrate reaction, temperature and time all provide according to company.

To be connected through double digestion pET-23b carrier with same through the PCR product of double digestion, coat the Luria-Bertani substratum (Tryptones 1% that contains 50 μ g/ml Ampicillin Trihydrates after connecting product transformed into escherichia coli (Top10) bacterial strain, sodium-chlor 0.5%, yeast powder 0.5%, agar 2%) on the solid plate, cultivate 12h, picking mono-clonal for 37 ℃; Mono-clonal inserted in the liquid Luria-Bertani substratum contain 50 μ g/ml Ampicillin Trihydrates cultivate, extract plasmid; Plasmid is carried out bacterium colony PCR checking with above-mentioned forward primer NdeI-SignalP-F and reverse primer XhoI-SignalP+ATG-R, and the result obtains the correct amplified production of size, and the recombinant plasmid that preliminary proof makes up is correct; Then this recombinant plasmid is sent to the order-checking of Shanghai Ying Jun Bioisystech Co., Ltd, the result shows, between the Ned of pET-23b I and Xho I restriction enzyme site, insert the csnW2 gene shown in the SEQ ID NO2, and direction of insertion is correct, so further the recombinant plasmid of proof structure is correct.

With above-mentioned recombinant plasmid transformed coli strain BL21 (DE3) (available from U.S. Novagen company), the operation steps that provides according to the said firm is carried out chitoanase CsnW2 abduction delivering and purifying then.

Embodiment 6csnW2 gene recombinant expressed in pichia spp X33

With the recombinant plasmid pMD19-T-csnW2 among the embodiment 4 is template, with following primer to carrying out pcr amplification.Primer is to as follows: forward primer XhoI-Sig-F:(GACCTCGAGAAAAGATATAGTGTCCCTGCCAACC), reverse primer XbaI-Sig+His-R:(GACTCTAGAAGCGCTAAGTTTAGCAAC), what the forward primer underscore marked is restriction enzyme Xho I site, and what the reverse primer underscore marked is restriction enzyme Xba I site.Archaeal dna polymerase (DRR200A) is available from precious biotech firm, and the PCR reaction system is operated according to the description of product that company provides.The PCR reaction conditions: 94 ℃ of pre-sex change 5 minutes, 94 ℃ sex change 30sec-50 ℃ annealing is extended 1min for 30sec-72 ℃ then, 30 circulations, last 72 ℃ are extended 10min.With PCR product Xho I and XbaI double digestion, agarose gel electrophoresis reclaims the PCR product that enzyme is cut.To purchase in the product pPICZ of American I nvitrogen company α A carrier Xho I and Xba I double digestion, agarose gel electrophoresis reclaims enzyme and cuts carrier.Xho I and Xba I all purchase in precious biotech firm, the description of product operation that the system of enzyme-to-substrate reaction, temperature and time all provide according to company.

To be connected through double digestion pPICZ α A carrier with same through the PCR product of double digestion, coat on the Luria-Bertani substratum solid plate that contains 50 μ g/ml Ampicillin Trihydrates after connecting product transformed into escherichia coli Top10 bacterial strain, cultivate 12h, picking mono-clonal for 37 ℃; Mono-clonal inserted in the liquid Luria-Bertani substratum contain 50 μ g/ml Ampicillin Trihydrates cultivate, extract plasmid; With plasmid forward primer XhoI-Sig-F(GACCTCGAGAAAAGATATAGTGTCCCTGCCAACC) and reverse primer XbaI-Sig+His-R(GACTCTAGAAGCGCTAAGTTTAGCAAC) carry out bacterium colony PCR checking, the result obtains the correct amplified production of size, and the recombinant plasmid that preliminary proof makes up is correct; Then this recombinant plasmid is sent to the order-checking of Shanghai Ying Jun Bioisystech Co., Ltd, the result shows, between the Xho I of pPICZ α A and Xba I restriction enzyme site, insert the csnW2 gene shown in the SEQ IDNO2, and direction of insertion is correct, so further the recombinant plasmid that makes up of proof is correct.

Above-mentioned recombinant plasmid is carried out linearizing with SacI, carry out electricity according to Invitrogen company Pichia anomala expression handbook then and change X33 and screening over to, obtain the transformant of high copy.Carry out chitoanase CsnW2 abduction delivering and purifying with the operation steps that the said firm provides, the result as shown in Figure 1, the chitoanase CsnW2 behind the purifying is single band on running gel, and the molecular weight of position and prediction matches.

The zymologic property analysis of embodiment 7 chitoanase CsnW2

(1) pH and temperature are to the influence of enzymic activity

With quality is 50mM sodium-acetate-acetate buffer solution (the pH scope is 3.6-6.0) that chitosan (deacetylation 90%) substrate of 1g is dissolved in the different pH values of 100ml, being mixed with mass concentration is 1% substrate, and press the 1000:1(volume ratio with the CsnW2 enzyme liquid of purifying) mixed after, 40 ℃ of reactions 30 minutes, survey enzyme activity by aforesaid DNS method.The result shows that CsnW2 reaches maximum vigor when pH4.0, and the optimal reaction pH that shows CsnW2 is 4.0(such as Fig. 2)

Under optimal pH, be that the CsnW2 enzyme liquid of 1% chitosan substrate and purifying is by the 1000:1(volume ratio with mass concentration) mixed, differing temps (30 ℃-70 ℃) reaction 30 minutes, survey enzyme activity respectively by aforesaid DNS method.The result shows that CsnW2 reaches maximum vigor in the time of 50 ℃, and the optimal reactive temperature that shows CsnW2 is 50 ℃ (as Fig. 3).

Under optimal pH and optimum temperuture, be that the CsnW2 enzyme liquid of 1% chitosan substrate and purifying is by the 1000:1(volume ratio with mass concentration) mixed afterreaction 30 minutes, survey enzyme activity by aforesaid DNS method.With purchasing in the protein content of the quantification of protein kit measurement CsnW2 of Beijing Suo Laibao company enzyme liquid, the result shows that it is 1852U/g that recombinant C snW2 lives to the ratio of chitosan.

(2) pH and temperature are to the influence of enzyme stability

Will differing temps (30 ℃-70 ℃) down CsnW2 enzyme liquid and the mass concentration of thermal treatment after 30 minutes be that 1% chitosan substrate solution (pH4.0) is pressed the 1000:1(volume ratio) mixed, measuring residual enzyme then under optimum temperuture lives, being defined as 100% relative vigor (relativie activity) without the work of heat treated enzyme liquid enzyme, the result shows that CsnW2 has thermostability preferably being lower than under 40 ℃ the temperature.

Will be at 30 ℃, different pH(pH3-10) CsnW2 enzyme liquid behind the preincubate 24h and mass concentration are that 1% chitosan substrate solution (pH4.0) is pressed the 1000:2(volume ratio) mixed, measuring residual enzyme then under optimum temperuture lives, be defined as 100% relative vigor (relativie activity) with the enzyme liquid enzyme work of handling without pH, the result is presented in the scope of pH4～10, the CsnW2 enzyme is lived and is still kept more than 60%, shows that CsnW2 is wider to pH value tolerance range.

(3) the substrate preference of CsnW2

With the CsnW2 of purifying is that chitosan, tobacco brown spot pathogen and three kinds of different substrates of carboxymethyl cellulose of 1% are by the 1000:1(volume ratio with mass concentration respectively) mixed, then at 50 ℃, whether reaction is 30 minutes under the pH5.0 condition, by having reducing sugar to produce to determine its activity in the aforementioned DNS method measured reaction liquid.The result shows with the carboxymethyl cellulose to be that substrate is significantly not active.

Embodiment 8 metal ions are to the active influence of CsnW2

To mass concentration is to add the different metal ion of final concentration 10mM in the 1% chitosan substrate (pH4.0), as: Na+, K+, Mg2+, Ca2+, Co2+, Mn2+, Fe3+, Cu2+or Ag+ (AgNO3), press the 1000:1(volume ratio with the CsnW2 enzyme liquid of purifying then) mixed, and, survey enzyme activity by aforesaid DNS method 40 ℃ of reactions 30 minutes.The activity (being set at 100%) of control group CsnW2 when not adding any metal ion.The result shows that Mg2+ and Mn2+ can increase CsnW2 activity (about 1-2 doubly); Work presents restraining effect to enzyme for Fe3+, Cu2+ and Ag+.

The high performance liquid chromatography (HPLC) of embodiment 9CsnW2 degrade chitosan products therefrom is analyzed

With mass concentration be 1% chitosan (pH4.0) and the CsnW2 of purifying press the 1000:1(volume ratio) mixed, react down at 50 ℃ then, choose different enzymolysis times (1h, 2h, 5h, product 10h) carries out efficient liquid phase chromatographic analysis.High performance liquid chromatography adopts ion-exchange chromatography: CarboPac PA1 (DIONEX, Column No.35391,4 * 250mm, I.D.with particle size of10 μ m); Detector: Coulochem II (ESA, USA); Column temperature: 30 ℃; Moving phase: 90mM NaOH; Flow velocity: 0.4mL/min.

Wherein 10 hours high-efficient liquid phase chromatogram of enzymolysis as shown in Figure 4.Color atlas shows the oligochitosan that a series of different polymerization degrees are arranged in 10 hours the product of CsnW2 degrade chitosan, and this result has proved that fully CsnW2 belongs to the inscribe chitoanase.Therefore, inscribe chitoanase CsnW2 can be used to the preparation and the field relevant with degradation of chitosan of oligochitosan, comprises chemical industry, agricultural, food, feed interpolation, medicine and marine alga genetic engineering etc.

Claims (6)

1. the bacterial strain of Aspergillus, it is excellent aspergillus W-2, classification name: excellent aspergillus Aspergillus clavatus, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number: CGMCC NO.7018, preservation date on December 18th, 2012.

2. inscribe chitosanase gene csnW2 who derives from the described bacterial strain of claim 1 (Aspergillus clavatus), its nucleotide sequence has one of following feature:

1) has Yeast Nucleic Acid (RNA) sequence of SEQ ID NO.1 in the sequence table or thymus nucleic acid (DNA) sequence of SEQ ID NO.2;

2) thymus nucleic acid (DNA) sequence of coding SEQ ID NO.3 aminoacid sequence;

3) have with sequence table in the homology of thymus nucleic acid (DNA) sequence that limited of SEQ ID NO.2 reach 90% and more than, and proteinic thymus nucleic acid (DNA) sequence of the degrade chitosan of encoding.

3. according to the inscribe chitoanase of the described inscribe chitosanase gene coding of claim 2, its amino acid sequence coded has one of following feature:

1) 1-262 or the 18-262 amino acids residue sequence that SEQ ID NO.3 begins from aminoterminal in the sequence table;

2) 1-262 that the SEQ ID NO.3 in the sequence table is begun from aminoterminal or 18-262 amino acids residue sequence have the active protein of degrade chitosan through after the replacement of amino-acid residue, disappearance, the interpolation one or two or more kinds;

3) with sequence table in the homology of the aminoacid sequence that limited of SEQ ID NO.3 reach 90% and above and have the active protein of degrade chitosan.

4. a method for preparing inscribe chitoanase as claimed in claim 3 is characterized in that: be that the described inscribe chitosanase gene of claim 2 is cloned into recombinant expression vector, import host cell, obtain recombinant expressed inscribe chitoanase;

The expression vector of described recombinant expressed inscribe chitoanase is meant coli expression carrier, Yeast expression carrier, Bacillus subtilus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungus expression vector, plant expression vector, insect expression vector or mammalian cell expression vector;

The host cell of described recombinant expressed inscribe chitoanase, be meant that e. coli host cell is (as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α etc.), the yeast host cell is (as Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis etc.), the Bacillus subtilus host cell is (as Bacillus subtilis R25, Bacillus subtilis9920 etc.), milk-acid bacteria host cell (as Lactic acid bacteria COCC101 etc.), actinomycetes host cell (as Streptomyces spp. etc.), filamentous fungal host cell is (as Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans etc.), insect cell is (as Bombyx mori, Antharaea eucalypti etc.), mammalian cell (as Chinese hamster ovary cell CHO, immature hamster kidney cell BHK, Chinese hamster pneumonocyte CHL etc.) a kind of in.

5. the application of an inscribe chitoanase as claimed in claim 3 in degradation of chitosan is characterized in that: have one of following purposes or more than two kinds;

1) be used to rupture chitosan or chitinous glycosidic link obtain oligochitosan or chitin oligo saccharide;

2) be used for the degrading chitin and the chitosan component of shrimp and crab shells or insect shell, extracting pigment or albumen wherein, pigment and albumen wherein can be applied to food and other research.

6. application as claimed in claim 5 is characterized in that: described inscribe chitoanase CsnW2 is used for the application of collaborative fracture chitosan glycosidic link aspect with after other chitoanases, deacetylase and chitin-binding protein mix.

Images