CN110272884A - A kind of chitinase and its gene for chitin oligo saccharide preparation - Google Patents

Abstract

The present invention provides a kind of chitinase and its gene for chitin oligo saccharide preparation, and by the method for its clonal expression in Escherichia coli and yeast cells, the chitinase gene nucleotide sequence is as shown in SEQ ID NO.1；The amino acid sequence of chitinase is as shown in SEQ ID NO.2；Recombinant vector containing the gene is that chitinase gene is connected to escherichia coli plasmid or yeast plasmid；The cell of the gene is converted by the recombinant vector and is obtained；The cell containing chitinase gene is comprising the nucleic acid molecule or the Escherichia coli converted with the recombinant vector or comprising the nucleic acid molecule or the pichia yeast converted with the recombinant vector.

Description

A kind of chitinase and its gene for chitin oligo saccharide preparation

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of chitinase and its base for chitin oligo saccharide preparation Cause.

Technical background

Marine environment can be provided with the new compound of huge applications potentiality, such as the enzyme with potential market value, It is to rarely have to see in marine sediment although constantly generating shell-fish waste in marine environment.These phenomenons are said The bright microorganism abundant for producing chitinase widely distributed in marine environment, these are produced from microorganisms of marine environment Raw chitinase hydrolyzes chitin, completes processing of the nature to chitin.It is reported that marine bacteria Alteromonas Sp. strain 0-7, Micrococcus sp. AG84, Pseudoalteromonas sp. DL-6, Vibrio Chitin degrading can be several by furnissii, Aeromonas caviae and Pseudoalteromonas sp. DXK012 Fourth oligosaccharides is for its metabolism or by other microorganisms as unique carbon nitrogen source.In addition to this, from South Korea's Jizhou Island coastal area The circumscribed chitinase that generates of the isolated Vibrio sp. 98CJ11027 of bryozoan be purified after be used to produce chitin Disaccharides.It is reported that have some bacteriums, including Acinetobacter ASK18, Alcaligenes faecalis AU02, Vibrio (Aestuarianus, Flavobacterium, Shewenella and Exiguobacterium) etc. is from sea Isolated, these environmental samples in foreign waste, coastal area sedimentary soil, marine product industrial wastewater and shell-fish waste It is all the main source of chitinase.

Chitin (Chitin) is called chitin, chitin, crab shell element or bright angle glutelin, is to be present in nature to remove The relatively rich sugar of the second major class except cellulose is to be connected by 2-acetylamino-2-deoxy-D-glucose (GlcNAc) with β-Isosorbide-5-Nitrae glycosidic bond High molecular polymer made of connecing, not soluble in water and most organic solvents, is dissolved in concentrated hydrochloric acid and the concentrated sulfuric acid, is fungal cell Wall, the principal structural component of insect exoskeleton and crustacean shell.Chitinase (Chitinase, EC 3.2.2.14) is glycosyl Hydrolase, it is characterised in that β-Isosorbide-5-Nitrae glycosidic bond of the N-acetylglucosamine in hydrolysis chitin chain.Chitinase it is most suitable PH value is generally 1.0-8.0, isoelectric point 3.1-10.0.The optimal reactive temperature of most of chitinases is 40-60 °C, and heat is steady It is qualitative good, it is generally relatively stable under the conditions of 40-60 °C.Many metal ions especially heavy metal ion such as Ag+, Cu2+, Zn2+, Hg2+, Co2+ and Fe3+ etc. have different degrees of influence to chitinase activity.It is several according to conserved amino acid sequences type Fourth matter enzyme belongs to 18,19 and 20 family's glycoside hydrolases.According to the similitude of bacterium chitinase catalyst structure domain, by 18 Family's chitinase is further divided into tri- subfamilies of A, B, C.It include one section in the catalyst structure domain of subfamily A chitinase Chitinase insert structure domain (CID) is the key structural feature of subfamily A.

Chitinase is the general name for one group of enzyme that single-minded degradation chitin is chitin oligo saccharide or monosaccharide, the portion acted on according to it Position is divided into circumscribed chitinase and endochitinase.Circumscribed chitinase is generally divided into chitobiose enzyme and N- acetyl group Portugal again Glycosidase, endochitinase random hydrolysis chitin chain generate chitin oligo saccharide (GlcNAc) n, n > 2.Chitobiose enzyme hydrolysis is several One end of fourth matter polysaccharide chain or chitin oligo saccharide generate 2-acetylamino-2-deoxy-D-glucose dimer (GlcNAc) 2, N- acetyl group glucose Glycosides enzyme can then hydrolyze one end of chitin polysaccharide chain or (GlcNAc) 2 generates 2-acetylamino-2-deoxy-D-glucose (GLcNAc) list Body.

Up to the present, the chitinase of the separate sources successful expression in Escherichia coli, Pichia pastoris.For example, 2018, Lee et al. successful expression in e. coli bl21 (DE3) derived from Microbulbifer thermotolerans The salt tolerant chitinase mtch509 of DAU221；Li Shujiang et al. is by the chitin in Streptomyces sampsonii KJ40 Enzyme gene chiKJ406136 expression in e. coli bl21 (DE3), recombinant protein have antifungic action；Lee's height et al. exists A kind of new chitinase gene is cloned in Streptomyces albolongus ATCC 27414, and in Escherichia coli Successful expression in BL21.2019, Menghiu et al. successful expression in Pichia pastoris derived from Bacillus The chitinase A of licheniformis DSM8785.

Escherichia expression system is studied clearer and easy to operate, and growth cycle is short, however, in Escherichia coli The protein of middle heterogenous expression is easily formed inclusion body.The expression of endocellular enzyme needs mechanical damage to discharge from cell, this can Enzyme inactivation or enzymatic activity can be caused to reduce, limit the large-scale production and application of chitinase.In addition, Pichia pastoris operation is multiple Miscellaneous, growth cycle is longer but has the advantages that high copy, high density fermentation, expressing gene is stablized in host genome, Therefore select suitable host also most important.

Summary of the invention

The main purpose of the present invention is to provide a kind of chitinases and its gene for chitin oligo saccharide preparation, and will The method of its clonal expression in Escherichia coli and yeast cells provides the new enzyme source with potential using value.

To achieve the above object, using following technical scheme:

Chitinase of the invention, the gene nucleotide series of the chitinase are as shown in SEQ ID NO.1；Chitinase Amino acid sequence is as shown in SEQ ID NO.2.

Recombinant vector containing chitinase gene is that chitinase gene is connected to escherichia coli plasmid or yeast plasmid Recombinant vector；The preferred recombinant vector is pET-28a (+)-chi1602Or pPIC9k-chi1602。

The cell of gene containing chitinase selects Bacillus coli cells or yeast cells is recipient cell；The cell by The recombinant vector is converted and is obtained；The cell is comprising the nucleic acid molecule or the large intestine bar converted with the recombinant vector Bacterium includes the nucleic acid molecules or the pichia yeast converted with the recombinant vector.

Specifically:

The present invention relates to chitinase producing strains and clone's chitinase gene are screened from marine environment.One of embodiment, The present invention extractsMicrobulbifer sp.The genomic DNA of bacterial strain obtains chitinase gene full length sequence through sequencing, claims Bechi1602。

The invention further relates to comprising describedchi1602The recombinant vector of coded sequence, by various expression commonly used in the art The recombinant vector of carrier preparation, wherein the coded sequence does not include the endogenous signal peptides sequence of derived microbial.Embodiment party It will be the present invention without the coded sequence of endogenous signal peptides in one of formulachi1602Coded sequence warpSacI andNot The bis- enzymes of I After cutting withSacI andNot PET-28a (+) carrier of I double digestion connects, and obtains large intestine recombinant expression carrier pET-28a (+)-chi1602.It, will be without the present invention of endogenous signal peptides coded sequence in another embodimentchi1602Coded sequence warpSnaBI andAvrAfter II double digestion withSnaBI andAvrThe pPIC9k carrier of II double digestion connects, and obtains yeast recombinant expression and carries Body pPIC9k-chi1602。

The present invention is also prepared comprising the present inventionchi1602The cell of coded sequence.In one of embodiment, the cell is It is converted building with aforementioned present invention recombinant vector.The recipient cell preferably various cells conducive to gene product expression, Such cell is well known in the art and commonly uses, such as various Bacillus coli cells and yeast cells.In embodiments of the present invention One of in, select e. coli bl21 (DE3) and pichia yeast X33 building expresschi1602Recombinant cell.

The present invention also provides expressionchi1602Method, comprising: culture it is described previously comprising the present inventionchi1602It compiles The cell or the transformed cell of code sequence, induce its expression, harvest expression product, further include the step of purified expression product Suddenly.In one of embodiment, the present invention is by the inclusion of the present inventionchi1602The yeast (such as pichia yeast X33) of coded sequence Fermentation purifies to have obtained the destination protein of pure enzyme form by anion-exchange chromatography to produce chitinase Chi1602.

The present invention using genetic engineering means be prepared for can high efficient expression and secrete chitinase Chi1602 recombination it is raw Strain is produced, good chitinase Chi1602 product is obtained.The present invention is special by enzyme activity determination, characterization analysis and substrate Anisotropic analysis, it was demonstrated that Chi1602 of the invention has good hydrolysis ability to tobacco brown spot pathogen.

The present invention has the advantages that the present invention provides a kind of new chitinase gene, and the chitin of heterogenous expression Enzyme can keep high activity and stability within the scope of extensive pH, can generate chitobiose with efficient degradation tobacco brown spot pathogen And 2-acetylamino-2-deoxy-D-glucose, there is application value.

Detailed description of the invention

Fig. 1Microbulbifer sp.The Chi1602 in source is on plasmid pMD18-T, pET-28a (+) and pPIC9k Recon structure chart.

Fig. 2 Microbulbifer sp.The SDS-PAGE of the Chi1602 in source.M: protein Marker；1: crude enzyme liquid； 2: recombinant chitinase Chi1602 DEAE column purification product.

Fig. 3Microbulbifer sp.The optimal pH and pH stability of the Chi1602 in source.A:pH is to recombinase The activity influence of Chi1602；B: recombinase Chi1602 pH stability.

Fig. 4Microbulbifer sp.The optimum temperature and temperature stability of the Chi1602 in source.A: temperature is to recombination The activity influence of enzyme Chi1602；B: the temperature stability of recombinase Chi1602.

Fig. 5Microbulbifer sp.The Chi1602 catabolite in source is analyzed.A: degradation substrate is colloid chitin Matter；B: degradation substrate is N- acetylation chitobiose.N1:N- acetyl-D-aminoglucose (GlcNAc) N2:N- acetylation shell two Sugar ((GlcNAc) 2.

Specific embodiment

The present invention is absolutely proved below according to specific embodiment.

Experimental material and reagent

1, bacterial strain and carrier:

E. coli bl21 (DE3), Top10 and expression vector pET-28a (+) are purchased from Novagen company, pichia yeast X33 and Expression vector pPIC9k is purchased from Invitrogen company (Carlsbad, CA, USA).

2, enzyme and other biochemical reagents:

Restriction enzyme is purchased from New England Biolabs company, and DNA Maker is purchased from TaKaRa company, Protein Maker is purchased from Quan Shi King Company.

3, culture medium:

The culture medium used:

(1) LB liquid medium: peptone (Tryptone) 10.0 g, NaCl 10.0 g, yeast extract (Yeast Extract) 5.0 g, distilled water are settled to 1 L, 121 °C of 20 min of sterilizing.

(2) LB solid medium is 2% (w/v) agar powder of addition, 121 °C of 20 min of sterilizing in LB liquid medium.

(3) MD culture medium: weighing 15.0 g agar powders in 800 mL distilled water, and 121 °C of 20 min of sterilizing are cooled to 60 °C or so, sequentially add 10 × YNB, 100 mL, 500 × biotin, 2 mL, 20% (w/v) glucose, 100 mL.

(4) YPD culture medium: yeast powder 1% (w/v), peptone 2% (w/v), 121 °C of 20 min of sterilizing, when use, are added 2% (w/v) glucose.Solid medium is that the agar powder of 2% (w/v) is added in YPD fluid nutrient medium.

(5) YPM culture medium: yeast powder 1% (w/v), peptone 2% (w/v), 121 °C of 20 min of sterilizing, when use, are added 2% (v/v) methanol.

4, Measurement for Biochemistry used in the present invention is the routine techniques in this field.In the examples below, Unless specifically indicated, all experimental implementations are carried out according to the related Sections in following laboratory manual or document or partially, comprising: [beauty] J. Sha's nurse Brooker etc., Molecular Cloning:A Laboratory guide；Zhao Yongfang etc., Measurement for Biochemistry principle and its application (second Version)；Zhu's inspection etc., Biochemistry Experiment [M].

5. all relevant enzyme activity, enzyme activity, enzymatic activity each mean chitinase Chi1602 activity in the present invention, adopt It is measured and calculates with DNS method and according to the method.

1 chitinase gene of embodimentchi1602Acquisition

(1)Microbulbifer sp.The separation and Extraction of genomic DNA:

Take 1.5 mL thalline culturesMicrobulbifer sp.(be purchased from Chinese Sea Microbiological Culture Collection administrative center) in In one sterilizing Ep pipe, 12000 rpm are centrifuged 1 min, abandon supernatant, collect thallus.

400 μ L lysates (40 mM Tris- acetic acid, 20 mM sodium acetates, 1 mM EDTA, 1% SDS, pH 7.8) is added It mixes, is placed in 37 DEG C of 1 h of water-bath.

Then the sodium chloride solution of 200 μ L l5 mol/L is added, is centrifuged 15 min in 13000 rpm after mixing.

Supernatant is taken, with phenol extraction 2 times, chloroform 1 time.

Add two volumes dehydrated alcohol, 1/10 volume potassium acetate (3 M, pH 8.0), after -20 DEG C of 1 h of preservation, 13000 Rpm is centrifuged 15 min, abandons supernatant, and precipitating is washed 2 times with 70% ethyl alcohol；After being placed in drying at room temperature, it is dissolved in 50 μ L TE solution, 4 DEG C are set to save backup.

(2) PCR obtains target gene

ThroughMicrobulbifer sp.Gene order-checking analysis, obtains a chitinase gene sequence, according to gene order point It She Ji not two couples of upstream and downstream primer P1/ P2 and P3/P4.Primer P1 and P2 contain respectivelySacI HeNotI restriction enzyme site, primer P3 Contain respectively with P4SnaBI andAvrII restriction enzyme site.Primer is synthesized by Shanghai Bo Shang Biotechnology Co., Ltd, primer sequence It is as follows:

P1:5'-TTCGAGCTCCTGCAGGTATACGTGGATGG-3'

P2:5'-GTTGCGGCCGCATTGATTGCTGTGCATGGCTTCAAT-3'

P3:5'-CAGTACGTACTGCAGGTATACGTGGATGG-3'

P4:5'-CTCCCTAGGTTGATTGCTGTGCATGGCTTCAAT-3'

The present embodiment PCR reaction system is 50ul (upstream and downstream primer each 1uL, dNTP 1uL, genome 1uL, archaeal dna polymerase 1uL, ddH₂O 45uL), program are as follows: 94 DEG C of 4 min of initial denaturation；94 DEG C of 30 s of denaturation, 52 DEG C of 30 s of annealing, 72 DEG C extend 2 Min, cyclic amplification 30 times；10 min of last 72 DEG C of extensions.It takes PCR product to carry out electrophoresis detection after amplification, and recycles solidifying Target gene in glue.

(4) cDNA is subcloned:

PCR amplification prepares double-strand cDNA, is inserted on carrier pMD18-T by the method for double digestion, obtains recombinant plasmid pMD18-T-chi1602(as shown in Figure 1) is transformed into recipient bacterium with chemical transformationE.coliTop 10 is containing 100 mg/ 37 DEG C of overnight incubations on the LB plate of mL Amp.Picking monoclonal colonies are inoculated into the LB liquid that 2 ml contain 100 mg/mL Amp In body culture medium, 37 DEG C of 200 rpm cultivates 6-10 h, and 10000 rpm are centrifuged 10 min and collect thallus, extracts plasmid, uses respectivelySacⅠ/NotI HeSnaB I/AvrII carries out double digestion, and recycling target gene is spare, and (plasmid extracts and glue recycling is used respectively The E.Z.N.A. Plasmid Mini Kit I and E.Z.N.A. Gel Extraction Kit kit of OMEGA company).It will Gained target gene carries out determined dna sequence (Invitrogen company), and is compared in ncbi database.

Analysis is the result shows that the coded sequence of gained chitinase shares 1623 bp(SEQ ID NO:1), coding is without letter The maturation protein of number peptide, the maturation protein contain 541 amino acid (SEQ ID NO:2).According in ncbi database homology The result of comparison primarily determines that obtained genetic fragment is chitinase gene.

Expression and amplification of the 2 chitinase coding gene chi1602 of embodiment in Escherichia coli

By obtained warp in embodiment 1- (4)Sec I andNotThe target gene of I double digestion, with processSec I andNotI is bis- PET-28a (+) plasmid of digestion connects, and obtains recombinant plasmid pET-28 (a)-chi1602(as shown in Figure 1).

It is thin to be added to e. coli bl21 (DE3) competence that 100 uL are prepared for the Plasmid DNA for taking 10 uL to build In born of the same parents, shakes up and be placed on ice, 30 min of ice bath；It is placed in thermal shock 45s in 42 DEG C of water-baths；Centrifuge tube is quickly moved to mixture of ice and water Middle 2 min of ice bath；800 uL LB liquid mediums are added in every pipe, in 1 rpm of h(80 rpm~200 that recovers on 37 DEG C of shaking tables)； 4000 rpm are centrifuged 5 min, abandon 800 uL supernatants, and remainder mixes；Applying plate, (LB-agar plate contains 100 ug/mL Amp), it is incubated overnight for 37 DEG C, picking recon to LB liquid medium, it is positive with the method validation of bacterium solution PCR to bacterium solution muddiness Recon, PCR product are detected through 1.0% (w/v) agarose gel electrophoresis, and purpose band size is 1600 bp or so, with implementation In example 1 fromMicrobulbifer sp.In obtained original genechi1602Sequence size it is consistent, the positive will be accredited as The bacterium solution of recon sends out sequencing, the correctness of further verifying purpose gene order and insertion point.

The insertion correct recombinant escherichia coli strain BL21 (DE3) of gene is taken, is inoculated in 200 ml LB culture solutions (1000 ml triangular flasks, 100 ug/mL Amp), 37 DEG C of 200 rpm shake culture to OD₆₀₀For 0.6-0.8, IPTG is added and lures (final concentration of 1 mmol/L) is led, 20 DEG C of 200 rpm is followed by shaking culture 10 h.10000 rpm of culture solution is taken to be centrifuged 10 min, Thallus is collected, isometric sterile water suspension thalline again is added, 12000 rpm are centrifuged 10 min, take 1/20 body of precipitating The Tris-HCl buffer suspension thalline of product pH 7.4,20 mM, carry out ultrasonic disruption, broken condition are as follows: 60% power, interval 5 s are crushed 10 min, stop 10 min, then broken 10 min.Supernatant measurement recombination enzyme activity is collected in 12000 rpm centrifugation, And pass through the expression quantity of SDS-PAGE electrophoretic analysis destination protein.

The building of 3 yeast recombinant expression carrier of embodiment

By warp obtained in embodiment 1-(4)SnaBI andAvrThe target gene of II double digestion, with processSnaBI andAvr The pPIC9k plasmid of II double digestion connects, and obtains recombinant plasmid pPIC9k-chi1602(as shown in Figure 1).

Recombinant plasmid is transferred toE.coliTop10 competent cell is coated on the LB screen plate containing 100 mg/mL Amp On be incubated overnight, picking recon is identified with the method for bacterium solution PCR, if positive recombinant, sends out sequencing, and sequencing result is into one Step proves the correctness of objective gene sequence and insertion point.The selection insertion correct bacterium solution of gene, referring to OMEGA Plasmid The extraction step of Mini Kit extracts recombinant plasmid, saves backup in -20 °C.

4 pichia yeast fermenting and producing recombinase Chi1602 of embodiment

Recombinant plasmid pPIC9k- prepared by embodiment 3chi1602ThroughSac I single endonuclease digestion obtains linearization plasmid pPIC9k- chi1602。

The recombinant plasmid pPIC9K- for taking 20 μ L to linearizechi1602It is added to the Pichia pastoris X33 competence of 80 μ L In cell, gently piping and druming is mixed；Mixed liquor is added in the electric revolving cup of 2 mm pre-cooling, electric revolving cup is put into warmed-up 30 min's Electroporation, electric shock are primary；After the completion of electric shock, the sorbierite for rapidly joining 1 mL pre-cooling gently blows and beats suspended bacteria in electric revolving cup Bacterium solution is sucked out into 1.5 mL EP pipes of sterilizing in liquid, 30 °C of 60 min of standings recovery；6000 × g of bacterium solution is centrifuged 2 min, It draws after thallus is resuspended in 80 μ L supernatants and is coated on MD screen plate；30 °C of inversions are cultivated 3 days or so, until transformant is grown.? Growing on MD plate is the positive clone molecule containing recombinant plasmid.

The pichia yeast X33 bacterial strain positive clone molecule of picking recombinant plasmid pPIC9k-chi1602 conversion is to 10 mL YPD Fluid nutrient medium is incubated overnight.By 10.0%(v/v) additive amount bacterium solution is added in 10 mL YPM culture mediums, 30 °C, 200 rpm constant-temperature table cultures 3 days, every 24 h add the methanol induction expression of final concentration of 2.0% (v/v).By bacterium solution 13000 Rpm is centrifuged 5 min, takes supernatant measurement chitinase activity and carries out SDS-PAGE analysis, as a result as shown in Figure 2.Recombination Unanimously, the expression of Chi1602 is (with hair for the SDS-PAGE molecular weight of Chi1602 and its theoretical molecular weight (59.47 kDa) The enzyme activity of zymotic fluid supernatant indicates) reach 1.266 U/ml, this explanationMicrobulbifer sp.The chitinase gene in sourcechi1602It is expressed and is accumulated in pichia yeast.

The purifying of 5 recombinase Chi1602 of embodiment

Bulk fermentation culture solution is prepared according to 4 method of embodiment, 10000 rpm are centrifuged 10 min and remove thallus, and supernatant is taken to make For crude enzyme liquid.Crude enzyme liquid is diluted 5 times with buffer solution A (Tris-HCl of the pH 8.0 of 50 mM), is added with the flow velocity of 1mL/min It is downloaded in DEAE cellulose column, (contains 1 mol to column equilibration, then with buffer solution B with the albumen of buffer solution A elution weak binding L^-1NaCl 50 mM pH 8.0 Tris-HCl) destination protein eluted with the flow velocity of 1 mL/min, collect eluent, SDS-PAGE measures destination protein purity.

After the completion of purifying, the chitinase specific activity of acquisition is increased to 5.0 U/ of pure enzyme from 3.94 U/mg of crude enzyme liquid Mg, purification are 1.27 times, yield 34.72%.SDS-PAGE result such as Fig. 2.

The biochemical property of 6 recombinase Chi1602 of embodiment characterizes

1, the measurement of optimal reaction pH and the pH stability of recombinase

Recombinase optimal reaction pH measurement: by recombinase after purification under the conditions of 50 DEG C, Yu Butong pH(pH 3.0,4.0, 5.0, enzyme activity 6.0,7.0,8.0,9.0,10.0,11.0) is surveyed in substrate, to determine its optimal reaction pH.

Recombinase pH Stability Determination: by purified recombinase different pH value (pH 3.0,4.0,5.0,6.0,7.0, 8.0, after 9.0,10.0,11.0) water-bath keeps the temperature 60 min at 37 DEG C in buffer, take enzyme solution in optimal pH and at a temperature of point Its enzyme activity is not measured.Recombinase Chi1602 reacts under the conditions of pH 4.0-9.0 as the result is shown can keep 85% or more most High activity (shown in Fig. 3 a), while there is good pH stability within the scope of pH 4.0-9.0, experimental result is as shown in Figure 3b.

2, the measurement of recombinase optimal reactive temperature and thermal stability

The measurement of recombinase optimal reactive temperature: by recombinase after purification in optimal pH substrate, in different temperatures (30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C) under the conditions of reaction assay enzyme activity, with determine its optimal reactive temperature.

Recombinase thermal stability determination: by purified recombinase in different temperatures (30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C) under 1 h of water-bath isothermal holding, measure its remaining enzyme activity respectively under the conditions of optimal pH and temperature.Chi1602 as the result is shown Optimal reactive temperature is between 50 DEG C to 60 DEG C.Experimental result is as shown in Figure 4.

3, the substrate specificity Journal of Sex Research of recombinase

Compound concentration is 0.5%(w/v) tobacco brown spot pathogen, fine powder chitin, chitosan, sodium carboxymethylcellulose (CMC-Na), Measurement recombination enzyme activity under optimum reaction conditions.It is set as 100% by the enzyme activity of tobacco brown spot pathogen of substrate, calculates other substrates Enzyme activity.The results show that Chitinase has stringent substrate specificity to tobacco brown spot pathogen.Experimental result such as 1 institute of table Show.

Table 1Microbulbifer sp.-G27The substrate specificity of the chitinase Chi1602 in source.

7 recombinase Chi1602 of embodiment degradation tobacco brown spot pathogen analysis

Chitin oligo saccharide is the derivative of chitosan, and unlike chitin, chitin oligo saccharide has due to the length that its short chain obtains There is good water solubility, be easy to be absorbed by organisms, and can complete natural degradation in vivo.If using mode appropriate by first The degradation of shell element, the chitin oligo saccharide of available different polymerization degree and acetyl degree, existing research show that the chitin of different structure is few Sugar has different pharmacological action and biological activity, is improving immunity of organisms, anti-oxidant, antibacterial and anti-tumor aspect Gradually show advantage.Therefore, the chitin oligo saccharide that can be directly absorbed by the body is prepared by degradation chitin, just becomes one Extremely significant research.Recombinase Chi1602 in this patent using production is come tobacco brown spot pathogen of degrading.

Reaction mixture is 900 μ L, 1% (w/v) tobacco brown spot pathogen and 100 μ L, 0.5 UmL^-1Purifying chitin Enzyme then boils 10 min and terminates reaction in 50 DEG C of incubation different times (1 h, 2 h, 6 h, 12 h and 24 h).Using HPLC The hydrolysate of recombinase is analyzed, liquid-phase chromatographic analysis uses nh 2 column, Composition distribution: 30 °C of column temperature, mobile phase For 70% (v/v) acetonitrile, flow velocity is 0.5 mLmin^-1, sample volume is 20 μ L.Mark product are 2-acetylamino-2-deoxy-D-glucose (GlcNAc) and N- acetylation chitobiose ((GlcNAc)₂.Experimental result is as shown in figure 5, degradation tobacco brown spot pathogen final product is (GlcNAc)₂And GlcNAc, recombinase Chi1602 have circumscribed chitinase activity and N- acetyl group glucoside enzyme activity simultaneously Property.

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

SEQUENCE LISTING

<110>University of Fuzhou

<120>a kind of chitinase and its gene for chitin oligo saccharide preparation

<130> 6

<160> 6

<170> PatentIn version 3.3

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gaaggctggg catggactga ggcttggtcc gatctgggtg cctgtaccgg cagctcgtcg 180

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agcggatcgt ccaccagctg cgctggtcta ccggtgtggg atgcggcaac agtttatgtc 300

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gcagagaaac tgacccacat ctattacgcc ttcggcaatg tgcagaacgg ccagtgcacc 660

atcggcgatt cctatgccga ttacgaccgt ttctacagtg cggcagagag tgtggatggc 720

caggcggata cctgggatgc cggcgcactg cgcggcagtt tcaaccagtt gcgcaaactg 780

aagcagatgt acccgcacat caaagtgctg tggtcctttg gtggctggac ctggtccggc 840

ggcttcggcc aggcgtcgca gaacccgcag gcattcgcgg attcctgtta caacctgctg 900

cacgacccgc gctgggacgg cgtgtttgac ggtatcgacc tcgactggga ataccccaat 960

gcctgcggtc tcagctgcga cagcagcggc tattcatcac tgaccaacgt catggcggcc 1020

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caa 1623

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Val Gly Gly Pro Tyr Ala Pro Gly Glu Gly Trp Ala Trp Thr Glu Ala

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Trp Ser Asp Leu Gly Ala Cys Thr Gly Ser Ser Ser Ser Ser Ser Ser

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Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Gly Gly Ser Ser

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85 90 95

Thr Val Tyr Val Gly Gly Asn Ala Val Gln His Ser Ser Ala Lys Tyr

100 105 110

Thr Ala Lys Trp Trp Thr Gln Gly Asp Asn Pro Ser Glu Ser Gly Glu

115 120 125

Tyr Gly Val Trp Arg Asn Asp Gly Asn Cys Ser Gly Gly Ser Ser Ser

130 135 140

Ser Ser Ser Ser Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Ser

145 150 155 160

Ser Ser Ser Ser Ser Ser Gly Ala Ser Ser Ser Gly Gly Ser Arg Leu

165 170 175

Gly Gly Tyr Phe Val Gln Trp Gly Val Tyr Gln Arg Asn Tyr His Val

180 185 190

Lys Asn Ile Val Thr Ser Gly Ser Ala Glu Lys Leu Thr His Ile Tyr

195 200 205

Tyr Ala Phe Gly Asn Val Gln Asn Gly Gln Cys Thr Ile Gly Asp Ser

210 215 220

Tyr Ala Asp Tyr Asp Arg Phe Tyr Ser Ala Ala Glu Ser Val Asp Gly

225 230 235 240

Gln Ala Asp Thr Trp Asp Ala Gly Ala Leu Arg Gly Ser Phe Asn Gln

245 250 255

Leu Arg Lys Leu Lys Gln Met Tyr Pro His Ile Lys Val Leu Trp Ser

260 265 270

Phe Gly Gly Trp Thr Trp Ser Gly Gly Phe Gly Gln Ala Ser Gln Asn

275 280 285

Pro Gln Ala Phe Ala Asp Ser Cys Tyr Asn Leu Leu His Asp Pro Arg

290 295 300

Trp Asp Gly Val Phe Asp Gly Ile Asp Leu Asp Trp Glu Tyr Pro Asn

305 310 315 320

Ala Cys Gly Leu Ser Cys Asp Ser Ser Gly Tyr Ser Ser Leu Thr Asn

325 330 335

Val Met Ala Ala Met Arg Ala Arg Phe Gly Asp Glu Leu Val Thr Ala

340 345 350

Ala Ile Thr Ala Asp Gly Thr Ala Gly Gly Lys Ile Asp Ala Ala Asp

355 360 365

Tyr Ala Gly Ala Ser Gln Tyr Val Asp Phe Tyr Asn Val Met Thr Tyr

370 375 380

Asp Phe Phe Gly Ala Phe Asp Ala Asp Gly Pro Thr Ala Pro His Ser

385 390 395 400

Ala Leu Tyr Asp Tyr Pro Gly Ile Pro Asn Ala Gly Phe Tyr Ser Asp

405 410 415

Arg Ala Ile Gln Ala Leu Lys Asn Lys Gly Val Pro Ala Ser Lys Leu

420 425 430

Asn Leu Gly Ile Gly Phe Tyr Gly Arg Gly Trp Thr Gly Val Ser Gln

435 440 445

Ser Ala Pro Gly Gly Ser Ala Thr Gly Ala Ala Ala Gly Thr Tyr Glu

450 455 460

Pro Gly Ile Glu Asp Tyr Lys Val Leu Lys Asn Thr Cys Pro Ser Thr

465 470 475 480

Gly Leu Val Ala Gly Thr Ala Tyr Ala Phe Cys Gly Asn Asn Trp Trp

485 490 495

Ser Tyr Asp Thr Pro Ser Thr Ile Ala Gly Lys Met Gln Tyr Ile Ala

500 505 510

Asp Gln Asp Leu Gly Gly Ser Phe Phe Trp Glu Leu Ser Gly Asp Thr

515 520 525

Gly Asn Gly Glu Leu Ile Glu Ala Met His Ser Asn Gln

530 535 540

<210> 3

<211> 29

<212> DNA

<213> P1

<400> 3

ttcgagctcc tgcaggtata cgtggatgg 29

<210> 4

<211> 36

<212> DNA

<213> P2

<400> 4

gttgcggccg cattgattgc tgtgcatggc ttcaat 36

<210> 5

<211> 29

<212> DNA

<213> P3

<400> 5

cagtacgtac tgcaggtata cgtggatgg 29

<210> 6

<211> 33

<212> DNA

<213> P4

<400> 6

ctccctaggt tgattgctgt gcatggcttc aat 33

Claims (9)

1. a kind of chitinase, it is characterised in that: the amino acid sequence of the chitinase is as shown in SEQ ID NO.2.

2. chitinase gene according to claim 1, it is characterised in that: the gene coding is described in claim 1 several Fourth matter enzyme, nucleotide sequence is as shown in SEQ ID NO.1.

3. a kind of recombinant vector comprising chitinase gene as claimed in claim 2.

4. recombinant vector according to claim 3, it is characterised in that: the recombinant vector is recombination escherichia coli plasmid pET-28a(+)-chi1602Or recombination yeast pPIC9k-chi1602。

5. a kind of cell comprising chitinase gene described in claim 2.

6. cell according to claim 5, it is characterised in that: the cell is carried comprising chitinase gene or with recombination The Escherichia coli of body conversion include chitinase gene or the Pichia pastoris converted with the carrier.

7. a kind of preparation method of recombinant vector as claimed in claim 3, it is characterised in that: connect gene described in claim 2 It is connected on pET-28a (+) carrier, obtains large intestine recombinant expression carrier pET-28a (+)-chi1602；Or it will be described in claim 2 Gene is connected to pPIC9k carrier, obtains yeast recombinant expression carrier pPIC9k-chi1602。

8. a kind of preparation method of chitinase as described in claim 1, it is characterised in that: the described culture includes chitinase The cell of gene induces fourth matter enzyme gene expression, harvests expression product, the purified purpose egg for obtaining the pure enzyme form of chitinase It is white.

9. chitinase as described in claim 1 prepares chitin oligo saccharide for tobacco brown spot pathogen of degrading.