CN104762307A - Chitinase gene chiC and encoded protein and application thereof - Google Patents

Chitinase gene chiC and encoded protein and application thereof Download PDF

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CN104762307A
CN104762307A CN201510219342.7A CN201510219342A CN104762307A CN 104762307 A CN104762307 A CN 104762307A CN 201510219342 A CN201510219342 A CN 201510219342A CN 104762307 A CN104762307 A CN 104762307A
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chic
chitinase
gly
ala
chitin
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CN104762307B (en
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王晓辉
张庆芳
迟乃玉
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Dalian University
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Dalian University
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Abstract

The invention relates to a chitinase gene chiC and encoded protein and application thereof, and belongs to the technical field of genetic engineering. According to the chitinase gene chiC and the encoded protein and the application thereof, pseudoalteromonas sp. DL-6 serves as a research object, the chitinase gene chiC with a nucleotide sequence showed in SEQ ID No 1 is cloned for the first time, and a high-activity chitinase is obtained. The preparation method of the high-activity chitinase is simple, the production cost is low, a product is easy to conserve, the appropriate hydrolysis temperature is 30 DEG C, The enzyme-decomposed PH is 9.0, high degradation activity of a colloidal chitin can reach to 1.569 +/- 0.017 U/ml, the chitinase chiC degrades an aipha chitin and a beta chitin to generate a chitobiose, and industrial production prospect is possessed.

Description

Chitinase gene chiC and proteins encoded thereof and application
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of chitinase gene chiC, particularly a kind of derive from new strains (Pseudoalteromonas sp.DL-6) (the CGMCC No.8580) of Pseudoalteromonas chitinase gene chiC and proteins encoded and application.
Background technology
Chitin (chitin), be commonly called as chitin, it is a kind of natural polysaccharide that can extract from shrimp and crab shells the like waste, its structure is that 2-acetylamino-2-deoxy-D-glucose (50 ~ 100%) and a small amount of D-glucosamine pass through β-1,4-glycosidic link (0 ~ 50%) is formed by connecting, the chitin that occurring in nature is synthesized by organism is every year 100-1000 hundred million tons, is that in ocean environment, content is the abundantest, and occurring in nature is only second to cellulosic second largest biomass source.Chitin degrading product is the chitin oligo saccharide, chitin list carbohydrates and their derivative etc. of high added value, there is strengthening immunity, antitumor, antibacterial, hypotensive and reduce the biological activity such as cholesterol, be widely used in the various fields such as medicine, agricultural, industry and food.
At present, it is chemical method that chitin prepares the method that chitin oligo saccharide generally adopts, and namely removes salt, albumen etc. with strong acid, removes lipid or remove ethanoyl etc. with highly basic.But the power consumption of this technique is large, the molecular weight distribution of reaction process and product is difficult to control, and sixty-four dollar question gives off a large amount of spent acid bucks in production process, causes serious environmental pollution.Therefore, find a kind of green, the efficiently method of high yield high-quality chitin oligo saccharide more and more to be paid attention to.Chitinase (chitinase, chi, EC3.2.1.14) refers to that single-minded degrade chitin is the general name of one group of enzyme of chitin oligo saccharide or monose.Occurring in nature, chi is extensively present in bacterium, virus, fungi, insect, plant and animal, and this enzyme has important physiological function, the chi of different sources, its catalytic mechanism and physiological function may difference very large.Bacteriogenic chitinase degradable chitin produces carbon source and the energy, obtains own growth, the required nutriment of breeding, ensures the chitinous recycle of occurring in nature simultaneously; Mykose enzyme can realize division and the Morphogenesis of own cells; The arthropods secretion chitinases such as insect meet the needs of casting off a skin and growing; Virus chitinase produces in pathogenesis; Plant secretion chitinase is but the self-protection in order to defend the invasion and attack of sex pheromone to carry out; Some protozoons need chitinase to digest the peritrophic membrane of insect.The chitinase function that the mankind produce not yet is determined, with the pathogenic agent in chitin source in opposed body or may play physiological function in carbohydrate metabolism process by inference.
Chitin is the most rich in natural resources of ocean environment, the marine bacteria of 10% relies on chitin as Carbon and nitrogen sources according to estimates, ocean has high salt (3.5%), high pressure (﹥ 100Mpa), low temperature, low light according to extreme ecotope such as, oligotrophic and unglazed photograph, localized hyperthermia's (400 DEG C) etc., and marine low temperature chi has the biological characteristics such as high enzymatic activity and high catalytic efficiency, the efficient kindliness of structure and thermolability under low temperature.Ecotope complicated and changeable and genetic mechanism and genetic construction are given marine low temperature chi and are produced and distinctively have the different polymerization degree (degree of polymerization, DP) chitin oligo saccharides.Chitin is mainly derived from marine organism, as shrimp, crab and algae and other mollusks are, therefore gathers very abundant chitin resource in ocean.If chitin resource processes bad, not only cause waste but also contaminate environment.Marine low temperature chitinase degrade chitin production chitin oligo saccharide and derivative thereof explain the basic law of ocean environment chitin material cycle; be not only the development and utilization of new type marine microorganism resource, the protection and treatment of ocean environment provides important theoretical foundation, and for the market of developing chitin industry, there is using value.
Summary of the invention
The technical problem that the present invention solves is to provide a kind of chitinase gene chiC with nucleotide sequence shown in SEQ ID No.1, and constructs chiC prokaryotic expression carrier, obtains highly active chitinase.
For solving the problems of the technologies described above, the design that the present invention adopts is: with Pseudoalteromonas (Pseudoalteromonassp.DL-6) (CGMCC No.8580) bacterial strain for research object, clone and prokaryotic expression chitinase chiC gene, for the product of acquisition high reactivity chitinase and the function with chitinase provides new approaches.
The technical solution used in the present invention is:
A kind of chitinase gene chiC is the nucleotide sequence shown in SEQ ID No.1.
The amplification method of above-mentioned chitinase gene chiC, for template, carries out pcr amplification with primer pair chiC-F and chiC-R with Pseudoalteromonas strain (Pseudoalteromonas sp.DL-6) genomic dna; Upstream primer chiC-F:5 '-CCC gGATCCgGCGCCTTCTACACCAAGCATTAATTGG-3 ', downstream primer chiC-R:5 '-CCG cTCGAGaAGTGCTAACCATACATCGG-3 '.
Second object of the present invention is a kind of chitinase of being expressed by above-mentioned chitinase gene chiC of request protection.
Described chitinase, has the aminoacid sequence as shown in SEQ ID No.4.
3rd object of the present invention is the expression and purification method of a kind of chitinase chiC of request protection, and step is as follows:
(1) with the nucleotide sequence of PCR method amplification as shown in SEQ ID No.1;
(2) build E. coli cloning vector pMD19-T-chiC: the DNA sequence dna of pcr amplification and the same restriction enzyme of pMD19-T simple carrier are carried out double digestion, digestion products, through reclaiming purifying, uses T 4dNA ligase connects the digestion products of purifying, obtains cloning vector pMD19-T-chiC;
(3) Recombinant protein expression carrier pET28a-chiC is built: by cloning vector pMD19-T-chiC Plastid transformation to E.coli DH5 α, obtain positive transformant, with restriction enzyme BamHI and XhoI, chiC gene fragment is cut from pMDl9-T-chiC plasmid, be connected on pET-28a expression vector, be converted into E.coli BL21 (DE3), cut Screening and Identification positive transformant by bacterium colony PCR, enzyme, obtain recombinant expression vector pET28a-chiC;
(4) Recombinant protein expression bacterial strain BL21 (DE3)-pET28a-chiC is built: by recombinant expression vector pET28a-chiC Plastid transformation to E.coli BL21 (DE3), select positive transformant clone, obtain recombinant strains BL21 (DE3)-pET28a-chiC;
(5) external evoked expression: recombinant strains BL21 (DE3)-pET28a-chiC is accessed in the LB substratum containing kantlex, after 37 DEG C of incubated overnight 14h, inoculum size by 1% is inoculated in the LB substratum containing kantlex, works as OD 600nm=0.6-0.8, adds the IPTG that final concentration is 0.2mM, in 30 DEG C, and 100rpm low temperature induction 6h;
(6) purifying of chitinase chiC: in 4 DEG C after external evoked expression terminates, 5,000 × g centrifugal 5min collect thalline, with PBS buffer solution thalline three times, use the resuspended thalline of PBS damping fluid again, be placed on ultrasonication on ice three times, each 30s, every minor tick 1min, then in 4 DEG C, 12,000 × g centrifugal 20min collect supernatant liquor, be crude protein, crude protein Ni 2+-NTA post carries out affinitive layer purification, obtains chitinase chiC.
The present invention also asks to protect above-mentioned chitinase in degraded containing the application in chitin substrate.
The application of described chitinase chiC, suitable enzymatic hydrolysis condition is: hydrolysis temperature 30 DEG C, and pH value is 9.0.
Described chitinase chiC has high degrading activity to tobacco brown spot pathogen and reaches 1.569 ± 0.017U/mL.
Described chitinase chiC degraded α chitin and β chitin generate chitobiose.
There is a product for the function of chitinase, there is the aminoacid sequence as shown in SEQ ID No.4.
The invention has the beneficial effects as follows:
1, present invention obtains the sequence of chitinase gene chiC, this sequence is clone in Pseudoalteromonas (Pseudoalteromonas sp.DL-6) first;
2, the present invention constructs chiC prokaryotic expression carrier, successfully obtain the zymoprotein that molecular weight is about 92.659kD, obtain and have highly active enzyme, and preparation process is simple, for chiC functional study provides short-cut method;
3, in the present invention, chitinase chiC cold-adapted enzyme is lived high, and the optimum temperature of reaction of enzymolysis is 30 DEG C, and live higher at pH=6.0 ~ 9.0 enzyme, when pH=9.0 reaches the highest, and production cost is low, and product is easily preserved;
4, in the present invention, chitinase chiC has higher degrading activity to tobacco brown spot pathogen, generates chitobiose, have certain industrial production prospect to α chitin and β chitin degrading.
Accompanying drawing explanation
Fig. 1 is the pcr amplification product agarose gel electrophoresis detected result of chiC gene of the present invention;
Fig. 2 is the SDS-PGAE detected result of chiC albumen of the present invention, M in figure: protein standard molecular weight; 1 ~ 3 is BL21/chiC-pET28aSDS-PGAE detected result: 1 is the full bacterium of broken wall; 2 is ultrasonication supernatant liquor; 3 is broken wall precipitation; 4 ~ 6 are BL21/pET28a detected result: 4 is the full bacterium of broken wall; 5 is supernatant liquor after broken wall; 6 is broken wall postprecipitation;
Fig. 3 is the SDS-PGAE detected result of chiC protein purification of the present invention, M in figure: protein standard molecular weight; 1: supernatant after recombinant bacterium broken wall; 2: after recombinant bacterium fragmentation, supernatant is through the effluent liquid of ni-sepharose purification; The imidazoles wash-out of 3:20mmol collects liquid; The imidazoles wash-out of 4:150mmol collects liquid; The imidazoles wash-out of 5:300mmol collects liquid; 1 ~ 5 applied sample amount is 10 μ L;
Fig. 4 is typical curve and the typical curve equation of chiC albumen;
Fig. 5 is enzyme typical curve alive and typical curve equation;
Fig. 6 be in the present invention temperature on the impact of chitinase chiC activity; In figure, A: chitinase chiC active optimum temperuture; B: chitinase chiC temperature stability;
Fig. 7 be in the present invention pH on the impact of chitinase chiC activity; In figure, A: chitinase chiC optimal pH; B: chitinase chiC pH stability;
Fig. 8 is the kinetic constant analysis of chiC albumen of the present invention;
Fig. 9 is the substrate specificity of chiC albumen of the present invention;
Figure 10 is chiC protein degradation product of the present invention analysis; In figure, A: α chitin; B: β chitin.
Embodiment
Below by embodiment, the present invention is described further, but is not used in and limits the scope of the invention.Experimental technique in embodiment, if no special instructions, is ordinary method; Used experimental raw mainly comprises: competent escherichia coli cell E.coli DH5 α, E.coli BL21 (DE3), and pMD19-T Simple is purchased from TaKaRa company; T4DNA ligase enzyme, restriction enzyme BamHI and XhoI, protein molecular weight standard are purchased from TaKaRa company; Ni 2+-NTA resin is purchased from Novagen company; Shrimp shell α type chitin, squid β type chitin available from Sigma; IPTG, kantlex, acrylamide sodium laurylsulfonate, glycine are purchased from Sangon Biotech (Shanghai) Co., Ltd., and other conventional chemical reagent is commercially available analytical pure.
The amplification of embodiment 1 chiC gene
The present invention, according to the gene order design primer of the Pseudoalteromonas chitinase that NCBI has reported, obtains the conserved sequence of Pseudoalteromonas (Pseudoalteromonas sp.DL-6) chitinase chiC by the method for polymerase chain reaction (PCR).This bacterial strain in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preservation date: on December 13rd, 2013, its deposit number is CGMCC No.8580.
Utilize round pcr with the genomic dna of Pseudoalteromonas (Pseudoalteromonas sp.DL-6) bacterial strain for template, adopt Primer5.0 software design primer, be respectively: upstream primer chiC-F (SEQ ID No.2): 5 '-CCC gGATCCgGCGCCTTCTACACCAAGCATTAATTGG-3 ' is designed with BamHI restriction enzyme site; Downstream primer chiC-R (SEQ ID No.3): 5 '-CCG cTCGAGaAGTGCTAACCATACATCGG-3 ' is designed with XhoI restriction enzyme site.
PCR reaction system:
Genomic DNA template 2.5ng
10×LA PCR Buffer(Mg 2+plus) 5μL
DNTPMixture (each 2.5mM) 8μL
Upstream primer (20 μMs) 1μL
Downstream primer (20 μMs) 1μL
TaKaRa LA Taq(5U/μL) 0.5μL
RNase Free dH 2O up to 50μL
PCR reaction conditions: 94 DEG C of 2min, 1 circulation; 94 DEG C of 30s, 55 DEG C of 30s, 68 DEG C of 7min, 30 circulations; 68 DEG C of 15min, 1 circulation.PCR reaction terminates to detect with 1% agarose gel electrophoresis, as shown in Figure 1, has bright single band, be goal gene chiC at 2631bp place.Its nucleotide sequence is (Genbank accession number is: KF234017) as shown in SEQ ID No.1.ChiC of the present invention encodes 876 amino acid, and molecular weight is about 92.659kD, has the aminoacid sequence as described in SEQID No.4.
The preparation of embodiment 2 E. coli cloning vector pMDl9-T-chiC
1, the connection of chiC gene and pMDl9-T
Purifying recovery is carried out to the PCR primer that embodiment 1 obtains, use TaKaRa company test kit (TaKaRa DNALigation Kit<Mighty Mix>), the PCR primer of the chiC reclaimed is connected with pMD19-T simple cloning vector.
Ligation system is:
The PCR primer of chiC 2μL
pMD19-TSimple(50ng/μL) 1μL
Ligation Mix(50ng/μL) 5μL
dH 2O 2μL
The condition of ligation is: 16 DEG C of connections of spending the night, and builds cloning vector pMDl9-T-chiC.
2, the conversion of cloning vector (pMDl9-T-chiC)
(1) get 100 μ L competent escherichia coli cell E.coli DH5 α of freeze thawing on ice, in the ligation liquid of above-mentioned 10 μ L, softly mix;
(2) place in ice after 30 minutes, at 42 DEG C of Heat thermostability 1min, then shift in ice immediately and place 1min;
(3) the SOC nutrient solution of 900 μ L is added, 37 DEG C of shaking culture 1h;
(4) get 100 μ L and be coated with LB/Kana media surface, 37 DEG C of incubated overnight, screening positive transformant.
LB/Kana substratum: add in 950mL deionized water: Tryptones 10g, yeast extract 5g, NaCl 10g, shake container is until solute dissolves.PH to 7.0 is adjusted by 5N NaOH solution (about 0.2mL).Be settled to 1L with deionized water, add 15g agar.Autoclave sterilization is cooled to room temperature.Mix after adding 1mL kantlex (kanamycin, kana, 50mg/mL), 4 DEG C of preservations.
3, the extraction of cloning vector (pMDl9-T-chiC)
With TaKaRa company test kit (TaKaRa MiniBEST Plasmid Purification), extract cloning vector (pMDl9-T-chiC).
The preparation of embodiment 3 coli expression carrier pET28a-chiC
1, the preparation of chiC gene
Recombinant plasmid pMDl9-T-chiC BamHI and XhoI built two kinds of digestion with restriction enzyme qualifications, reaction system is as follows:
pMDl9-T-chiC(30ng/μL) 10μL
BamHI(5U/μL) 2μL
XhoI(5U/μL) 2μL
10×M buffer 10μL
dH 2O up to 100μL
Reaction system is enzymolysis 2-3h in 37 DEG C of water-baths, and product detects through 1% agarose gel electrophoresis.Enzymolysis product TaKaRa company test kit TaKaRa Agarose Gel DNA Purification Kit Ver.2.0, reclaims gene chiC.
2, the preparation of expression vector pET-28a
Utilized by expression vector pET-28a same restriction enzyme BamHI and XhoI to carry out double digestion qualification, reaction system is as follows:
pET-28a(20ng/μL) 20μL
BamHI(5U/μL) 2μL
XhoI(5U/μL) 2μL
10×M buffer 10μL
dH 2O up to 100μL
Reaction system is enzymolysis 2-3h in 37 DEG C of water-baths, and product detects through 1% agarose gel electrophoresis.Enzymolysis product TaKaRaAgarose Gel DNA Purification Kit Ver.2.0, reclaims expression vector pET-28a.
3, the structure of RT-PCR expression vector pET28a-chiC
Use DNALigation Mix test kit (purchased from Takara company), chiC gene subclone to the same enzyme produced by BamHI and XhoI digestion with restriction enzyme is cut in the expression vector pET-28a of process, reaction system: cloning vector (pMDl9-T-chiC) digestion products chiC gene (30ng/ μ L) 1 μ L, pET-28a (20ng/ μ L) 2 μ L, 5 μ L LigationMix, 2 μ L dH 2o.Reaction conditions is: 16 DEG C of connections of spending the night.
Embodiment 4 builds Recombinant protein expression bacterial strain
1, the conversion of recombined pronucleus expression plasmid pET28a-chiC
(1) get 100 μ L competent escherichia coli cells E.coli BL21 (DE3) of freeze thawing on ice, in the ligation liquid of above-mentioned 10 μ L, softly mix;
(2) place in ice after 30 minutes, at 42 DEG C of Heat thermostability 1min, then shift in ice immediately and place 1min;
(3) the SOC nutrient solution of 900 μ L is added, 37 DEG C of shaking culture 1h;
(4) get 100 μ L to be coated with dull and stereotyped (LB/Kana), 37 DEG C of incubated overnight screening positive transformants;
(5) extraction of recombinant prokaryotic expression vector pET28a-chiC, extracts expression vector pET28a-chiC with TaKaRa company test kit (TaKaRa MiniBESTPlasmid Purification).
2, Recombinant protein expression bacterial strain is built
(1) cultivation is planted: E.coli BL21 (DE3) cell containing recombinant prokaryotic expression vector pET-28a recombinant prokaryotic expression vector pET28a-chiC and empty fusion expression vector pET-28a being chosen 1-2 single colony inoculation is in the 2mL LB/Kana substratum of 50 μ g/mL to containing kantlex final concentration, 37 DEG C of incubated overnight.
(2) main cultivation: cultivate after 14 hours and be transferred in 100mL LB/Amp substratum by 1% inoculum size, 37 DEG C of shaking culture are to the OD of nutrient solution 600nmreach 0.6-0.8.
(3) abduction delivering: add IPTG 100rpm low temperature induction expression 6h at 30 DEG C that final concentration is 0.2mM.
(4) full cell is collected: in 4 DEG C, 5,000 × g, centrifugal 10min collects thalline, and with the PBS suspended bacteria somatocyte of 300 μ L, gets 200 μ L bacteria suspension ultrasonication 10s/2 ~ 3 time after mixing, until liquid-transparent, take out 50 μ L as full cell sample.
(5) extracting of soluble protein: by the nubbin of above-mentioned bacterium liquid in 12,000 × g, 4 DEG C of centrifugal 5min, get 50 μ L supernatant liquors and be soluble protein.
(6) extracting of soluble albumen: add 150 μ L PBS damping fluids in step (5) precipitation, fully vibration mixing is soluble albumen.
(7) target protein of SDS-PAGE electrophoresis detection expression, is shown in shown in accompanying drawing 2.
The purifying of embodiment 5 recombinant chitinase chiC albumen
1, a large amount of abduction deliverings of chiC albumen
(1) picking recombination bacillus coli bacterium colony access 50mL is containing the LB nutrient solution of kantlex (50 μ g/mL), in 37 DEG C of incubated overnight in 250mL shaking flask;
(2) by the LB nutrient solution of 50mL overnight culture access 500mL containing kantlex (50 μ g/mL), in 37 DEG C of shaken overnight cultivations in 2L shaking flask, to mid-log phase (OD 600nm=0.6-0.8);
(3) add final concentration be the IPTG of 0.2mM at 30 DEG C, 100rpm low temperature induction target protein 6h;
(4) in 4 DEG C with 5,000 × g, centrifugal 5min collects thalline (about 3.5g);
(5) bacterial sediment is resuspended in the PBS damping fluid of 8 times of volumes, 4 DEG C of ultrasonic disruption 10s × 3 time, altogether until liquid-transparent;
(6) in 4 DEG C with 12,000 × g, centrifugal 20min collects supernatant liquor and carries out the research of chiC protein purification.
2, chiC albumen passes through Ni 2+-NTA resin carries out large scale purification
(1) Ni is cleaned with intermediate water 2+-NTA resin;
(2) Ni is cleaned with PBS 2+-NTA resin;
(3) by the fusion rotein crude extract upper prop of a large amount of abduction delivering;
(4) with the recombinant protein that 10 times of volume 20mM-300mM bufferA/ imidazoles wash-out (Elute) combine, every part of 800 μ L distribute and collect, monitoring OD 280, carry out 10%SDS polyacrylamide gel electrophoresis with the albumen of 20 μ L equal portions, as shown in Figure 3, analyze the distribution situation of chiC albumen, obtain recombinant chitinase chiC purifying protein.
The preparation of embodiment 6 chiC reference protein solution
Reference protein solution is prepared by the step that plasmid pET28a replaces pET28a-chiC to carry out embodiment 1 and embodiment 6.
The mensuration of embodiment 7 chiC protein concentration
One, the mensuration of protein standard curve
BCA-100 determination of protein concentration test kit (enhancement type) is used to carry out protein determination.First according to testing sample number, the mixed solution (50 parts of reagent A+1 part of reagent B) of BCA A and BCA B is prepared, each reaction use 200 μ L after mixing.3 replicate(determination)s of each response preparation.0.5mg/mL BSA standard and BCA A+B mixture is added, mixing by the order of table 1.
Table 1 BSA typical curve is tested
Test condition: 37 DEG C are incubated 30 minutes, cool to room temperature, measure the absorbance value of 570nm, according to absorbance, simulate typical curve, calculate the protein concentration of testing sample according to typical curve.Protein standard curve and equation as shown in Figure 4,
Two, the mensuration of chiC protein concentration
Get the chiC albumen of appropriate concentration, add the 50mM Tris-HCl (pH9.0) of certain volume, cumulative volume is 20 μ L, adds Mix (A+B) 200 μ L, and 37 DEG C are incubated 30 minutes, cool to room temperature, measure the photoabsorption of 570nm.Show that the concentration of chiC protein solution is 1.186mg/mL according to Fig. 4 Equation for Calculating.
The chitinase enzyme of embodiment 8 chiC albumen is lived
One, the preparation of tobacco brown spot pathogen: get 10.0g powdery α chitin and be placed in mortar, slowly add 100mL, the strong phosphoric acid of 85% in stink cupboard, continuous glass stick fully stirs simultaneously.Mortar preservative film is sealed, spends the night at 4 DEG C.Then rinsing with a large amount of intermediate waters, until pH is centrifugal after about 5.0, is 1L with damping fluid (as 0.1mol/L phosphoric acid buffer, pH7.0) constant volume.Be mixed with 1% tobacco brown spot pathogen solution, keep in Dark Place for subsequent use in refrigerator.
Two, the foundation of enzyme activity determination typical curve
Adopt Tripotassium iron hexacyanide method to measure chiC enzymic activity, get 1mL sample solution, add 1mL Tripotassium iron hexacyanide reagent (preparation 1L 0.5M sodium carbonate solution, adds the 0.5g Tripotassium iron hexacyanide), in boiling water, boil 10min, be then cooled to room temperature with tap water, measure OD 420nm, table 2 specific as follows.With 2-acetylamino-2-deoxy-D-glucose (mmol/L) for X-coordinate, OD 420nmabsorbancy is ordinate zou, calculates 2-acetylamino-2-deoxy-D-glucose typical curve.Typical curve and equation are as shown in Figure 5.
Table 2 Tripotassium iron hexacyanide method measures reducing sugar content
Three, the chitinase activity of chiC albumen
Get crude enzyme liquid 0.1mL and 0.9mL, 1% tobacco brown spot pathogen mixing, after being incubated 30min at 20 DEG C, reaction mixture being heated in boiling water bath and within 10 minutes, stops enzyme reaction, 15,000 × g centrifugal 5min, get supernatant 0.5mL, add 1mL, Schales reagent, boiling water bath 10min, cooling.With the enzyme liquid of deactivation as a control group, to contrast as reference, under 420nm, absorbancy is measured.Calculate reducing sugar content according to typical curve, thus it is alive to calculate enzyme.Each gradient arranges 3 repetitions, averages.
Enzyme activity unit definition (U): under these conditions, per minute discharges the enzyme amount needed for 1 μm of ol reducing sugar (in 2-acetylamino-2-deoxy-D-glucose).Be 6.947U/mL according to Fig. 5 Equation for Calculating chiC albumen chitinase activity.
The research of embodiment 9 chiC albumen zymologic property
The zymologic property of the chitinase chiC albumen that embodiment 5 obtains is measured, comprising optimum temperuture, temperature stability, optimal pH, pH stability, metal ion and chemical reagent to the impact of chiC protease activity, is that substrate carries out kinetic constant mensuration and substrate specificity Journal of Sex Research with tobacco brown spot pathogen.
(1) enzyme activity determination
Specifically see embodiment 8.
(2) temperature is on the impact of chitinase chiC
1. optimal reactive temperature: be under the condition of 8.0 (50mmol/L Tris-HCl damping fluids) in pH value, with 3.5mg/mL tobacco brown spot pathogen for substrate, in the temperature range of 4 DEG C-70 DEG C, measure enzyme live, according to enzyme relative reactivity curve plotting at different temperatures, determine the optimal reactive temperature of enzyme.As shown in Figure 6A, the optimum temperuture of chiC is 30 DEG C to result.
2. the thermostability of chiC: be incubated 1h under enzyme liquid being placed in respectively differing temps (4 DEG C-70 DEG C), then detect remnant enzyme activity under optimal reaction pH and optimal reactive temperature, and compare with untreated enzyme is alive, calculate relative reactivity.As shown in Figure 6B, chiC is at lower than the temperature of 30 DEG C after preincubate 24h, and enzyme is lived and remained on more than 60% for result.
(3) pH is on the impact of chitinase chiC
1. optimal reaction pH value: enzyme temperature of reaction alive is 30 DEG C, with 3.5mg/mL tobacco brown spot pathogen for substrate, is 50mmol/L damping fluid (pH 3.0 ~ 5.0 phosphoric acid-citric acid, pH 6.0 ~ 10.0Tris – HCl, the pH 11.0 ~ 12.0Na of 3-12 in pH value 2hPO 4– NaOH) work of middle mensuration enzyme, according to the relative reactivity curve plotting of enzyme under different pH value, determine the optimal reaction pH value of enzyme.As shown in Figure 7 A, the optimal pH of chiC is pH9.0 to result.
2. the pH stability of chiC: the damping fluid (pH 3.0 ~ 5.0phosphate-citrate, pH 6.0 ~ 10.0Tris – HCl, the pH 11.0 ~ 12.0Na that enzyme liquid are placed in respectively the different pH of 50mmol/L 2hPO 4– NaOH) in, at 30 DEG C, place 24h, then under optimal reaction pH and optimal reactive temperature, detect remnant enzyme activity, and compare with untreated enzyme is alive, calculate relative reactivity.As shown in Figure 7 B, chiC, at 30 DEG C, after different pH preincubate 24h, starts to improve in more than pH6 activity result, in the maintenance more than 75% alive of pH>8.0 enzyme, shows chi C meta-alkalescence.1M Tris-HCl (pH8.0): 6.057g Tris, 29.22g NaCl, constant volume is transferred to 8.0 to 1L, HCl, and the used time is diluted to desired concn.
(4) metal ion and chemical reagent are on the impact of chitinase chiC
KCl, NaCl, CaCl that final concentration is 1mmol/L or 5mmol/L is added in reaction system 2, MgCl 2, MnCl 2, CuCl 2, ZnCl 2, CoCl 2, FeSO 4and FeCl 3, then measure enzyme at the standard conditions and live, and compare with the enzyme not adding any metal ion is alive, calculate relative reactivity.EDTA, urea, PMSF, DTT, SDS, Triton X-100 and Tween-20 that final concentration is 1mmol/L or 5mmol/L is added in reaction system, then measure enzyme at the standard conditions to live, and compare with enzyme when not adding any chemical reagent is alive, calculate relative reactivity, the results are shown in Table 3, in reaction solution, add the K of 1mM and 5mM as known from Table 3 +, Na +, Mg 2+, Mn 2+, Ca 2+, 1mM EDTA and PMSF have promoter action to enzymatic reaction, 1mM and 5mM urea and DTT is little on enzymatic reaction impact, and when adding the Cu of 1mM and 5mM 2+, Zn 2+, Fe 2+, Fe 3+, Co 2+, SDS, Triton X-100 and Tween-20 have restraining effect to enzymatic reaction.
Table 3 metal ion and chemical reagent are on the impact of chiC albumen
(5) mensuration of enzyme kinetics parameter
With the pure enzyme liquid of certain volumetric molar concentration ([E]), with the substrate reactions of different concns, under standard assay conditions, detect initial reaction speed, because k cat=Vmax/ [E], wherein [E] is known, can calculate k cat, finally obtain Km and k cat.Concrete measuring method: dilute the substrate solution of substrate solution tobacco brown spot pathogen to a series of different concns (2 ~ 10mg/mL) with 50mmol/L Tris-HCl damping fluid (pH8.0); The substrate solution getting 0.9mL different concns mixes with the chiC of 0.1mL proper concn, is placed in 30 DEG C of water-bath 2h and mixes.To boil the enzyme liquid of deactivation as a control group, to contrast as reference calculates initial reaction speed according to typical curve.The results are shown in Figure 8, the Km value that kinetic constant analysis shows chiC for 3.47mg/mL, Vmax be 12.32 μm of ol/min.
(6) substrate specificity of chiC
1mL reaction system comprises the chiC enzyme of 5 μMs, α-the chitin of 2mg/mL, β-chitin, tobacco brown spot pathogen, β-chitin nanowhisker, Microcrystalline Cellulose and chitosan 6 kinds substrate, at 30 DEG C of reaction 24h, then survey reducing sugar content (concrete with reference to embodiment 8).With the enzyme liquid of deactivation as a control group, 3 repetitions are set.The results are shown in Figure 9, chiC more easily degrades tobacco brown spot pathogen as seen from the figure, enzymic activity reaches 1.569 ± 0.017U/mL, be followed successively by α chitin, β chitin, chitosan, β chitin nanowhisker and Microcrystalline Cellulose, corresponding enzymic activity is respectively 1.422 ± 0.044U/mL, 1.34 ± 0.019U/mL, 1.249 ± 0.027U/mL, 1.179 ± 0.013U/mL and 0.971 ± 0.018U/mL.ChiC prefers to the higher degrading activity of tobacco brown spot pathogen and illustrates that the combination and the enzymic catalytic reaction that are more conducive to chitinase activity site occur because the tobacco brown spot pathogen after acid hydrolysis process destroys the chitinous crystalline structure of powdery.
The degraded product analysis of embodiment 10 chiC albumen
Matrix Assisted Laser Desorption time-of-flight mass spectrometry (MALDI-TOF/TOF 5800) is utilized to detect the product of chiC effect chitin substrate generation.1mL reaction system: 5mmol/L chiC, 2mg/mL α chitin and β chitin, is dissolved in 50mmol/L Tris-HCl damping fluid (pH 9.0), reacts 24h in 30 DEG C of water-baths.Getting the supernatant liquor point that 0.5 μ L handles well is added in the special sample panel of MALDI TOF mass spectrograph, naturally dry at ambient temperature (about needing 3min), dropping 0.5 μ L matrix (2 on it, 4-dihydroxy benzophenone, DHB) solution covers, dry rear for subsequent use with method, each sample repeats 3 holes.Adopt N_2 laser source, wavelength is 337nm, linear cationic detecting pattern; Acceleration voltage is 15000V; Time of lag is 300ns; Laser intensity is 7000Hz; Each sample spot establishes laser to shoot 40 points, each shooting 50 times at random, and m/z acquisition range is 1000D ~ 20000D.Data Explorer 4.0 software of instrument configuration is adopted to carry out base correction and acquiescence smoothing processing to mass spectrum.As shown in Figure 10, chiC degraded α chitin and β chitin mainly generate chitobiose is primary product.
<110> University Of Dalian
<120> chitinase gene chiC and proteins encoded thereof and application
<130> 2015
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2631
<212> DNA
<213> Pseudoalteromonas sp. DL-6
<400> 1
atgaatatta aacaattaag cgcagctatg ggtgttgcac tatttgccgg ttctgtatcg 60
gcagcgcctt ctacaccaag cattaattgg gaaccgcagc aatattcgtt tgttgaggta 120
gatcttgagg gaaacggctc ttataagcag ctagttaccc gtgttgagca agttaatatc 180
aacattgagt ggagtgcatg gagtggtgat ggtggcgata gctacaaagt gtactttgat 240
gacatgctag tcaatgaagg cacgcttgct gctggttcta aaagtggcac tatcacattc 300
ccttatgata aagcgggccg ccatacaatg tatgttgagc tatgtgaagg tggcacaacg 360
tgtgctcgca gtgcaggcaa accaattgta attgctgaca ccgacggcgg acacctagca 420
ccattaccta tggatgttga cccaaataac cgtgacattg gtatcaaaca aggtttagtt 480
acaggcgctt actttgttga gtggggaatt tatggccgtg attatgatgt aacgaatatg 540
ccagcacaaa atctgagtca tattttgtat ggctttattc caatttgtgg tgaaaacgca 600
tcgttatcgg gtggtcctaa gcgtgcgctt gatactgcct gcgccggctc tgcagattac 660
gaagtggtta ttcacgaccc atgggcagca gtacaaaaag cactgccagg ggttgatgca 720
aaagatccta tccgtggtac ttactctcag ctaatggcgc ttaaacagcg ttacccagat 780
attaaaattt taccatctgt aggtggttgg acgttatcag acccattcgg tggctttacc 840
aataaagcta accgcgacac ctttgttgcc tcaatggaag aatttttaag aacatggaaa 900
ttctatgatg gtgtagatat tgactgggaa ttcccaggtg gtgacggccc caacccagac 960
attggcgatc caatcaacga tggcccagcc tatgtggcgc taatgcaaga gctacgcgct 1020
atgcttgata aacttgaagc agaaacgggc cgtacttacg agttaacctc agccattggt 1080
gcaggttacg ataaaattga agatgtagat taccaagccg caagccagta tatggattac 1140
atttttgcca tgacctatga cttttatggc gcatggagta acgtaaccgg acaccaaaca 1200
gcactttact gtggcgaaca tatgagtgtt ggtcaatgta atggtaccgg gcttgatgaa 1260
aatggcgaac ctcgtaaagg ccctgcttat accaccgata atgcagtgca gctgttactt 1320
gcgcaaaatg taccatcgaa aaaaatcgtc gtgggcacag ctatgtatgg ccgtggttgg 1380
gaaggtgttt acccgcaaaa tgcagcaatt gatggtaacc caatgaccgc ccctggtaat 1440
ggcccgttaa aaggctctac ggcacaaggc gtatgggaag atggggttat tgattacaaa 1500
ggcgttaaag caaatatgat tggtgcggcg ggcaccggta ttaatggttt tgaagtgggt 1560
tatgatgagc aagcgcaagc cgcttatgta tggaatcgtg caaccggtaa actaattact 1620
tatgatagcc gtaagtcagt actggctaag ggcgcgtatg ttaatcagta taatttaggt 1680
ggtttatttg catgggaaat agatgctgat aatggtgata ttctaaatgc tatgcatgat 1740
ggtttaggag gggtagttgc tccgccaaca aataaaaagc cagttgtttc tgtgtcttca 1800
tcagtgtctg ttaattcggg tgagagtatc acagtaacag cttctgcaac cgatgccgat 1860
aacgatccac ttagctttag ctggagtgct gataacgcac ttgtagtatc tggacaaaat 1920
agcgcatcat tagtcattac agcgccaaca gtgactgcag atacacagta tgtggcaacc 1980
gttgcggtat cagatggtga ggcaaccgtt aatcgtgatg ttgttgttaa tgttattgcg 2040
ccaacatcag gtggtgaaaa cacagctcct agcgttgatg ctatcgctaa tatcagcgtt 2100
gaagaaggcg catcaacatc agttgctgtt gtggcaagcg atgctcaaaa cgatgcagtt 2160
acatatacgt ggacagtacc agctggttta acgttagtgg gtagtggttc aaatgtaact 2220
attgaggctg gcgcagttga tgcagataca gatttcacag tgtctgttgc agttagtgat 2280
ggcgcattaa ccacaacgca aagctttagc gtaacagtta caaacgttga tactacaaat 2340
ccacctacag gaagctggga tgcgagtgtt gcatacgttg gtggtgatgt cgttacttat 2400
aacggcgttg aatataaagc aaagtggtgg actcaaggcg agcgtcctga tttaggtggt 2460
gcatgggaag caacaacaca acctacagat ggtacgggtg tcgcagtttg gcagccaacg 2520
gctatttata acagtggtga tgaagtttct tatcaaggta ataagtacca agctaaatgg 2580
tggactcaag ggaacgaacc tggttcaacc gatgtatggt tagcacttta a 2631
 
<210> 2
<211> 37
<212> DNA
<213> synthetic
<400> 2
cccggatccg gcgccttcta caccaagcat taattgg 37
 
<210> 3
<211> 29
<212> DNA
<213> synthetic
<400> 3
ccgctcgaga agtgctaacc atacatcgg 29
 
<210> 4
<211> 876
<212> PRT
<213> Pseudoalteromonas sp. DL-6
<400> 4
Met Asn Ile Lys Gln Leu Ser Ala Ala Met Gly Val Ala Leu Phe Ala
1 5 10 15
Gly Ser Val Ser Ala Ala Pro Ser Thr Pro Ser Ile Asn Trp Glu Pro
20 25 30
Gln Gln Tyr Ser Phe Val Glu Val Asp Leu Glu Gly Asn Gly Ser Tyr
35 40 45
Lys Gln Leu Val Thr Arg Val Glu Gln Val Asn Ile Asn Ile Glu Trp
50 55 60
Ser Ala Trp Ser Gly Asp Gly Gly Asp Ser Tyr Lys Val Tyr Phe Asp
65 70 75 80
Asp Met Leu Val Asn Glu Gly Thr Leu Ala Ala Gly Ser Lys Ser Gly
85 90 95
Thr Ile Thr Phe Pro Tyr Asp Lys Ala Gly Arg His Thr Met Tyr Val
100 105 110
Glu Leu Cys Glu Gly Gly Thr Thr Cys Ala Arg Ser Ala Gly Lys Pro
115 120 125
Ile Val Ile Ala Asp Thr Asp Gly Gly His Leu Ala Pro Leu Pro Met
130 135 140
Asp Val Asp Pro Asn Asn Arg Asp Ile Gly Ile Lys Gln Gly Leu Val
145 150 155 160
Thr Gly Ala Tyr Phe Val Glu Trp Gly Ile Tyr Gly Arg Asp Tyr Asp
165 170 175
Val Thr Asn Met Pro Ala Gln Asn Leu Ser His Ile Leu Tyr Gly Phe
180 185 190
Ile Pro Ile Cys Gly Glu Asn Ala Ser Leu Ser Gly Gly Pro Lys Arg
195 200 205
Ala Leu Asp Thr Ala Cys Ala Gly Ser Ala Asp Tyr Glu Val Val Ile
210 215 220
His Asp Pro Trp Ala Ala Val Gln Lys Ala Leu Pro Gly Val Asp Ala
225 230 235 240
Lys Asp Pro Ile Arg Gly Thr Tyr Ser Gln Leu Met Ala Leu Lys Gln
245 250 255
Arg Tyr Pro Asp Ile Lys Ile Leu Pro Ser Val Gly Gly Trp Thr Leu
260 265 270
Ser Asp Pro Phe Gly Gly Phe Thr Asn Lys Ala Asn Arg Asp Thr Phe
275 280 285
Val Ala Ser Met Glu Glu Phe Leu Arg Thr Trp Lys Phe Tyr Asp Gly
290 295 300
Val Asp Ile Asp Trp Glu Phe Pro Gly Gly Asp Gly Pro Asn Pro Asp
305 310 315 320
Ile Gly Asp Pro Ile Asn Asp Gly Pro Ala Tyr Val Ala Leu Met Gln
325 330 335
Glu Leu Arg Ala Met Leu Asp Lys Leu Glu Ala Glu Thr Gly Arg Thr
340 345 350
Tyr Glu Leu Thr Ser Ala Ile Gly Ala Gly Tyr Asp Lys Ile Glu Asp
355 360 365
Val Asp Tyr Gln Ala Ala Ser Gln Tyr Met Asp Tyr Ile Phe Ala Met
370 375 380
Thr Tyr Asp Phe Tyr Gly Ala Trp Ser Asn Val Thr Gly His Gln Thr
385 390 395 400
Ala Leu Tyr Cys Gly Glu His Met Ser Val Gly Gln Cys Asn Gly Thr
405 410 415
Gly Leu Asp Glu Asn Gly Glu Pro Arg Lys Gly Pro Ala Tyr Thr Thr
420 425 430
Asp Asn Ala Val Gln Leu Leu Leu Ala Gln Asn Val Pro Ser Lys Lys
435 440 445
Ile Val Val Gly Thr Ala Met Tyr Gly Arg Gly Trp Glu Gly Val Tyr
450 455 460
Pro Gln Asn Ala Ala Ile Asp Gly Asn Pro Met Thr Ala Pro Gly Asn
465 470 475 480
Gly Pro Leu Lys Gly Ser Thr Ala Gln Gly Val Trp Glu Asp Gly Val
485 490 495
Ile Asp Tyr Lys Gly Val Lys Ala Asn Met Ile Gly Ala Ala Gly Thr
500 505 510
Gly Ile Asn Gly Phe Glu Val Gly Tyr Asp Glu Gln Ala Gln Ala Ala
515 520 525
Tyr Val Trp Asn Arg Ala Thr Gly Lys Leu Ile Thr Tyr Asp Ser Arg
530 535 540
Lys Ser Val Leu Ala Lys Gly Ala Tyr Val Asn Gln Tyr Asn Leu Gly
545 550 555 560
Gly Leu Phe Ala Trp Glu Ile Asp Ala Asp Asn Gly Asp Ile Leu Asn
565 570 575
Ala Met His Asp Gly Leu Gly Gly Val Val Ala Pro Pro Thr Asn Lys
580 585 590
Lys Pro Val Val Ser Val Ser Ser Ser Val Ser Val Asn Ser Gly Glu
595 600 605
Ser Ile Thr Val Thr Ala Ser Ala Thr Asp Ala Asp Asn Asp Pro Leu
610 615 620
Ser Phe Ser Trp Ser Ala Asp Asn Ala Leu Val Val Ser Gly Gln Asn
625 630 635 640
Ser Ala Ser Leu Val Ile Thr Ala Pro Thr Val Thr Ala Asp Thr Gln
645 650 655
Tyr Val Ala Thr Val Ala Val Ser Asp Gly Glu Ala Thr Val Asn Arg
660 665 670
Asp Val Val Val Asn Val Ile Ala Pro Thr Ser Gly Gly Glu Asn Thr
675 680 685
Ala Pro Ser Val Asp Ala Ile Ala Asn Ile Ser Val Glu Glu Gly Ala
690 695 700
Ser Thr Ser Val Ala Val Val Ala Ser Asp Ala Gln Asn Asp Ala Val
705 710 715 720
Thr Tyr Thr Trp Thr Val Pro Ala Gly Leu Thr Leu Val Gly Ser Gly
725 730 735
Ser Asn Val Thr Ile Glu Ala Gly Ala Val Asp Ala Asp Thr Asp Phe
740 745 750
Thr Val Ser Val Ala Val Ser Asp Gly Ala Leu Thr Thr Thr Gln Ser
755 760 765
Phe Ser Val Thr Val Thr Asn Val Asp Thr Thr Asn Pro Pro Thr Gly
770 775 780
Ser Trp Asp Ala Ser Val Ala Tyr Val Gly Gly Asp Val Val Thr Tyr
785 790 795 800
Asn Gly Val Glu Tyr Lys Ala Lys Trp Trp Thr Gln Gly Glu Arg Pro
805 810 815
Asp Leu Gly Gly Ala Trp Glu Ala Thr Thr Gln Pro Thr Asp Gly Thr
820 825 830
Gly Val Ala Val Trp Gln Pro Thr Ala Ile Tyr Asn Ser Gly Asp Glu
835 840 845
Val Ser Tyr Gln Gly Asn Lys Tyr Gln Ala Lys Trp Trp Thr Gln Gly
850 855 860
Asn Glu Pro Gly Ser Thr Asp Val Trp Leu Ala Leu
865 870 875

Claims (10)

1. a chitinase gene chiC is the nucleotide sequence shown in SEQ ID No.1.
2. the amplification method of a chitinase gene chiC as claimed in claim 1, it is characterized in that, for template, pcr amplification is carried out with primer pair chiC-F and chiC-R with Pseudoalteromonas strain (Pseudoalteromonas sp.DL-6) genomic dna; Upstream primer chiC-F:5 '-CCC gGATCCgGCGCCTTCTACACCAAGCATTAATTGG-3 ', downstream primer chiC-R:5 '-CCG cTCGAGaAGTGCTAACCATACATCGG-3 '.
3. the chitinase of a chitinase gene chiC expression as claimed in claim 1.
4. chitinase according to claim 3, is characterized in that, has the aminoacid sequence as shown in SEQ ID No.4.
5. an expression and purification method of chitinase chiC, it is characterized in that, step is as follows:
(1) with the nucleotide sequence of PCR method amplification as shown in SEQ ID No.1;
(2) build E. coli cloning vector pMD19-T-chiC: the DNA sequence dna of pcr amplification and the same restriction enzyme of pMD19-T simple carrier are carried out double digestion, digestion products, through reclaiming purifying, uses T 4dNA ligase connects the digestion products of purifying, obtains cloning vector pMD19-T-chiC;
(3) Recombinant protein expression carrier pET28a-chiC is built: by cloning vector pMD19-T-chiC Plastid transformation to E.coli DH5 α, obtain positive transformant, with restriction enzyme BamHI and XhoI, chiC gene fragment is cut from pMDl9-T-chiC plasmid, be connected on pET-28a expression vector, be converted into E.coli BL21 (DE3), cut Screening and Identification positive transformant by bacterium colony PCR, enzyme, obtain recombinant expression vector pET28a-chiC;
(4) Recombinant protein expression bacterial strain BL21 (DE3)-pET28a-chiC is built: by recombinant expression vector pET28a-chiC Plastid transformation to E.coli BL21 (DE3), select positive transformant clone, obtain recombinant strains BL21 (DE3)-pET28a-chiC;
(5) external evoked expression: recombinant strains BL21 (DE3)-pET28a-chiC is accessed in the LB substratum containing kantlex, after 37 DEG C of incubated overnight 14h, inoculum size by 1% is inoculated in the LB substratum containing kantlex, works as OD 600nm=0.6-0.8, adds the IPTG that final concentration is 0.2mM, in 30 DEG C, and 100rpm low temperature induction 6h;
(6) purifying of chitinase chiC: in 4 DEG C after external evoked expression terminates, 5,000 × g centrifugal 5min collect thalline, with PBS buffer solution thalline three times, use the resuspended thalline of PBS damping fluid again, be placed on ultrasonication on ice three times, each 30s, every minor tick 1min, then in 4 DEG C, 12,000 × g centrifugal 20min collect supernatant liquor, be crude protein, crude protein Ni 2+-NTA post carries out affinitive layer purification, obtains chitinase chiC.
6. a chitinase as claimed in claim 3 is being degraded containing the application in chitin substrate.
7. the application of chitinase chiC according to claim 6, is characterized in that, suitable enzymatic hydrolysis condition is: hydrolysis temperature 30 DEG C, pH value is 9.0.
8. the application of chitinase chiC according to claim 6, is characterized in that, described chitinase chiC has high degrading activity to tobacco brown spot pathogen can reach 1.569 ± 0.017U/ml.
9. the application of chitinase chiC according to claim 6, is characterized in that, described chitinase chiC degraded α chitin and β chitin generate chitobiose.
10. there is a product for the function of chitinase, there is the aminoacid sequence as shown in SEQ ID No.4.
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CN109897861A (en) * 2019-02-20 2019-06-18 中国水产科学研究院黄海水产研究所 A kind of Portunus trituberculatus Miers chitinase gene and its recombinant expression protein and application
CN109897861B (en) * 2019-02-20 2019-11-08 中国水产科学研究院黄海水产研究所 A kind of Portunus trituberculatus Miers chitinase gene and its recombinant expression protein and application
CN109897859A (en) * 2019-03-11 2019-06-18 大连大学 Polysaccharide cracks monooxygenase gene PsLMPO10A and its coding albumen and function
CN109971741A (en) * 2019-03-27 2019-07-05 大连大学 Expression and purification method of the cold-active chitinase gene chiC in Kluyveromyces lactis
CN110029097A (en) * 2019-03-28 2019-07-19 南京工业大学 A kind of glycoside hydrolase CmNAGase and its clonal expression and application
CN109810966A (en) * 2019-03-28 2019-05-28 南京工业大学 A kind of chitinase CmChi6 gene and its clonal expression and application
CN109810966B (en) * 2019-03-28 2021-05-18 南京工业大学 Chitinase CmChi6 gene and cloning expression and application thereof
CN110029097B (en) * 2019-03-28 2022-09-02 南京工业大学 Glycoside hydrolase CmNAGase and clone expression and application thereof
CN111235133A (en) * 2019-09-30 2020-06-05 广西民族大学 Chitinase gene of chitin-like paenibacillus and cloning expression and application thereof
CN111235133B (en) * 2019-09-30 2023-09-22 广西民族大学 Bacillus chitin-philic chitinase gene and clone expression and application thereof
CN112941054A (en) * 2021-01-08 2021-06-11 南京工业大学 Chitinase CmChi4 and cloning expression and application thereof
CN113528490A (en) * 2021-06-07 2021-10-22 昆明理工大学 Chitinase, coding gene and application thereof
CN113528490B (en) * 2021-06-07 2023-05-02 昆明理工大学 Chitinase, coding gene and application thereof
CN113717963A (en) * 2021-10-13 2021-11-30 广西科学院 Chitinase and AfChi18 gene as well as expression method and application thereof
CN113717963B (en) * 2021-10-13 2023-05-09 广西华仁医学科技集团有限公司 Chitinase and AfChi18 gene and expression method and application thereof

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