CN109810966A - A kind of chitinase CmChi6 gene and its clonal expression and application - Google Patents
A kind of chitinase CmChi6 gene and its clonal expression and application Download PDFInfo
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- CN109810966A CN109810966A CN201910242877.4A CN201910242877A CN109810966A CN 109810966 A CN109810966 A CN 109810966A CN 201910242877 A CN201910242877 A CN 201910242877A CN 109810966 A CN109810966 A CN 109810966A
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- Prior art keywords
- chi6
- chitinase
- cmchi6
- gene
- chitin
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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to a kind of chitinasesCmChi6 gene and its clonal expression and application.The present invention clones the circumscribed chitinase with the nucleotide sequence as shown in SEQ ID No.1 for the first timeCmChi6 gene, and the circumscribed chitinase of high activity is obtained, preparation method is simple, and production cost is low, and product is easy to maintain, and suitable hydrolysis temperature is 45 DEG C, and enzymatic hydrolysis optimal pH is 7.4, and the good thermal stability at 25-50 DEG C, 33 h still keep 60% or more enzyme activity；Optimal pH 7.4, in pH7.0-10.0,33h enzyme activity still keeps 75% or more；Ba2+It is rightCmChi6 vigor has activation slightly；CmChi6 has high degrading activity to tobacco brown spot pathogen, and chitobiose can be prepared with the enzyme, and high conversion rate, energy consumption is few, environmental-friendly, has important economic value in industrialization degradation chitin application.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of chitinaseCmChi6 gene and its clonal expression
With application.
Background technique
Chitin (Chitin): also known as chitin, chitin, glutelin, chitin, chitin etc. are a kind of nitrogenous
Polysaccharide material is the second largest biopolymer abundant of nature, is widely distributed, and is that many lower animals especially segmental appendage is dynamic
The important component of the shells such as object such as shrimp, crab, insect and the component part of rudimentary plant mushroom cell membrane, lichens, green alga, ferment
Also contain in female, jellyfish and squid body.Annual biosynthesis amount is more than 10,000,000,000 tons according to estimates, is a kind of huge renewable
Resource.
Chitin is the straight chain to be got up by monomer N-acetylglucosamine (GlcNAc) by β -1,4- glucosides key connection
Polymer, molecular weight is mostly 1,000,000 or more.The catabolite of chitin is the chitin oligo saccharide of high added value, chitin monosaccharide
And corresponding derivative etc., all there is strengthen immunity, antitumor, antibacterial, blood pressure lowering and reduce sterol and beauty and other effects, extensively
Applied to various fields such as medicine, agricultural, industry and food.At present.It is mainly acid-hydrolysis method that chitin, which prepares chitin oligo saccharide,.Acid
Hydrolyze method, which often utilizes concentrated acid, to be Glucosamine by chitin degrading, and N-acetylglucosamine is obtained after acetylation；Or
Chitin oligo saccharide is obtained using diluted acid degradation chitin.But the energy consumption of the method technique is big, and product yield is low, product bioactivity is poor.
Especially seriously polluted outstanding problem, therefore industrial production is badly in need of a kind of degradation chitin of green, environmentally friendly, efficient high yield high-quality
Matter method.
Chitinase (Chitinase, EC3.2.1.14): being a kind of glycoside hydrolase, bacterium, fungi, plant and low
Animal can generate.In general it is an enzyme system, includes a variety of isodynamic enzymes.Difference in the way of cutting glycosidic bond can divide
For circumscribed chitinase, endochitinase and N- acetyl glucosaminidase enzyme.Circumscribed chitinase is non-reduced from chitin sugar chain
Chitin degrading is (GlcNAc) by end2 , endochitinase and circumscribed chitinase are different, can chance mechanism in
Any one glycosidic bond of chitin long-chain, and it is hydrolyzed into chitin oligo saccharide；And N-acetylglucosamine glycosides enzyme cannot be made
For chitin, can only hydrolyze chitin oligo saccharide is monosaccharide GlcNAc.Chitinase it is this by chitin degrading be chitin oligo saccharide or
Monosaccharide, reaction condition is mild, environment-friendly and green, and contamination-free generates, and raw material and the lower characteristic of cost input, can meet industry
Change the needs of low cost and high yield.Therefore, have the chitinase of the catalytic capability of high value for degradation by excavating
Chitin resource is of great significance.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of chitinasesCmchi6Gene and its clone
Expression and application.The gene source is in strainChitinolyticbacter meiyuanensis SYBC-H1(ATCC BAA-
2140T), recombinant chitinaseCmChi6 enzyme activity is higher, up to 1.00 ± 0.05U.45 DEG C of the optimum temperature of the enzyme, in 25-50
DEG C when good thermal stability, 33 h still keep 60% or more enzyme activity；Optimal pH 7.4, in pH7.0-10.0,33h enzyme activity is still kept
75% or more, showCmChi6 high stability；Ba2+It is rightCmChi6 vigor has faint activation；CmChi6 hydrolyzes glue
Body chitin product is mainly (GlcNAc)2, with a small amount of GlcNAc；Hydrolysis oligosaccharides mode is typical excision enzyme.
To solve prior art problem, the technical scheme adopted by the invention is as follows:
A kind of chitinaseCmChi6 gene, amino acid sequence is as shown in SEQ ID No.4.
A kind of chitinaseCmChi6 gene, nucleotide sequence is as shown in SEQ ID No.1.
A kind of recombinant expression carrier contains chitinase described in the codingCmThe nucleotide sequence of Chi6 gene
Recombinant expression carrier.
A kind of genetically engineered host cell, the genetically engineered transformation of host cells are as described above
Recombinant expression carrier.
Above-mentioned chitinaseCmThe clonal expression of Chi6 gene, comprising the following steps:
Step 1, nucleotide sequence shown in PCR amplification SEQ ID No.1；
Step 2, plasmid vector such as pColdI- is constructed Cmchi6
The DNA sequence dna of PCR amplification and pColdI carrier are subjected to double enzymes with same restriction enzyme NdeI and HindIII
It cuts, uses T4The digestion products for the purifying that DNA ligase ligase is cut back to close obtain plasmid vector pCooldI- Cmchi6；
Step 3, clone strain pColdI- is constructedCmchi6- E.coliTrans1T1
By plasmid vector pColdI-Cmchi6Conversion is extremelyE.coliTrans 1T1, obtains positive transformant, sieves through bacterium colony PCR
It selects and is sequenced and be determined as aimed strain；
Step 4, building expression bacterial strain pColdI- Cmchi6-BL21(DE3)
By clone strain pColdI- Cmchi6- E.coliThe plasmid that Trans1T1 is extracted convert toE.coli BL21
(DE3), positive transformant Direct Cloning culture is selected, recombinant strains pColdI- is obtainedCmchi6-BL21(DE3)；
Step 5, external evoked expression
By recombinant strains pCooldI-Cmchi6- BL21 (DE3) is accessed in the LB culture medium of 5ml benzyl containing ammonia mycin, in
After 25-40 DEG C is incubated overnight 10-20h, it is inoculated by 1% inoculum concentration in the LB culture medium of the 100ml mycin of benzyl containing ammonia, works as OD600=
The IPTG of final concentration of 0.5mM-1 mM is added in 0.4-0.6, in 16-25 DEG C, 150-250rpm low temperature induction 20-24h；
Step 6, chitinase obtains
In 4 DEG C after external evoked expression, 6000-12000rpm is centrifuged 7-10min and collects thallus, is washed with PBS buffer solution
Three times, then with PBS buffer solution thallus is resuspended in thallus, be placed on ultrasonication on ice, each 10-15min, 2 seconds intervals 3 of ultrasound
Second, then in 4 DEG C, 6000-12000rpm is centrifuged 7-10min and collects supernatant to get crude enzyme liquidCmChi6。
It is as improved, the purifying of crude enzyme liquid
The imidazoles of the pure water, 50mM and the 500mM that are crossed using ultrasound successively clean protein purification instrument pipeline and nickel column, remove therein
Impurity.Supernatant filtering will be taken to have removed the crude enzyme liquid upper prop of impurity and smudge cells by centrifugation 5-10min.Then 50mM is used
Imidazoles get through pipeline, target protein has His label, therefore can be adsorbed in nickel column, foreign protein without His label and
Other impurity cannot be adsorbed in nickel column to be rinsed, and after target protein is all adsorbed in nickel column, use 500mM's instead
Imidazoles elutes target protein, pays attention to collecting target protein at this time, will finally collect obtained target protein sampling and carries out 8% SDS
Polyacrylamide gel electrophoresis, the result is shown in Figure 1 are purifiedCmChi6 enzyme solution.
It is that primer used in PCR amplification is upstream primer Chi-F:5 '-in step 1 as improved
CATATGATGGCCTTGTTGCCGTTGG -3 ', downstream primer: Chi-R:5 '-CCCAAGCTTCTACTTTGCCGCCGGAAT-
3’。
Above-mentioned chitinaseCmApplication of the Chi6 in enzymatic hydrolysis substrate containing tobacco brown spot pathogen.
It is that hydrolysis temperature is 45 DEG C in the application, and pH value 7.4, at 25-50 DEG C, thermal stability is good as improved
Good, 33 h still keep 60% or more enzyme activity；In pH7.0-10.0,33h enzyme activity still keeps 75% or more, shows CmChi6 stability
It is higher.
It is that the substrate containing tobacco brown spot pathogen can be changed to chitobiose, chitin trisaccharide, chitin tetrose, several as improved
Six sugar of fourth pentasaccharides and chitin.
The utility model has the advantages that
Compared with prior art, present invention has an advantage that
1, present invention obtains chitinasesCmThe nucleotide sequence of Chi6 gene, the sequence for the first time fromChitinolyticbacter meiyuanensis SYBC-H1(ATCC BAA-2140T) in clone, nucleic acid sequence and come
FromUnclutured bacteriumThe chitinase gene sequence similarity of gene ST35 is only 2.48%, amino acid sequence
Column withChitiniphilus shinanonensisIn the likelihood of glycoside hydrolase GH18 chitinase be also only
76.69%, therefore its primary structure is novel.And it is low in cost, molecular level is easy to operate, be easy to clonal expression, repeatability compared with
It is good, there is potential using value in the industry；
2, the present invention constructs chitinaseCmChi6 prokaryotic expression carrier successfully obtains the zymoprotein that molecular weight is about 40kD,
Protein expression effect is good, and enzyme activity is high, and preparation process is simple, isCmChi6 functional study provides short-cut method；
3, chitinase good thermal stability at 25-50 DEG C in the present invention, 33 h still keep 60% or more enzyme activity, digest most suitable
Suitable reaction temperature is 45 DEG C;Reach highest in the enzyme activity of pH=7.4, in pH7.0-10.0,33h enzyme activity still keeps 75% or more, table
It is brightCmChi6 high stability.Therefore the reaction condition of the enzyme is mild, and adaptation pH range is wide, has industrialization potential value；
4, chitinase, and can be to chitin trisaccharide to the sugar of chitin six to tobacco brown spot pathogen degrading activity with higher in the present invention
It all degrades, but non-degradable chitobiose, and chitin odd number oligosaccharides is degraded to disaccharides and a small amount of monosaccharide, chitin even number oligosaccharide
Degradable is disaccharides, therefore is a kind of typical excision enzyme, is obtained in chitin oligo saccharide in industrialization degradation chitin with good
Good production application prospect.
Detailed description of the invention
Fig. 1 is chitinase of the present inventionCmThe SDS-PAGE testing result of Chi6, wherein M: protein standard molecular weight, 1-
ForCmThe supernatant that chi6 albumen is centrifuged, 2- areCmPrecipitating that Chi6 albumen is centrifuged, 3- is after purificationCmChi6 egg
It is white；
Fig. 2 is temperature in the present invention to chitinaseCmThe active influence of Chi6；Wherein (a) is optimum temperature, (b) steady for temperature
It is qualitative；
Fig. 3 is pH in the present invention to chitinaseCmThe active influence of Chi6；In figure, (a) is optimal pH；It (b) is pH stability；
Fig. 4 is metal ion pairCmThe active influence of Chi6；
Fig. 5 is chitinase of the present inventionCmChi6 degradation tobacco brown spot pathogen product interpretation of result；
Fig. 6 is chitinase of the present inventionCmChi6 degradation chitin oligo saccharide product situation, wherein (a) chitobiose, (b) chitin three
Sugar, (c) chitin tetrose, (d) chitin pentasaccharides, (e) six sugar of chitin.
Specific embodiment
The present invention is described further below by embodiment, but is not used in and limits the scope of the invention.Implement
Experimental method in example is unless otherwise specified conventional method.
Used material, reagent etc., unless otherwise specified, commercially obtain in following examples.
Quantitative test in following examples, is respectively provided with three repeated experiments, and results are averaged.
Tobacco brown spot pathogen as used in the following examples is prepared as follows: chitin powder being taken to be added
It is stirred well to paste in concentrated hydrochloric acid, after standing 1-2h, solution liquid is added slowly to 20% ethanol solution of 2l volumn concentration
In, side edged is vigorously stirred, and is stood overnight, and the tobacco brown spot pathogen of precipitation is obtained, and it is fixed with buffer solution to be adjusted to after corresponding pH value again
Appearance.
1 chitinase of embodimentCmThe amplification of Chi6 gene
The present invention passes through polymerase chain reaction according to the gene order design primer of reported SYBC-H1 chitinase
(PCR) method obtains SYBC-H1 chitinaseCmThe conserved sequence of Chi6.
It is gifted using round pcr with the Kui of Southern Yangtze University HaoChitinolyticbacter meiyuanensis
The genomic DNA of SYBC-H1 is respectively as follows: upstream primer using Primer5.0 software Design primers for templateChi- F(SEQ
ID No.2): 5 '-CCCAAGCTTCTACTTTGCCGCCGGAAT-3 ' design has NdeI restriction enzyme site；Downstream primerChi-R
(SEQ ID No.3): 5 '-CCCAAGCTTCTACTTTGCCGCCGGAAT-3 ' design has HindIII restriction enzyme site.
PCR reaction system:
PCR reaction condition: 95 DEG C of initial denaturations, 2min；95 DEG C of denaturation 20s, 52 DEG C of annealing, 20s, 72 DEG C of extensions, 2min, altogether
30 circulations；Extend after 72 DEG C, 5min, last 4 DEG C of heat preservations.
PCR reaction terminates have a bright band, as chitinase at 2019bp with the detection of 1% agarose gel electrophoresisCmChi6 gene.Its nucleotide sequence is as shown in SEQ ID No.1.Chitinase gene of the present inventionCmchi6Coding
403 amino acid, molecular weight about 40kD have the amino acid sequence as described in SEQ ID No.4.
2 plasmid vector pColdI- of embodimentCmchi6And clone strain pColdI-Cmchi6- E.coliTrans1T1
Preparation
1, purification and recovery is carried out to the PCR product that embodiment 1 obtains, uses TaKaRa company kit (TaKaRa DNA
Ligation Kit<Mighty Mix>)；
2、Construct plasmid vector pColdI- Cmchi6
The DNA sequence dna of PCR amplification and pColdI carrier are subjected to double enzymes with same restriction enzyme NdeI and HindIII
It cuts, the recovered purifying of digestion products, and uses T4DNA ligase connection, obtains plasmid vector pCooldI- Cmchi6；
3, clone strain pColdI- is constructedCmchi6- E.coliTrans1T1
By plasmid vector pColdI-Cmchi6Conversion is extremelyE.coliTrans 1T1:(1) take 20 μ l competence of freeze thawing on ice
In the connection reaction solution of 10 μ l of cell Trans 1T1, Yu Shangshu, it is gently mixed uniformly；(2) after standing 30min on ice, 42 DEG C
Then Heat thermostability 45s places rapidly 2min on ice；(3) add 800 μ l of LB culture medium, 37 DEG C of shaking table cultures in superclean bench
1h；(4) 4000g centrifugation medium is coated on the LB plate containing ammonia benzyl mycin, and 37 DEG C are incubated overnight, and screens positive transformants
Son, and guarantee is sequenced and obtains aimed strain, then go to fluid nutrient medium culture；(5) it is extracted with TaKaRa company kit big
Measure recombinant plasmid pColdI-Cmchi6。
Coupled reaction system:
Connect the condition of reaction are as follows: 16 DEG C of connections overnight, construction recombination plasmid pColdI-Cmchi6。
Embodiment 3
Construct recombinant strains pColdI-Cmchi6- E.coli BL21(DE3)
1, strain culturing: the recombinant plasmid pColdI- that will have been constructed Cmchi6It is extracted from cloning host, and will recombination
Plasmid is transformed into againE.coli.BL21(DE3) in competent cell.Step of converting in conversion operation such as above-described embodiment 2.Most
1-2 single colonie of whole picking is inoculated into the 5mlLB culture medium containing ammonia benzyl mycin final concentration of 0.2%, and 37 DEG C are incubated overnight.
2, it main culture: is transferred in 100ml LB/Amp culture medium after culture 12h by 1% inoculum concentration, 37 DEG C of shake cultures
To OD600=0.4-0.6。
3, inducing expression: the IPTG induction somatic cells of final concentration of 0.05mM are added, 200rpm low temperature lures at 18 DEG C
Lead 20h.
4, collect full cell: in 4 DEG C, 6000rpm, centrifugation 8-10min collects thallus, and with Yu Chao after 10ml PBS mixing
Sonication handles 10min, until liquid presentation is transparent.
5, soluble protein enzymeCmThe extraction of Chi6: above-mentioned bacterium solution is taken into supernatant i.e. in 12000rpm, 4 DEG C of centrifugation 5min
For soluble protein, i.e., enzyme of the present inventionCmChi6.Precipitating is generally smudge cells and a small amount of background expresses albumen.
6, the destination protein of SDS-PAGE electrophoresis detection expression, as shown in attached drawing 1.
Embodiment 4
ChitinaseCmThe enzyme activity determination of Chi6
1, DNS preparation of reagents
Sodium potassium tartrate tetrahydrate 182.0g is weighed, 0.5l pure water is added, is dissolved by heating at 50 DEG C, 3.5- dinitrosalicylic is then added
Acid (DNS) 6.3g, sodium hydroxide 21.0g, phenol 5.0g, stirring is to being completely dissolved, and after cooling plus pure water is settled to 1.0l, is protected from light
It saves, it is spare after placement 7d.
2, the foundation of enzyme activity determination standard curve
Using DNS(3,5 dinitrosalicylic acids) method measurementCmChi6 enzyme activity.Take respectively glucose standard (1mg/ml) 0,0.2,
0.4,0.6,0.8,1.0ml complements to 1.0ml with pure water, accurate respectively that DNS reagent 1ml is added, boiling water bath adds in 4ml pipe
Hot 5min, flowing water is cooling, measures absorbance under 540nm wavelength.Establish glucose standard curve.
3, chitinaseCmThe determination of activity of Chi6
Crude enzyme liquid 0.1ml is taken, 0.5ml tobacco brown spot pathogen is mixed with 0.4mlPBS buffer, at 37 DEG C after insulation reaction 30min,
DNS is added and heats 5min in boiling water bath and terminates and reacts, 12000rpm is centrifuged 5min, takes supernatant.Using the enzyme solution of inactivation as pair
According to group, absorbance is measured at 540nm.Content of reducing sugar is calculated according to standard curve, to calculate enzyme activity.
Enzyme activity unit defines (U): under the above conditions, discharging 1 μm of ol reduced sugar per minute (with N- acetyl-D-
Glucosamine meter) needed for enzyme amount.Finally obtaining its enzyme activity is 1.0 ± 0.05U, and enzyme activity is significantly higher.
Embodiment 5
CmChi6 zymology Quality Research
The chitinase that embodiment 3 is obtainedCmChi6 zymologic property is measured, including optimum temperature, temperature stability, most
The suitable influence of pH, pH stability, metal ion to enzyme activity, is measured by substrate of tobacco brown spot pathogen.
(1) enzyme activity determination
It is specifically shown in embodiment 4.
(2) temperature is to chitinaseCmThe influence of Chi6
1. optimal reactive temperature
Under conditions of pH value is 7.0 (Tris-HCl buffer), using 1% tobacco brown spot pathogen as substrate, reaction system includes
500 μ l tobacco brown spot pathogens, 400 μ l buffers and 100 μ lCmChi6 enzyme solution, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C,
50 DEG C, the interior reaction 30min of 55 DEG C of temperature gradients, reaction terminate to add 1mlDNS reagent into each reaction system immediately, boil
5min measures light absorption value under the ultraviolet-visible spectrophotometer of 540nm, determines under different temperaturesCmChi6 enzyme activity.According to
The relative activity of enzyme at different temperatures draws curve, determinesCmThe optimal reactive temperature of Chi6 enzyme.As a result as shown in Figure 2,CmThe optimum temperature of Chi6 is 45 DEG C.
2. chitinaseCmThe thermal stability of Chi6
Enzyme solution is respectively placed under different temperatures (20 DEG C -55 DEG C, be spaced 5 DEG C) and keeps the temperature 40h, then under optimal reactive temperature
Remnant enzyme activity is detected using DNS method, and is compared with untreated enzyme activity, relative activity is calculated.
As a result as shown in Fig. 2 (b), chitinaseCmChi6 good thermal stability at 25-50 DEG C, 33h still keep 60% with
Upper enzyme activity.
(3) pH is to chitinaseCmThe influence of Chi6
1. optimal pH
Enzyme activity reaction temperature is 45 DEG C, using 1% tobacco brown spot pathogen as substrate, in the 50mmol/L buffer (pH that pH value is 3-10
3.0~6.0 sodium citrate buffer solutions, 6.0~8.0 phosphate buffer of pH, pH 8.0-10.0 Glycine-NaOH are molten
Liquid) in measure enzyme activity, curve is drawn according to relative activity of the enzyme under different p values, determines the optimal reaction pH value of enzyme.Knot
Fruit as shown in Fig. 3 (a),CmThe optimal pH of Chi6 is 7.4.
②CmThe pH stability of Chi6
By enzyme solution be respectively placed in 50mmol/L difference pH buffer (3.0~6.0 sodium citrate buffer solution of pH, pH 6.0~
8.0 phosphate buffers, pH 8.0-10.0 Glycine-NaOH solution) in, it is placed at 45 DEG C for 24 hours, then most suitable
Remnant enzyme activity is detected under reaction temperature, and is compared with untreated enzyme activity, and relative activity is calculated.As a result such as Fig. 3 (b) institute
Show,CmChi6 is at 45 DEG C, and after different pH preincubates, in pH7.0-10.0, the enzyme activity for being incubated for the chitinase of 33h is still kept
75% or more, showCmChi6 high stability.
(4) metal ion is to chitinaseCmThe influence of Chi6
Be added into reaction system KCl, NaCl of final concentration of 10mmol/l, CaCl2, MgCl2, MnCl2, CuCl2,
Then ZnCl2, CoCl2 and FeCl3 measure enzyme activity at the standard conditions, and carry out with the enzyme activity for not adding any metal ion
Compare, calculates enzyme relative activity.
As a result see Fig. 3, it is known that Ba is added in reaction solution2+To chitinaseCmChi6 vigor has activation slightly.
6 chitinase of embodimentCmChi6 hydrocolloid chitin product analysis
(1) it samples: 0.1ml being added into reaction system CmChi6 enzyme solution, 0.4ml buffer, 0.5ml tobacco brown spot pathogen, most
Suitable temperature 45 C, under the conditions of pH7.4,200rpm reacts 8h.1h, 2h, 4h are reacted, 6h, 8h respectively sample 200 μ l, high temperature enzyme deactivation
It is living, -20 DEG C of freezings.
(2) product detection is analyzed: high performance liquid chromatograph is utilized, to filter with the ultrasonic acetonitrile crossed and water as mobile phase,
After flushing pipeline and pillar, with 75% acetonitrile and 25% water, the analysis of 1ml/min sample detection.After having sampled, column is protected with 90% methanol.
The result is shown in Fig. 5, it is known that reaction system has mainly generated (GlcNAc) after 6h2With a small amount of GlcNAc.It can push away
5 prime excision enzyme activity may be had by surveying the enzyme.
7 chitinase of embodimentCmChi6 hydrolyzes chitin oligo saccharide pattern analysis
(1) it samples: taking (GlcNAc)2-(GlcNAc)6Each 10 μ l of chitin oligo saccharide, it is each that 1 μ l is added CmChi6 enzyme solution, in most thermophilic
45 DEG C of degree, 200rpm reaction.Every 15min, 30min, 1h, 2h, 11h, 12h, 2 μ l are sampled, high temperature enzyme deactivation is living, -20 DEG C of freezings
Preservation.
(2) sample dilutes: by obtained sample after being centrifuged, diluting 10 times with ultrapure water, is put into liquid phase bottle interpolation pipe
It is interior, it is spare.
Hydrolysising product analysis: utilizing high performance liquid chromatograph, to filter the acetonitrile crossed with ultrasound and water as mobile phase, rinses
After pipeline and pillar, with 75% acetonitrile and 25% water, 1ml/min analyzes the sample detection diluted.After having sampled, with 90% first
Alcohol protects column.
The result is shown in Fig. 6, from 6(a) -6(e) figure, it can be seen thatCmThe non-degradable chitobiose of Chi6, but can by chitin trisaccharide,
Pentasaccharides is degraded to most disaccharides and a small amount of monosaccharide, and chitin tetrose and six sugar are all degraded to disaccharides, characteristic and chitin
It is consistent that matter excision enzyme from the non-reducing end digestion of chitin obtains disaccharides, therefore is typical excision enzyme.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Sequence table
<110>Nanjing University of Technology
<120>a kind of chitinase CmChi6 gene and its clonal expression and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1209
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgcgtctgc tgttatccct attgcttgct gccttgttgc cgttggccgt tgctgccgac 60
gagccggctc cggccaagcc cacgcctgcg gcatccaagc cgtatcgcgt ggtggcgtat 120
tacatttcgt gggccgcata cgctcgccgc tatttccctt cggatatcga cgcccgcaag 180
gtcacacaca tcaattttgc tttcgccgat atcaaggatg gagaggtcgt ggttggcgac 240
cccggtgtgg acacccgtgg ccccaacaac ctgaaggcgt tgagtgagtt gaaggcgaag 300
cacccccacc ttcgcaccat gatttcagtg ggtggatgga gttggtcgac ccatttctcc 360
gatgcagcac ttaccccggc ctcgcgcaag aaatttgccg acagcgccgt taccttcatt 420
cgccgcttca acatggatgg cgtcgatatc gactgggaat tcccggttga aggtgggccg 480
gataccatga agcatcggcc tgaggataag cagaactaca cgctgctcgc gaaggctttg 540
cgcgatgcgc tggatgcagc tggcaaggcc gatggcaaat actacgagct gtcgacggcg 600
gtctggggca acgacaagtt catccgtaat accgagatgg acaaggtagg gaagatcttc 660
gatttcatca acttgatgac ctacgactac aacggcacct ggaacagttt ttccggccac 720
cttgcgccgt tcaaacacga tcctgcgtat gacaagcccg gtatctcgcc gacgttcaat 780
gtggtcgata ccgtcgatgc gtacctgaag gcgggcgttc cggcagatcg actggtcgtc 840
ggcataccgc tctacggcta cagctggaaa ggctgcgcac cgcagcgcaa cggcgaatat 900
caggaatgca agggcaaggg tcgtggttcc tgggaggatg gcaatctcga cttcaccgag 960
atcgagaaga gcctgatcga caagaagggc ttcaagcgcc actggaacga ggtggccaag 1020
gtcgcttatc tctacaacga gaagaccggc gaattcatca gctatgaaga cccgcaagcg 1080
ctggcgctca aactggcgtt gatcaaggcc aagggactgg gcggcgccat gtactgggag 1140
atcacggcag atcgcaagca gacgctggtc aagcagatat ccgatgcgct gattccggcg 1200
gcaaagtag 1209
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
catatgatgg ccttgttgcc gttgg 25
<210> 3
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cccaagcttc tactttgccg ccggaat 27
<210> 4
<211> 402
<212> PRT
<213>amino acid (Abies alba)
<400> 4
Met Arg Leu Leu Leu Ser Leu Leu Leu Ala Ala Leu Leu Pro Leu Ala
1 5 10 15
Val Ala Ala Asp Glu Pro Ala Pro Ala Lys Pro Thr Pro Ala Ala Ser
20 25 30
Lys Pro Tyr Arg Val Val Ala Tyr Tyr Ile Ser Trp Ala Ala Tyr Ala
35 40 45
Arg Arg Tyr Phe Pro Ser Asp Ile Asp Ala Arg Lys Val Thr His Ile
50 55 60
Asn Phe Ala Phe Ala Asp Ile Lys Asp Gly Glu Val Val Val Gly Asp
65 70 75 80
Pro Gly Val Asp Thr Arg Gly Pro Asn Asn Leu Lys Ala Leu Ser Glu
85 90 95
Leu Lys Ala Lys His Pro His Leu Arg Thr Met Ile Ser Val Gly Gly
100 105 110
Trp Ser Trp Ser Thr His Phe Ser Asp Ala Ala Leu Thr Pro Ala Ser
115 120 125
Arg Lys Lys Phe Ala Asp Ser Ala Val Thr Phe Ile Arg Arg Phe Asn
130 135 140
Met Asp Gly Val Asp Ile Asp Trp Glu Phe Pro Val Glu Gly Gly Pro
145 150 155 160
Asp Thr Met Lys His Arg Pro Glu Asp Lys Gln Asn Tyr Thr Leu Leu
165 170 175
Ala Lys Ala Leu Arg Asp Ala Leu Asp Ala Ala Gly Lys Ala Asp Gly
180 185 190
Lys Tyr Tyr Glu Leu Ser Thr Ala Val Trp Gly Asn Asp Lys Phe Ile
195 200 205
Arg Asn Thr Glu Met Asp Lys Val Gly Lys Ile Phe Asp Phe Ile Asn
210 215 220
Leu Met Thr Tyr Asp Tyr Asn Gly Thr Trp Asn Ser Phe Ser Gly His
225 230 235 240
Leu Ala Pro Phe Lys His Asp Pro Ala Tyr Asp Lys Pro Gly Ile Ser
245 250 255
Pro Thr Phe Asn Val Val Asp Thr Val Asp Ala Tyr Leu Lys Ala Gly
260 265 270
Val Pro Ala Asp Arg Leu Val Val Gly Ile Pro Leu Tyr Gly Tyr Ser
275 280 285
Trp Lys Gly Cys Ala Pro Gln Arg Asn Gly Glu Tyr Gln Glu Cys Lys
290 295 300
Gly Lys Gly Arg Gly Ser Trp Glu Asp Gly Asn Leu Asp Phe Thr Glu
305 310 315 320
Ile Glu Lys Ser Leu Ile Asp Lys Lys Gly Phe Lys Arg His Trp Asn
325 330 335
Glu Val Ala Lys Val Ala Tyr Leu Tyr Asn Glu Lys Thr Gly Glu Phe
340 345 350
Ile Ser Tyr Glu Asp Pro Gln Ala Leu Ala Leu Lys Leu Ala Leu Ile
355 360 365
Lys Ala Lys Gly Leu Gly Gly Ala Met Tyr Trp Glu Ile Thr Ala Asp
370 375 380
Arg Lys Gln Thr Leu Val Lys Gln Ile Ser Asp Ala Leu Ile Pro Ala
385 390 395 400
Ala Lys
Claims (10)
1. a kind of chitinaseCmChi6 gene, which is characterized in that amino acid sequence is as shown in SEQ ID No.4.
2. chitinase according to claim 1CmChi6 gene, which is characterized in that nucleotide sequence such as SEQ ID
Shown in No.1.
3. a kind of recombinant expression carrier contains the chitinase encodingCmThe recombinant expression of the nucleotide sequence of Chi6 gene
Carrier.
4. a kind of genetically engineered host cell, genetically engineered transformation of host cells weight as described above
Group expression vector.
5. the chitinase stated based on claim 1CmThe clonal expression of Chi6 gene, which comprises the following steps:
Step 1, nucleotide sequence shown in PCR amplification SEQ ID No.1；
Step 2, e. coli plasmid vector pColdI- is constructed Cmchi6
The DNA sequence dna of PCR amplification and pColdI carrier are subjected to double enzymes with same restriction enzyme NdeI and HindIII
It cuts, T is used in the recovered purifying of digestion products4DNA ligase connection, obtains plasmid vector pCooldI- Cmchi6；
Step 3, escherichia coli cloning bacterial strain pColdI- is constructedCmchi6- E.coliTrans1T1
By plasmid vector pColdI-Cmchi6Conversion is extremelyE.coliTrans 1T1, obtains positive transformant, is screened and is surveyed
Sequence is determined as aimed strain；
Step 4, E. coli expression strains pColdI- is constructedCmchi6-BL21(DE3)
By clone strain pColdI- Cmchi6- E.coliThe plasmid that Trans1T1 is extracted convert toE.coliBL21
(DE3), positive transformant clone is selected, recombinant strains pColdI- is obtainedCmchi6-BL21(DE3)；
Step 5, external evoked expression
By recombinant strains pCooldI-Cmchi6- BL21 (DE3) access 5ml benzyl containing ammonia mycin shakes in pipe LB culture medium,
After 25-40 DEG C is incubated overnight, it is inoculated by 1% inoculum concentration in the LB culture medium of the 100ml mycin of benzyl containing ammonia, works as OD600=0.4-
0.6, the IPTG that final concentration of 0.5mM-1 mM is added is induced, in 16-25 DEG C, 150-250rpm Low- temperature culture 20-24h；
Step 6, chitinase obtains
In 4 DEG C after external evoked expression, 6000-12000rpm is centrifuged 7-10min and collects thallus, is washed with PBS buffer solution
Three times, then with PBS buffer solution thallus is resuspended in thallus, be placed on ultrasonication on ice, each 10-15min, 2 seconds intervals 3 of ultrasound
Second, then in 4 DEG C, 6000-12000rpm is centrifuged 7-10min and collects supernatant to get recombinant chitinaseCmChi6 crude enzyme liquid.
6. chitinase according to claim 5CmThe clonal expression of Chi6 gene, which is characterized in that PCR expands in step 1
Increasing primer used is upstream primerChi- F:5 '-CATATGATGGCCTTGTTGCCGTTGG-3 ', downstream primer:Chi- R:
5’-CCCAAGCTTCTACTTTGCCGCCGGAAT-3’。
7. being based on claim 5 gained chitinaseCmApplication of the Chi6 in enzymatic hydrolysis substrate containing tobacco brown spot pathogen.
8. application according to claim 7, which is characterized in that hydrolysis temperature is 45 DEG C in the application, pH value 7.4,
The good thermal stability at 25-50 DEG C, 33 h still keep 60% or more enzyme activity；Optimal pH 7.4, in pH7.0-10.0,33h
Enzyme activity still keeps 75% or more.
9. application according to claim 7, which is characterized in that Ba in the application2+It is rightCmChi6 vigor has faint
Activation.
10. application according to claim 7, which is characterized in that the substrate containing tobacco brown spot pathogen is chitin trisaccharide, chitin
Six sugar of tetrose, chitin pentasaccharides or chitin.
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| 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 |
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