CN104178473A - Preparation of novel hybrid chitinase - Google Patents
Preparation of novel hybrid chitinase Download PDFInfo
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- CN104178473A CN104178473A CN201310662769.5A CN201310662769A CN104178473A CN 104178473 A CN104178473 A CN 104178473A CN 201310662769 A CN201310662769 A CN 201310662769A CN 104178473 A CN104178473 A CN 104178473A
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- chitinase
- binding domain
- chi
- chitin binding
- chitin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2442—Chitinase (3.2.1.14)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01014—Chitinase (3.2.1.14)
Abstract
The invention relates to the technical field of gene recombination, and concretely relates to a method for preparing hybrid chitinase with increased yield and enhanced activity. The method comprises the following steps: designing a primer according to the full length sequence of chitinase gene, directionally cloning the primer into a pET-30a vector to obtain pETChiA, inserting a chitin binding domain or catalyzing domain at the carboxyl terminal of chitinase, and recombining to form an expression vector; transforming the expression vector into Escherichia coli, inducing expression, carrying out ultrasonic crushing on thalli obtained through re-suspending by using a Tris buffer solution, centrifuging, and collecting the obtained supernatant. A recombinant chitinase-containing solution obtained by collecting through an HIS nickel column has the advantages of high product purity, high yield, high activity, low cost, simple production process and stable product quality.
Description
One, technical field
The present invention relates to gene recombination technology field, be specially a kind of method that obtains the heterozygosis chitinase of output raising, increased activity.
Two, background technology
Due to traditional chemical agricultural chemicals, have the problems such as target organisms resistance, non-target organism toxicity, the mankind are in the urgent need to developing green agricultural chemicals.Chitin (chitin) is the linear polysaccharide that N-Acetyl-D-glucosamine is formed by connecting with β-Isosorbide-5-Nitrae-glycosidic link.Because chitin is structural constituent (exoskeleton and peritrophic membrane) important in insect body and not being present in high animal and plant body, so chitin is considered to unique target of environment friendly agricultural research and development.Wherein chitinase (chitinase, EC3.2.1.14) plays an important role in chitin degrading, has become a focus of domestic and international research at present.But the outstanding problem existing is at present exactly that chitinase activity is not high, the enzymic activity that therefore how to improve them just becomes problem in the urgent need to address.
Chitinase generally comprises a plurality of functional zone such as signal peptide (signal peptide), catalytic domain (catalytic domain), chitin binding domain, Saliva Orthana III type homologous region (Fibronectin typeIII like domain) and hinge area.Wherein, the similarity of catalytic domain is higher, and the differing greatly of other structural domains.In recent years, by methods such as disappearance structural domains, the function of each structural domain has been carried out to some researchs, found that chitin binding domain is huge to chitinase activity influence.
Chitin binding domain can help chitinase to be attached on chitin substrate, improves the enzyme concn of regional area, the physical structure between simultaneously can also partial destruction polysaccharide molecule, thus promote the degraded to substrate.According to the difference of chitin binding domain three-dimensional structure, can be divided into three classes: I, II and III type.The chitin binding domain of fungi and plant belongs to I type; The chitin binding domain of virus and insect belongs to II type; The chitin binding domain of bacterium belongs to III type.Whether the combination territory of this three types there are differences in function, not clear at present, but a large amount of research shows the chitinase that lacks chitin binding domain and the hydrolysis ability of soluble substrate is not changed or change little, but soluble chitinous hydrolysis ability is but reduced, and fungistatic effect, insecticidal effect have also reduced.On the other hand, on the chitinase without chitin binding domain, add chitin (or polysaccharide) and can increase chitinase in the concentration of substrate surface in conjunction with territory, thereby improve the chitinous ability of decomposing.As breathed out, hereby in wooden mould chitinase gene Chit42, there is no chitin calmodulin binding domain CaM, at the C of this gene expression product end, add the speed that chitin calmodulin binding domain CaM can significantly improve degrade chitin, and the insecticidal effect of Pyrausta nubilalis (Hubern). is improved to 30%.The chitin binding domain of carboxyl terminal interpolation silkworm chitinase, aphid chitinase and balsam pear chitinase that the gorgeous China of Southwestern University's model waits at beauveria bassiana chitinase Bbchitl has all increased heterozyme to the chitinous binding ability of crystal and hydrolytic activity.Compare with Bacillus licheniformis DSM13 chitinase (containing catalytic domain and chitin binding domain) the mutant BliGH of disappearance chitin binding domain, the heterozygosis chitinase BliGH-CeBD that replaces the chitin binding domain acquisition of DSM13 chitinase with cellulose binding domain has strengthened the stability of the adsorptive power of tobacco brown spot pathogen, catalytic capability and conformation.Interestingly chitin binding domain being added in proteolytic enzyme also to make proteolytic enzyme obtain in conjunction with chitinous ability.Should also be noted that at the chitinase C end interpolation external source or the chitin binding domain of self that contain chitin binding domain and still can improve chitinase activity.The ability of the chitinous efficiency of its association colloid and degrade chitin can be improved in the chitin land of adding the chitin land of paddy rice chitinase or again adding former chitinase as chitinase afterbody tobacco leaf moth.And find, along with the increase of chitin binding domain number, joint efficiency and degradation efficiency all can further improve, and show dosage effect.In the chitinase that dosage effect contains a plurality of chitin binding domains at some, be also found.As Aeromonas hydrophila JP101 chitinase 92 has the chitin binding domain of two repetitions, in the situation that they all exist, chitinase is respectively 12 times and 10 times of single chitin binding domain to the binding ability of powder chitin and tobacco brown spot pathogen.
Summary of the invention
Technical problem
The object of the invention is for current chitinase yield poorly, the problem such as activity is low, purity is low, a kind of method for creating of novel heterozygosis chitinase is provided.
Technical scheme
The present invention adopts following technical scheme to realize: (1) is according to chitinase gene Chi (chitinase, abbreviation Chi) full length sequence design primer, at primer 5 ' end, increase restriction enzyme site, chitinase gene sequence directed cloning is entered to pET30a carrier; Then the carboxyl terminal at chitinase inserts chitin binding domain.So, reassemble into expression vector pET-30a-Chi-ChBD, corresponding expression product is Chi-ChBD.(2) the expression vector pET-30a-Chi-ChBD building in step (1) is converted in e. coli bl21 (DE3), with the expression of IPTG induction recombination, centrifugal collection thalline.(3) with the resuspended thalline reaching of PBS damping fluid, ultrasonication, centrifugal collection supernatant liquor, obtains the solution that contains recombinant chitinase.(4), by HIS nickel ion affinity column on the solution that contains recombinant chitinase, with PBS damping fluid upper prop, 50mM imidazoles wash-out foreign protein, 250mM wash-out target protein, dialysed overnight, obtains the purifying product that contain recombinant chitinase.The building process of above-mentioned Chi expression vector is shown in shown in Figure of description 1,2.
In above-mentioned steps, the full length sequence of described chitinase Chi can obtain from various plants, microorganism, the present invention, through shaker test, utilizes round pcr from the clayey Serratia of high yield chitinase, to obtain highly active chitinase gene ChiC SEQ NO-1 as shown in sequence table; ChBD sequence is the chitin binding domain of clayey Serratia chitinase gene ChiC, and its sequence is SEQ NO-2 as shown in sequence table.
Utilize the heterozygosis chitinase specific activity wild type strain that gene recombination technology of the present invention obtains to improve several times, and the chitinase purity obtaining by HIS ni-sepharose purification is high, active high, cost is low, production technique is simple, and constant product quality plays an important role in the prevention and control of plant diseases, pest control.
Accompanying drawing explanation
Fig. 1 is the plasmid signal collection of illustrative plates of pET30a.
Fig. 2 is the plasmid construction schematic diagram of pET-30a-Chi-ChBD.
Fig. 3 is the electrophorogram of pcr amplification chitinase gene Chi.
Fig. 4 is NdeI and the HindIII double digestion electrophoretogram of pET-30a-Chi-ChBD.
Fig. 5 is purified product collection of illustrative plates.
Fig. 6 is for to utilize 2-Acetamido-2-deoxy-D-glucose (NAG) as concentration-absorbancy typical curve of color reaction standard substance.
Three, embodiment
Embodiment mono-: the construction and expression of Chi expression vector
(1) choose the clayey Serratia of the high yield chitinase of screening acquisition, utilize pcr clone to obtain Chi gene.This Chi full length gene sequence SEQ NO-1 as shown in sequence table.As follows according to Chi sequences Design Auele Specific Primer (comprising restriction enzyme site):
Upstream primer ChiF:GGG CAT ATG AGC ACA AAT AAC A
Downstream primer ChiR:GGG GCG GCC GCA GGC GAT GAG CTG CCA
PCR reaction system 50 μ l:
PCR reaction conditions is: 94 ℃ of 4min; 94 ℃ of 40s, 55 ℃ of 40s, 72 ℃ of 90s, 30 circulations; 72 ℃, 10min.
After reaction finishes, get PCR product and carry out 1% agarose gel electrophoresis test strip and be positioned at 1.4kb position, reclaim goal gene, with restriction enzyme NdeI, become sticky end with NcoI double digestion.Fig. 3 is the electrophoretogram of ChiC gene.
With restriction enzyme NdeI and NcoI double digestion pET30a, add PCR product and the T4DNA ligase enzyme of above-mentioned double digestion, 4 ℃ of connections are spent the night, and transform bacillus coli DH 5 alpha.With penbritin-LB plate screening, choose hickie clone.Extract plasmid DNA, utilize PCR and enzyme blanking method to identify and obtain positive colony.So just successfully obtain pET-30a-Chi carrier.
The primer of design amplification ChBD, comprises restriction enzyme site:
Upstream primer ChBDF:GGG GAG CTC AGC GGC CTGACC CCG GCC A
Downstream primer ChBDR:GGG AAG CTT GGC GAT GAG CTG CCA CAG GGT
PCR reaction system 50 μ l:
PCR reaction conditions is: 94 ℃ of 4min; 94 ℃ of 40s, 55 ℃ of 40s, 72 ℃ of 30s, 30 circulations; 72 ℃, 10min.
After reaction finishes, get PCR product and carry out 1% agarose gel electrophoresis test strip and be positioned at about 0.3kb position, reclaim goal gene, with restriction enzyme SacI, become sticky end with HindIII double digestion.Same, with restriction enzyme SacI and HindIII double digestion pET-30a-Chi carrier, after purifying, mix respectively, add T4DNA ligase enzyme, 4 ℃ of connections are spent the night, and transform bacillus coli DH 5 alpha.With penbritin-LB plate screening, choose hickie clone.Extract plasmid DNA, utilize PCR and enzyme blanking method to identify and obtain positive colony.So just successfully obtain pET-30a-Chi-ChBD efficient expression vector.Fig. 4 is NdeI and the HindIII double digestion electrophoretogram of pET-30a-Chi-ChBD.The building process of above-mentioned chitinase gene expression vector is counted as shown in accompanying drawing 1,2 as title, and the collection of illustrative plates of plasmid pET30a is from business-like test kit, and the sequence of amplification is inserted in multiple clone site wherein through design.
By correct pET-30a-Chi-ChBD plasmid transformation escherichia coli BL21DE3, with penbritin-LB plate screening, choose single spot clone, overnight incubation in the LB of 5mL liquid nutrient medium, according to 1:50, transfer, in shaking flask 37 ℃ to be cultured to OD600 be 0.4-0.6, adding IPTG to make its final concentration is 0.5mM, cultivates 4-8 hour for 25 ℃, centrifugal collection thalline, is stored in-20 ℃ or enter immediately next step purifying.
With PBS damping fluid, wash mixed bacterial sediment, with the resuspended about 500mL bacterium liquid precipitate of 20-50mL volume, (3s is ultrasonic under condition of ice bath, to carry out carrying out ultrasonic bacteria breaking, 5s intermittently, total length 40min), the centrifugal 25min of 12000rpm/min, collects supernatant liquor, and this supernatant liquor is the solution that contains Chi of high density.
Embodiment bis-: the evaluation of the purifying of Chi stoste and Chi sterling
In the solution that contains Chi of high density, according to nickel ion affinity column operation instruction absorb-elute Chi, by gained elutriant dialysed overnight, obtain highly purified Chi sterling.
Chi sterling authentication step is as follows: get Chi sterling 2 μ l, the sample preparation liquid that adds 4 μ l after diluting 10 times, boiling water bath 5min, get 5 μ l loadings and carry out SDS-PAGE electrophoresis, as shown in Figure 5, Far Left is protein molecular weight contrast, is respectively thereafter the front sample of purifying, and the Chi product of different time purification acquisition.Product can be for being further made into other finished products.
Determination of activity:
Adopt DNS Whitfield's ointment assay method to measure chitinase active.First prepare tobacco brown spot pathogen.Take 5g chitin, join gradually in the 200ml concentrated hydrochloric acid of ice precooling, stir at interval, after all dissolving, transfers in the 2L distilled water of ice precooling, after colloidal precipitation, supernatant liquor is carefully gone.Then add water, mix.So repeatedly until pH becomes 7.With the whole content of 20mmol/L PBS phosphoric acid buffer adjustment, be finally 1% (volume is 500mL).Then get 0.2mL liquid to be measured and mix with the phosphoric acid buffer that 0.8mL contains 1.0% tobacco brown spot pathogen, be positioned in 37 ℃ of thermostat water baths and be incubated 1h; Enzyme liquid after effect is positioned over to 10min in 100 ℃ of boiling water baths; Go to ice bath and add 3 of 1.0mL, 5-edlefsen's reagent, 100 ℃ of boiling water bath 10min after cooling; The centrifugal 10min of the cooling rear 6000rpm of room temperature; Get in the ultraviolet spectrophotometer of supernatant liquor under wavelength 540nm and survey absorbancy.Under these conditions, the required chitinase amount of generation 1 μ gNAG per hour is 1 enzyme activity unit.Utilizing 2-Acetamido-2-deoxy-D-glucose enzyme (NAG) is contrast as the standard substance of color reaction.Detected 2 samples after purifying, wherein the vigor of sample 1 is 2890U/mL, and the vigor of sample 2 is 4366U/mL.
Claims (3)
1. the initiative of novel heterozygosis chitinase, is characterized in that comprising the following steps:
(1) according to the full length sequence design primer of chitinase gene Chi, at primer 5 ' end, increase restriction enzyme site, chitinase gene sequence directed cloning is entered to pET30a carrier; Then the carboxyl terminal at chitinase inserts chitin binding domain.So, reassemble into expression vector pET-30a-Chi-ChBD, corresponding expression product is Chi-ChBD.
(2) the expression vector pET-30a-Chi-ChBD building in step (1) is converted in e. coli bl21 (DE3), with the expression of IPTG induction recombination, centrifugal collection thalline.
(3) with the resuspended thalline reaching of Tris damping fluid, ultrasonication, centrifugal collection supernatant liquor, obtains the solution that contains recombinant chitinase.
(4), by HIS nickel ion affinity column on the solution that contains recombinant chitinase, with Tris damping fluid upper prop, 50mM imidazoles wash-out foreign protein, 250mM wash-out target protein, dialysed overnight, obtains the purifying product that contain recombinant chitinase.
2. described in claim 1, chitin binding domain comprises the chitin binding domain of 1 or several dissimilar or same type.
3. described in claim 1, chitin binding domain includes but not limited to outside chitin binding domain, also comprises structural domain or the albumen such as catalytic domain, proteolytic enzyme.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110075809A (en) * | 2019-04-23 | 2019-08-02 | 南京工业大学 | A kind of powder chitin that improves is to the method and its application of the adsorption capacity of zymoprotein |
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| CN101613682A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active chitinase ChiC1 of Di Siwa mite and the application thereof of killing |
| CN101775405A (en) * | 2009-08-14 | 2010-07-14 | 杨霞 | Method for preparing recombinant chitinase |
| CN101921792A (en) * | 2010-05-13 | 2010-12-22 | 复旦大学 | Method for improving antibacterial activity of antifungal protein |
| CN102533819A (en) * | 2012-02-20 | 2012-07-04 | 西南大学 | Method for improving beauveria bassiana chitinase gene disease resistance and culturing disease resistance plants adopting method |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613682A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active chitinase ChiC1 of Di Siwa mite and the application thereof of killing |
| CN101775405A (en) * | 2009-08-14 | 2010-07-14 | 杨霞 | Method for preparing recombinant chitinase |
| CN101921792A (en) * | 2010-05-13 | 2010-12-22 | 复旦大学 | Method for improving antibacterial activity of antifungal protein |
| CN102533819A (en) * | 2012-02-20 | 2012-07-04 | 西南大学 | Method for improving beauveria bassiana chitinase gene disease resistance and culturing disease resistance plants adopting method |
Non-Patent Citations (1)
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| 登录号: ""GenBank Accession No：GU724603.1"", 《GENBANK》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110075809A (en) * | 2019-04-23 | 2019-08-02 | 南京工业大学 | A kind of powder chitin that improves is to the method and its application of the adsorption capacity of zymoprotein |
| CN110075809B (en) * | 2019-04-23 | 2021-10-22 | 南京工业大学 | Method for improving adsorption capacity of chitin powder to enzyme protein and application thereof |
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