CN108018245A - One plant of bacillus subtilis for producing chitosan enzyme and its application - Google Patents

One plant of bacillus subtilis for producing chitosan enzyme and its application Download PDF

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CN108018245A
CN108018245A CN201810030676.3A CN201810030676A CN108018245A CN 108018245 A CN108018245 A CN 108018245A CN 201810030676 A CN201810030676 A CN 201810030676A CN 108018245 A CN108018245 A CN 108018245A
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chitosan
enzyme
bacillus subtilis
chitosan enzyme
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王刚刚
苟艳
谢天
刘忠川
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Chengdu Institute of Biology of CAS
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    • C12Y302/01132—Chitosanase (3.2.1.132)

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Abstract

The invention belongs to biological technical field, and in particular to one plant of production chitosan enzyme bacillus subtilis and its application.Bacillus subtilis provided by the invention, its produced chitosan enzyme yield is high, enzyme activity is strong, has the protein structure different from common chitosan enzyme, the further investigation for chitosan enzyme molecular machinery provides new approaches.Common chitosan enzyme only has a preferable activity in alkalescent to faintly acid, and present invention discover that chitosan enzyme preferable activity is respectively provided with the conditions of alkalescent, neutrality, faintly acid and strong acidity.Meanwhile present invention discover that chitosan enzyme, not only may act on the chitosan that can be dissolved, may also act to insoluble high chitosan, eliminate the processing before degradation of chitosan；Reacting final product is two poly glucosamines and three poly glucosamines, and component is simple, easy to subsequently be processed further applying, dramatically saves on production cost.

Description

One plant of bacillus subtilis for producing chitosan enzyme and its application
Technical field
The invention belongs to biological technical field, and in particular to one plant of bacillus subtilis for producing chitosan enzyme and its application.
Background technology
Glucosamine is important medical precursor and industrial chemicals, and tool has been widely used.The shell in shrimp and crab shells source gathers Sugared (Chitosan) is to pass through β -1,4- glucosides key connection shapes by D- Glucosamines (or a small amount of 2-acetylamino-2-deoxy-D-glucose) Into polymer, the chitosan that produces every year of the whole world can produce substantial amounts of amino more than 100,000,000,000 tons by hydrolyzing chitosan Glucose.
At present, common chitosan hydrolyzate method has respectively：Acid hydrolysis, oxidative degradation, physical degradation methods and enzyme process water Solution.Wherein enzymic degradation chitosan has the advantages that environmental pollution is low, reaction condition is gentle, no coupling product, is before applying at present The most wide degradation of chitosan method of scape, has been gradually available for industrialized production in recent years, and the key of enzymatic hydrolysis chitosan exists In chitosan enzyme.
Although enzyme process prepares chitosan oligosaccharide, there are many advantages, existing chitosan enzyme generally existing：Applicable pH scopes It is narrow, dissolvable chitosan can only be acted on, the problems such as catabolite is mostly oligosaccharides of the pentasaccharides (G5) more than.Such as one entitled Itd is proposed in the report of " research overview of chitosanase from microogranism "：Existing all kinds of chitosan enzymes, majority is basic protein, most suitable PH is generally concentrated between 5~8, and the optimal pH for occasionally having several enzymes is 4, but its corresponding optimum temperature is higher, causes in reality The situation that energy consumption is higher in production application, the scope of application is not wide.
Therefore, develop that a kind of advantageous pH range is wide, optimum temperature is relatively relatively low, can be disaccharides by insoluble degradation of chitosan (G2), the chitosan enzyme of the oligosaccharide such as trisaccharide (G3), can effectively promote the deep exploitation of ocean Xia Xie sources chitosan, have weight The realistic meaning wanted.
The content of the invention
The object of the present invention is to provide one plant of bacillus subtilis for producing chitosan enzyme and its application.
For achieving the above object, the technical solution adopted in the present invention is：One bacillus subtilis, 2017 10 The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, strain name on 30th:Bacillus subtilis, Bacillus subtilis, MY002, deposit number is CGMCC No.14841.
Correspondingly, a bacillus subtilis, its 16SrDNA sequencing result is as shown in SEQ ID NO 1；Its gyrA is sequenced As a result if sequencing result is as shown in SEQ ID NO 2；Its gyrB sequencing result is as shown in SEQ ID NO 3.
Correspondingly, application of the bacillus subtilis in hydrolyzing chitosan.
Preferably, the pH scopes of the application are 2.5~7.5.
Preferably, the pH of the application is：3 or 6.
Preferably, the temperature range of the application is 35~65 DEG C.
A kind of correspondingly, chitosan enzyme CsnMY002, it is characterised in that：Its nucleotide sequence as shown in SEQ ID NO 4, Its amino acid sequence is as shown in SEQ ID NO 5.
Correspondingly, applications of the chitosan enzyme CsnMY002 in hydrolyzing chitosan.
Preferably, the pH scopes of the application are：2.5~7.5.
Preferably, the temperature of the application is 35~65 DEG C.
The invention has the advantages that：
1st, the present invention provides the bacillus subtilis of one plant of production chitosan enzyme, its produced chitosan enzyme to have yield High, the advantages that enzyme activity is strong.
2nd, present invention discover that chitosan enzyme, there is the protein structure different from common chitosan enzyme, the ammonia of its protein Base acid sequence TRDEWR fragments, helically structure；And this section of sequence is had no in other chitosan enzymes, relevant position is random Loop structures.This provides new approaches for the further investigation of chitosan enzyme molecular machinery.
3rd, common chitosan enzyme only has a preferable activity in weak acid to weak base, and present invention discover that chitosan enzyme Not only vigor is preferable under normal conditions, also has good enzyme activity under stronger acid condition.This causes answering for the enzyme Common chitosan enzyme is much larger than with scope and prospect.
4th, present invention discover that chitosan enzyme, not only may act on the chitosan that can be dissolved, may also act to insoluble High chitosan even undressed chitosan coarse raw materials, eliminate the processing before degradation of chitosan, dramatically saves on Production cost.
5th, present invention discover that the product of chitosan enzyme hydrolyzing chitosan be mainly two poly glucosamines and trimerization amino Glucose, product component is simple, easy to be processed further applying.
Brief description of the drawings
Fig. 1 is the hydrolysis circle blank control of non-inoculating strain；
Fig. 2 is the hydrolysis that bacterium is formed on Selective agar medium；
Fig. 3 is D-Glucose hydrochloride canonical plotting；
Fig. 4 is the 16SrDNA systematic evolution trees of bacterial strain MY002；
Fig. 5 is the gyrA systematic evolution trees of bacterial strain MY002；
Fig. 6 is the gyrB systematic evolution trees of bacterial strain MY002；
Fig. 7 is the chitosan enzyme gel permeation chromatography result of signal peptide；
Fig. 8 is chitosan enzyme CsnMY002 action effect comparison diagrams at different temperatures；
Fig. 9 is chitosan enzyme CsnMY002 action effect comparison diagrams under different pH；
Figure 10 is chitosan enzyme CsnMY002 hydrocolloid chitosan product analysis；
Figure 11 hydrolyzes insoluble high chitosan product analysis for chitosan enzyme CsnMY002.
Embodiment
Embodiment 1：Produce the screening of chitosan enzyme bacterial strain
1st, material prepares：
(1) pedotheque：Soil sample is gathered from Mianyang planting asparagus area.
(2) culture medium：
1) chitosan enzyme screening and culturing medium (g/L)：Colloid chitosan 2.0, K2HPO40.7, KH2PO40.3, (NH4)2SO4 5,MgSO4﹒ 7H2O 0.5, FeSO4﹒ 7H20 0.01, agar 12.0, pH=7.0；
2) broth bouillon (g/L)：Peptone 10.0, beef extract 3.0, NaCl 5.0, pH=7.0.
3) chitosan enzyme inducing culture (g/L)：Colloid chitosan 5.0, K2HPO40.7, KH2PO40.3, (NH4)2SO4 1.0,MgSO4﹒ 7H2O 0.5, FeSO4﹒ 7H20 0.01, pH=7.0；
4) LB culture mediums (g/L):Peptone 10.0, yeast extract 5.0, NaCl 10.0.
(3) reagent：
1) colloid chitosan:High poly- fine powder chitosan 10g is weighed, adds appropriate concentrated hydrochloric acid, and stirred on magnetic stirring apparatus Mixing 4~6h makes it fully dissolve, then cleans centrifugation (4000r/min) repeatedly with deionized water, eventually forms one side after jelly The general NaOH that is slowly added into of stirring adjusts pH to neutrality, that is, obtains colloid chitosan, saved backup in 4 DEG C.
2) 3,5- dinitrosalicylic acids (DNS):The distilled water of 50ml is taken to be heated in 80 DEG C of water-bath, after to be heated 3, the 5- dinitrosalicylic acids of the 2mol/L NaOH and 0.63g of 26.2ml are added, the winestone of 18.2g is added after dissolving to be mixed Sour potassium sodium, adds 0.5g crystalline phenols and 0.5g Na after stirring and dissolving2SO3, stirring and dissolving, is determined after to be cooled with distilled water Hold to 100ml, be stored in brown bottle, 4 DEG C save backup.
(4) kit, enzyme and cell
1) BamHI, SalI purchase are in New England Biolabs, Inc.；
2) purchase of plasmid extraction kit is in Beijing Quanshijin Biotechnology Co., Ltd；
3) purchase of T4 ligases is in Takara Bio.Inc.；
4) competent cell DH5 α and e. coli bl21 preserve for this laboratory.
2nd, bacterial strain primary dcreening operation
(1) weigh 5g soil samples to be put into the conical flask (250m) for filling 45ml distilled water, stirring and dissolving, boils 30min, so After take supernatant carry out gradient dilution (10-1,10-2,10-3,10-4,10-5Totally 5 concentration), respectively take 100ul to be respectively coated on shell In dextranase screening and culturing medium (each gradient setting 2 is parallel), 2~4d of culture in 37 DEG C of constant incubators is placed in.
(2) picking on selection culture plate growth and around have the bacterium colony of obvious hydrolysis, streak inoculation is in new Selection culture plate on, be placed in 37 DEG C of constant incubators and cultivate 2d, then choose bacterium colony streak inoculation, so carry out the screening of 3 generations, To ensure to obtain purebred bacterial strain.
(3) picking single bacterium falls within Liquid Culture in beef-protein medium after continuous 3 generation point is pure, then takes 5ul It is added on a diameter of 6mm filter papers, filter paper is placed on Selective agar medium, 37 DEG C of constant temperature incubation 2d, and hydrolysis circle is such as Fig. 1,2 institutes Show.The size of transparent circle is measured, as shown in table 1.
1 bacterium of table produces the size of hydrolysis circle
(4) bacterial strain progress conservation pure, and that hydrolysis can be produced will be divided, and stored at -80 DEG C.
3rd, bacterial strain secondary screening and chitosan enzyme enzyme activity determination
(1) 5umol/L D-Glucose hydrochloride standard curves are drawn
The centrifuge tube of 30 200ul is taken, is uniformly divided into 3 groups of equivalent, as shown in table 2, is separately added into the D- of 5 μm of ol/ml Glucosamine salt hydrochlorate titer, distilled water and 3,5- dinitrosalicylic acid (DNS) reagent, are configured to the D-Glucose of different content Hydrochloride standard reaction liquid.Each centrifuge tube is shaken up, is placed in boiling water bath accurate heating 5min colour developings, it is then fast in ice bath Quickly cooling but, after sufficiently cool, reuse microplate reader and measures light absorption value under 540nm wavelength.Three groups of data are averaged, are inserted In table 2.Standard curve is drawn using Excel, is D-Glucose hydrochloride canonical plotting, as shown in Figure 3.
2 D-Glucose hydrochloride standard curve of table
(2) DNS methods survey enzyme activity
1) bacterial strain fermentation liquor is obtained：By selected bacterial strain broth bouillon overnight incubation, as seed liquor, then by seed liquor It is inoculated in 5% inoculum concentration in chitosan enzyme inducing culture, in 37 DEG C, 2d is cultivated under the conditions of 220r/min, then 12000g, 30min are centrifuged, and take supernatant.
2) the 200ul steps 1) supernatant is taken, the phosphate buffer (6.1ml of 1.0% colloid chitosan is contained with 200ul 20mM Na2HPO4Solution and 3.9ml 20mM NaH2PO4Solution mixes, pH=7.0) mixing.37 DEG C keep the temperature 1h, then boiling water Bath 10min is reacted with terminating, then treats that ice bath cools down, and adds DNS reagents 400ul.Rapid ice bath cooling after boiling water bath 5min, 12000r/min centrifuges 5min, takes supernatant to survey absorbance (OD values) at wavelength 540nm.It is bent with D-Glucose hydrochloride standard Line is control, calculates the burst size U of reduced sugar：U=(3.6842* average absorbance values+0.3692) * 2.
Enzyme activity unit defines:Under these conditions, enzyme amount needed for generation 1umol reduced sugars is defined as 1 enzyme per hour Unit of activity.
The results are shown in Table 3 for the enzyme activity determination of bacterium.
The enzyme activity size of 3 bacterium of table
Embodiment 2：Bacterial strain is identified
1st, 16SrDNA sequencing identifications
The genomic DNA of strain to be tested is extracted, is then cloned with the universal primer of bacterial 16 S rDNA from genomic DNA 16SrDNA。
Universal primer is：
27F:AGA GTT TGA TCC TGG CTC AG；
1492R:TAC GGT TAC CTT GTT ACG ACT T。
PCR result Song Sheng works biotech firm is sequenced, by sequencing result in Gene Bank website (http:// Www.nicbi.nlm.nih.gov/ BLAST contrasts) are carried out, finds and downloads the gene order of reference culture, sequencing result is such as Shown in SEQ ID NO 1.The genetic distance between different strains is calculated using MEGA7.0, draws the phylogenetic tree of bacterium, such as Shown in Fig. 4.
Strain to be tested is compared with the 16SrDNA of bacillus type strain, with bacillus subtilis (Bacillus Subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and bacillus licheniformis (Bacillus Amyloliquefaciens genetic distance) is nearest, is respectively:0.002nd, 0.005 and 0.015.
2nd, gyrA and gyrB sequencing identifications
The A subunits and B subunits of gyrase, as special albumen, are guarded, every about 1,000,000 years gene evolutions in evolution 0.7~0.8%, the speed of its evolution 1% in 50,000,000 years every compared to 16SrDNA is fast, so it is often used in identification parent Edge relation is closer to the bacterial strain that can not be distinguished with 16SrDNA.Equally using the genomic DNA of strain to be tested as template, bacterium is expanded Gyrase A and 1 B gene.
(1) gyrA is expanded using universal primer, and primer is：
gyrA-F：5’-CAG TCA GGA AAT GCG TAC GTC CTT-3’；
gyrA-R：5’-CAA GGT AAT GCT CCA GGC ATT GCT-3’.
The sequencing result of gyrA is if sequencing result is as shown in SEQ ID NO 2.PCR sequencing results and this kind are not belonged to together The gyrase A genes of reference culture are compared, and build systematic evolution tree using MEGA 7.0, and calculate genetic distance, tie Fruit is as shown in Figure 5.Wherein strain to be tested and the genetic distance of bacillus subtilis (Bacillus subtilis) is nearest, is 0.010 (with the genetic distances of other bacterial strains more than 0.2).
(2) gyrB is according to 16SrDNA qualification results, according to drawing for the conserved regions design of the gyrB sequences of several sibling specieses Thing, primer are：
gyrB-F：5’-AAC AGC AAA GGC CTT CAC CA-3’；
gyrB-R：5’-GCA GAG TCA CCC TCT ACG ATA TA-3’.
The sequencing result of gyrB is if sequencing result is as shown in SEQ ID NO 3.PCR sequencing results and this kind are not belonged to together The gyrase 1 B gene of reference culture is compared, and builds systematic evolution tree using MEGA 7.0, and calculates genetic distance, ties Fruit is as shown in Figure 6.The genetic distance of strain to be tested and bacillus subtilis (Bacillus subtilis) is nearest, is 0.016, (exist with the genetic distance of Bacillus subtilis subsp.spizizenii for 0.087 with the genetic distance of other bacterial strains More than 0.2).
3rd, based on the above results, it is bacillus subtilis (Bacillus subtilis) to determine strain to be tested MY002.Should Bacterial strain is on October 30th, 2017, in China Committee for Culture Collection of Microorganisms's common micro-organisms center (China General Microbiological Culture Collection Center) preservation, strain name:Bacillus subtilis (Bacillus subtilis) MY002, deposit number are CGMCC No.14841.
Embodiment 3：Structure restructuring chitosan enzyme CsnMY002
1st, the recombination expression of chitosan enzyme and purifying
(1) chitosanase gene clone and sequencing
By to Genebank (https://www.ncbi.nlm.nih.gov/genbank/) in known withered grass gemma Bacillus chitosanase gene carries out Multiple Sequence Alignment, and design primer is as follows：
F:5’-CGC GGA TCC ATG AAA ATC AGT ATG CAA AAA GC-3’；
R:5’-A CGC GTC GAC TTA TTT GAT TAC AAA ATT ACC G-3’。
The full-length genome of the bacterial strain is extracted using bacterium complete genome DNA extracts kit, and is gathered as template Polymerase chain reacts (PCR), successfully clones the chitosanase gene (CsnMY002) of the bacterial strain, its gene nucleotide series As shown in SEQ ID NO 4, protein amino acid sequence is as shown in SEQ ID NO 5.
Sequencing result is shown：The chitosanase gene total length of bacillus subtilis MY002 is 834bp, encodes 277 ammonia altogether Base acid.Above-mentioned amino acid sequence is passed through into 4.1 Server (http of SignalP://www.cbs.dtu.dk/services/ SignalP/) it is predicted, 35 amino acid are signal peptide sequence before discovery.
Inventor has found：There is one section of special sequence in the amino acid sequence of CsnMY002：TRDEWR (corresponding sequences In SEQ ID NO 5：Thr Arg Asp Glu Trp Arg), form helical structure；And had no in other chitosan enzymes This section of sequence, its relevant position are random Loop structures.
(2) construction recombination plasmid
Using PET-28a as carrier, the recombinant plasmid for constructing signal peptide chitosan enzyme (has 6 group ammonia in albumen n end Acidity scale label).It is as follows to design primer：
F:5’-CGC GGA TCCGCG GGA CTG AAT AAA GAT CAA AAG-3’；
R:5’-A CGC GTC GAC TTA TTT GAT TAC AAA ATT ACC G-3’。
Wherein, the line base in above-mentioned primer introduces BamHI (GGA TCC) and SalI (GTC GAC) digestion position respectively Point.And using bacillus subtilis MY002 complete genome DNAs template, carry out PCR reactions.Returned using PCR product QIAquick Gel Extraction Kit Receive PCR product.
Meanwhile PET-28a plasmids are extracted using plasmid extraction kit, by it with PCR recovery products respectively in 37 DEG C of temperature Degree is lower overnight, carries out the reaction of BamHI, SalI double digestion, digestion recovery product then is pressed gene：Plasmid=10：1 (molar ratio) Linked system is built, 16h is connected under the conditions of 4 DEG C using T4 ligases.Linked system is transferred in competent cell DH5 α again, Positive colony is filtered out plasmid therein is transferred in expressive host e. coli bl21 again afterwards.
(3) expression and purification of chitosan enzyme (CsnMY002) is recombinated
The above-mentioned e. coli strain bl21 containing plasmid is inoculated in the LB culture mediums containing 60ug/ml kanamycins In, in 37 DEG C, culture is 0.4~0.6 to OD600 under the conditions of 220rpm, is then cooled to 16 DEG C, adds final concentration of 0.2mM IPTG carry out low temperature stay overnight, induced expression restructuring chitosan enzyme.Then 5600r/min, centrifugation 30min collect thalline.
Lysis buffer (50mM MES, 500Mm NaCl, 20mM imidazoles, pH=6.0) is prepared, precipitation is suspended with it, Ultrasound is recycled to carry out clasmatosis, broken rear solution centrifuges 30min under the conditions of 12000rpm, 4 DEG C, collects supernatant and carry out Ni Column affinity chromatography, is then eluted with elution buffer (50mM MES, 500Mm NaCl, 100mM imidazoles, pH=6.0).Parent The eluent obtained with chromatography recycles Resource S cation seperation columns to carry out ion-exchange chromatography, solidifying using Superdex 75 Glue Filter column carries out gel permeation chromatography.Stable and active high purity of shell dextranase is finally obtained.
Removing the chitosan enzyme gel permeation chromatography of signal peptide, the results are shown in Figure 7, and the relative molecular weight size of the enzyme is 27.42kDa。
The overnight incubation in shaking flask, then purified by affinity chromatography.The yield of the enzyme reaches 100mg/L.At 37 DEG C, pH Under the conditions of=6.0, enzyme activity is 547 ± 23U/mg (as shown in table 4).
Table 4 recombinates the enzyme activity determination result of chitosan enzyme
From the above it can be seen that：It is high, active high to recombinate chitosan enzyme CsnMY002 yield.
Embodiment 4：The optimum reaction conditions analysis of bacillus subtilis MY002 chitosan enzymes
1st, optimal reactive temperature is analyzed
(1) buffer solution is prepared：The MES buffer solutions (pH=6) of 50mM
(2) enzyme preparation is prepared：Using Bradford methods, measure and recombinate chitosan enzyme after purification described in embodiment 3 The concentration of CsnMY002.31.25 μ g enzyme liquids are taken, it is dense to 500 μ l, final enzyme preparation with buffer solution (50mM MES, pH=6) polishing Spend for 0.0625 μ g/ μ l.
(3) substrate solution is prepared：10ml colloids chitosan (1%, w/v) is taken, supernatant is removed after centrifugation (12000g, 5min) Liquid, adds the buffer solution (50mM MES, pH=6) of 9.5ml.
(4) measure：24 centrifuge tubes are taken, often the reaction system of pipe is 400 μ l, adds 380 μ l substrates into every pipe respectively Solution and 20 μ l enzyme preparations.10 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C are set, amounts to 8 Temperature Treatments, Each processing set 3 it is parallel.Each processing is incubated 5min under each temperature gradient respectively, then with boiling water boiling 10min with end Only react.After cooling, then 400 μ l DNS are separately added into, then 5min colour developings is boiled in boiling water, and cooled down rapidly.After cooling, 5min is centrifuged under 12000g rotating speeds, removes chitosan precipitation.
(5) result：Each reaction system respectively takes 200 μ l supernatants to measure light absorption value, further according to D-Glucose hydrochloride standard Curve, calculates the burst size of reduced sugar, so as to embody the hydrolysing activities of restructuring chitosan enzyme CsnMY002 at each temperature.Knot Fruit is as shown in Figure 8.
The results show：Chitosan enzyme CsnMY002 is recombinated at 35 DEG C~65 DEG C, is respectively provided with preferable enzyme activity, optimal reaction Temperature is 50 DEG C.To be easy to operate, temperature is all using 37 DEG C in following experiments；In actual production, institute can be according to circumstances selected Need temperature.
2nd, the analysis of optimal pH
(1) buffer solution is prepared：The glycine-HCI buffer solution (pH=2~4) of 50mM, the MES buffer solutions (pH=5 of 50mM ~6), the Tris-HCl buffer solutions (pH=7~9) of 50mM.
(2) according to the method in step 1, MES buffer solutions is changed to above-mentioned three groups of buffer solutions respectively, prepare three groups of difference pH The enzyme preparation and substrate solution of value, corresponding three pH processing, each handle set three it is parallel.And the assay method of step 1 is pressed, Reacted under the conditions of 37 DEG C, calculate the burst size of reduced sugar, show that hydrolysis of the restructuring chitosan enzyme CsnMY002 under different pH is lived Property.The results are shown in Figure 9.As can be seen from the figure：Chitosan enzyme CsnMY002 is recombinated in pH=2.5~7.5, is had good Activity, and in pH=3, activity is optimal；In pH=6, good activity.This and the chitosan enzyme high activity reported at present It is significantly different that section is concentrated mainly on pH=5~7.The present invention restructuring chitosan enzyme CsnMY002 not only can alkalescent, Hydrolyzing chitosan substrate under neutral and solutions of weak acidity, can also have an effect under the conditions of acidity is stronger.
Embodiment 5：Recombinate the analysis of chitosan enzyme CsnMY002 hydrolysis properties
1st, using solvable chitosan as substrate.
(1) be separately added into the colloid chitosan solution (1%, w/v) of 90 μ l into 7 centrifuge tubes, under 12000g rotating speeds from Heart 5min, except supernatant, with 50mM, the MES buffer solutions of pH=6 are resuspended.It is 0.5mg/ that concentration is added in 6 centrifuge tubes thereto again The chitosan enzyme CsnMY002 solution (the MES buffers of 50mM, pH=6) of ml, as experimental group；Add in 1 centrifuge tube Enter the MES buffer solutions of equivalent 50mM, pH=6, as blank group.
(2) by above-mentioned experimental group respectively at 37 DEG C, the hydrolysis for carrying out 30s, 1min, 3min, 5min, 10min, 1h is anti- Should；Blank group is placed in 1h under the same terms.Then 10min is boiled in boiling water to terminate reaction.
(3) G1~G6 is loaded in mixture into standard items point sample in silica gel plate (60 F of Silica gel254, 1.05554.0001, purchase In Merck KGaA company) as standard reference (std in Figure 10)；Question response terminates and then takes 1 μ l respectively from each processing Centrifuged supernatant, point sample carry out TLC analyses in above-mentioned silica gel plate.The expansion system that TLC is used for：Ethyl acetate/ethanol/water/ 28% ammonium hydroxide=5/5/4/0.5 (v/v), is fully deployed after about 2h, and 0.1% ninhydrin is immersed after silica gel plate is air-dried In (solvent is ethanol), then 5min colour developings are toasted at 90 DEG C.Results of hydrolysis is as shown in Figure 10.
(4) the results show：Reaction 30s or so can detect G3, G4, G5, G6, longer with the progress of reaction Chitosan oligosaccharide is gradually hydrolyzed into shorter oligosaccharides, and final hydrolysate is G2 and G3.
Wherein, G1, G2, G3 described in full text etc. represents oligosaccharide (oligosaccharides), the digital representation Glucosamine behind G The degree of polymerization.Such as：G1 is Glucosamine, and G2 is two poly glucosamines, and G3 is three poly glucosamines.
2nd, using insoluble high chitosan as substrate.
Operating procedure is identical with step 1.Wherein, colloid chitosan solution equivalent is replaced with insoluble chitosan to suspend Liquid, its preparation method are：Powdered chitosan 0.2g is weighed, adds 10ml buffer solutions (50mM MES, pH=6.0)；Fully shaking, Powder is set to suspend.Blank group is not provided with, hydrolysis time is respectively 4h, 8h, 12h.Results of hydrolysis is as shown in figure 11.
The results show：It is relatively slow to the hydrolysis of insoluble high chitosan to recombinate chitosan enzyme, after being incubated overnight Detect the formation of hydrolysate, its product remains as G2 and G3.
The above results are shown：Present invention discover that chitosan enzyme CsnMY002 not only solvable chitosans of hydrolyzable, may be used also Hydrolyze insoluble high chitosan.Meanwhile the oligosaccharides of G5, G6 even more long-chain is generally with the end-product of conventional chitosan enzyme Mixture except that：The short chain sugar of all G3 and G2 of hydrolysis end-product of chitosan enzyme CsnMY002, composition is simpler, The result determined easy to subsequent applications, the protein structure that it is special that this is probably enzyme itself.
Present invention discover that chitosan enzyme, there is new protein structure, it is lower to operating environment requirements, and have difference In the action effect of other chitosan enzymes, there is broader practice prospect, also for chitosan enzyme molecular mechanism deeper into grind Study carefully and provide theoretical foundation.
Sequence table
<110>Chengdu Inst. of Biology, Chinese Academy of Sciences
<120>One plant of bacillus subtilis for producing chitosan enzyme and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1455
<212> DNA
<213>Bacillus subtilis (Bacillus subtilis MY002)
<400> 1
gccattgcgg cgtgctatac atgcagtcga gcggacagat gggagcttgc tccctgatgt 60
tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120
gaaaccgggg ctaataccgg atggttgttt gaaccgcatg gttcaaacat aaaaggtggc 180
ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240
accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa 420
gaacaagtac cgttcgaata gggcggtacc ttgacggtac ctaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540
cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg 600
agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag 660
cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720
actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 780
gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg 840
cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900
ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960
gtcttgacat cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag 1020
gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080
gcaacccttg atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac 1140
aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200
acgtgctaca atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa 1260
atctgttctc agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt 1320
aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac cttttaggag ccagccgccg 1440
aaggtgaaca agaga 1455
<210> 2
<211> 959
<212> DNA
<213>Bacillus subtilis (Bacillus subtilis MY002)
<400> 2
tcccgtcagc atgagcgtta tcgtgtccgt gctcttccag atgttcgaga cggtttaaaa 60
ccggttcata gacggatttt gtatgcaatg aatgatttag gcatgacaag tgacaagcct 120
tataaaaaat ccgcgcgtat cgttggagaa gttatcggga aataccaccc gcacggtgat 180
tcagcggtat atgaatccat ggtcagaatg gctcaggatt tcaactaccg ttatatgctc 240
gttgacggtc acggaaactt cggttctgtt gacggagact cagcggcggc catgcgttat 300
acagaagcaa gaatgtctaa aatctcaatg gagattcttc gtgacatcac aaaagacaca 360
atcgattacc aggataacta tgacgggtca gaaagagaac ctgtcgttat gccttcaagg 420
ttcccgaatc tgctcgtgaa cggtgctgcc ggcattgcgg taggtatggc aacaaacatt 480
cctccgcacc agctgggaga aatcattgac ggtgtacttg ctgtcagtga gaatccggac 540
attacaattc cagagcttat ggaagtcatt ccagggcctg atttcccgac cgcgggtcaa 600
atcttgggac gcagcggtat ccggaaagca tacgaatcag gccgaggctc tatcacgatc 660
cgggcaaaag ctgagatcga acaaacatct tcgggtaaag aaagaattat cgttacagag 720
ttaccttacc aagtgaataa ggcgaaatta attgagaaaa ttgctgatct cgtaagggac 780
aaaaagatag agggtatcac agatctgcgt gatgagtcag atcgtacagg tatgagaatt 840
gtcattgaaa tcagacgcga cgccaatgca aatgtcatct taaacaatct gtacaaacaa 900
actgctctac aaacatcttt tggcatcaac ctgcttgcac ttgtgatggc cagccgaaa 959
<210> 3
<211> 1142
<212> DNA
<213>Bacillus subtilis (Bacillus subtilis MY002)
<400> 3
ccggagcatg tcgaatagta ttgacgaagc ccttgccggt tattgtacgg atatcaatat 60
ccaaatcgaa aaagacaaca gtatcacggt tgtagataat ggccgcggta ttccagtcgg 120
tattcatgaa aaaatgggcc gtcctgcggt agaagtcatt atgacggtgc ttcatgccgg 180
aggaaaattt gacggaagcg gctataaagt atccggagga ttacacggtg taggtgcttc 240
ggtcgtaaac gcactatcca cagagcttga tgtgacggtt caccgtgacg gtaaaattca 300
ccgccaaacc tataaacgcg gagttccggt tacagacctt gaaatcattg gcgaaacgga 360
tcatacagga acgacgacac attttgtccc ggaccctgaa attttctcag aaacaaccga 420
gtatgattac gatctgcttg ccaaccgcgt gcgtgaatta gcctttttaa caaagggcgt 480
aaacatcacg attgaagata aacgtgaagg acaagagcgc aaaaatgaat accattacga 540
aggcggaatt aaaagttatg tagagtattt aaaccgctct aaagaggttg tccatgaaga 600
gccgatttac attgaaggcg aaaaggacgg cattacggtt gaagtggctt tgcaatacaa 660
tgacagctat acaagcaaca tttactcgtt tacaaacaac attaacacgt acgaaggcgg 720
tacccatgaa gctggcttca aaacgggcct gactcgtgtt atcaacgatt acgccagaaa 780
aaaagggctt attaaagaaa atgatccaaa cctaagcgga gatgacgtaa gggaagggct 840
gacagcgatt atttcaatca aacaccctga tccgcagttt gagggccaaa caaaaacaaa 900
gctgggcaac tcagaagcac ggacgatcac cgatacgtta ttttctacag cgatggaaac 960
atttatgctg gaaaatccag atgcagccaa aaaaattgtc gataaaggct taatggcggc 1020
aagagcaaga atggctgcga aaaaagcccg tgaactaaca cgccgtaaga gtgctttgga 1080
aatttccaac ttgcccggta agttagcgga ctgctctcaa aagatccgag catccccgag 1140
tg 1142
<210> 4
<211> 834
<212> DNA
<213>Bacillus subtilis chitosan enzyme (CsnMY002)
<400> 4
atgaaaatca gtatgcaaaa agcagatttt tggaaaaagg cagcgatctc attacttgtt 60
ttcaccatgt tttttaccct gataatgagc gaaacggttt ttgcggcggg actgaataaa 120
gatcaaaagc gccgggcgga acagctgaca agtatctttg aaaacggcac aacggagatc 180
caatatggat atgtagagcg attggatgat gggcgaggct atacatgcgg acgggcaggc 240
tttacaacgg ctaccgggga tgcattggaa gtagtggaag tatacacaaa ggcagttccg 300
aataacaaac tgaaaaagta tctgcctgaa ttgcgccgtc tggccaagga agaaagcgat 360
gatacaagca atctcaaggg attcgcttct gcctggaagt cgcttgcaaa tgataaggaa 420
tttcgcgccg ctcaagacaa agtaaatgac catttgtatt atcagcctgc catgaaacga 480
tcggataatg ccggactaaa aacagcattg gctagagctg tgatgtacga tacggttatt 540
cagcatggcg atggtgatga ccctgactct ttttatgcct tgattaaacg tacgaacaaa 600
aaagcgggcg gatcacctaa agacggaata gacgagaaga agtggttgaa taaattcttg 660
gacgtacgct atgacgatct gatgaatccg gccaatcatg acacccgtga cgaatggaga 720
gaatcagttg cccgtgtgga cgtgcttcgc tctatcgcca aggagaacaa ctataatcta 780
aacggaccga ttcatgttcg ttcaaacgag tacggtaatt ttgtaatcaa ataa 834
<210> 5
<211> 277
<212> PRT
<213>Bacillus subtilis chitosan enzyme (CsnMY002)
<400> 5
Met Lys Ile Ser Met Gln Lys Ala Asp Phe Trp Lys Lys Ala Ala Ile
1 5 10 15
Ser Leu Leu Val Phe Thr Met Phe Phe Thr Leu Ile Met Ser Glu Thr
20 25 30
Val Phe Ala Ala Gly Leu Asn Lys Asp Gln Lys Arg Arg Ala Glu Gln
35 40 45
Leu Thr Ser Ile Phe Glu Asn Gly Thr Thr Glu Ile Gln Tyr Gly Tyr
50 55 60
Val Glu Arg Leu Asp Asp Gly Arg Gly Tyr Thr Cys Gly Arg Ala Gly
65 70 75 80
Phe Thr Thr Ala Thr Gly Asp Ala Leu Glu Val Val Glu Val Tyr Thr
85 90 95
Lys Ala Val Pro Asn Asn Lys Leu Lys Lys Tyr Leu Pro Glu Leu Arg
100 105 110
Arg Leu Ala Lys Glu Glu Ser Asp Asp Thr Ser Asn Leu Lys Gly Phe
115 120 125
Ala Ser Ala Trp Lys Ser Leu Ala Asn Asp Lys Glu Phe Arg Ala Ala
130 135 140
Gln Asp Lys Val Asn Asp His Leu Tyr Tyr Gln Pro Ala Met Lys Arg
145 150 155 160
Ser Asp Asn Ala Gly Leu Lys Thr Ala Leu Ala Arg Ala Val Met Tyr
165 170 175
Asp Thr Val Ile Gln His Gly Asp Gly Asp Asp Pro Asp Ser Phe Tyr
180 185 190
Ala Leu Ile Lys Arg Thr Asn Lys Lys Ala Gly Gly Ser Pro Lys Asp
195 200 205
Gly Ile Asp Glu Lys Lys Trp Leu Asn Lys Phe Leu Asp Val Arg Tyr
210 215 220
Asp Asp Leu Met Asn Pro Ala Asn His Asp Thr Arg Asp Glu Trp Arg
225 230 235 240
Glu Ser Val Ala Arg Val Asp Val Leu Arg Ser Ile Ala Lys Glu Asn
245 250 255
Asn Tyr Asn Leu Asn Gly Pro Ile His Val Arg Ser Asn Glu Tyr Gly
260 265 270
Asn Phe Val Ile Lys
275

Claims (10)

  1. A 1. bacillus subtilis, it is characterised in that：On October 30th, 2017 is preserved in Chinese microorganism strain preservation management Committee's common micro-organisms center, strain name:Bacillus subtilis, Bacillus subtilis, MY002, deposit number are CGMCC No.14841。
  2. A 2. bacillus subtilis, it is characterised in that：Its 16SrDNA sequencing result is as shown in SEQ ID NO 1；Its gyrA is surveyed Sequence result is if sequencing result is as shown in SEQ ID NO 2；Its gyrB sequencing result is as shown in SEQ ID NO 3.
  3. 3. application of the bacillus subtilis described in claim 1 or 2 in hydrolyzing chitosan.
  4. 4. application of the bacillus subtilis as claimed in claim 3 in hydrolyzing chitosan, it is characterised in that：The application PH scopes are：2.5~7.5.
  5. 5. application of the bacillus subtilis as claimed in claim 3 in hydrolyzing chitosan, it is characterised in that：The application PH=3 or 6.
  6. 6. application of the bacillus subtilis as claimed in claim 3 in hydrolyzing chitosan, it is characterised in that：The application Temperature range is：35~65 DEG C.
  7. A kind of 7. chitosan enzyme CsnMY002, it is characterised in that：Its nucleotide sequence is as shown in SEQ ID NO 4, its amino acid Sequence is as shown in SEQ ID NO 5.
  8. 8. applications of the chitosan enzyme CsnMY002 in hydrolyzing chitosan described in claim 7.
  9. 9. applications of the chitosan enzyme CsnMY002 as claimed in claim 8 in hydrolyzing chitosan, it is characterised in that：It is described to answer PH scopes are：2.5~7.5.
  10. 10. applications of the chitosan enzyme CsnMY002 as claimed in claim 8 in hydrolyzing chitosan, it is characterised in that：It is described The temperature range of application is：35~65 DEG C.
CN201810030676.3A 2018-01-12 2018-01-12 One plant of bacillus subtilis for producing chitosan enzyme and its application Pending CN108018245A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112175921A (en) * 2020-10-23 2021-01-05 中国科学院成都生物研究所 Chitosanase mutant G21R and application thereof
CN113278546A (en) * 2021-05-13 2021-08-20 青岛海洋生物医药研究院股份有限公司 Bacillus subtilis LC1-1 capable of efficiently producing enzyme and enzyme production method and application thereof
CN113913450A (en) * 2021-10-29 2022-01-11 湖南省植物保护研究所 Method for expressing chitosanase by using rhodopseudomonas palustris, chitosanase, recombinant plasmid, recombinant bacterium, fermentation inoculant and application
CN114480537A (en) * 2021-12-21 2022-05-13 中国海洋大学 Method for preparing chitosan oligosaccharide with high polymerization degree

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001069975A (en) * 1999-09-03 2001-03-21 Hankyu Bio Ind Kk Chitosanase
KR100803095B1 (en) * 2006-11-10 2008-02-18 제주대학교 산학협력단 Gene coding chitosanase from bacillus subtilis ch2 expression vector comprising the gene transformant transfected with the expression vector and method for purification of protein produced by thereof
CN101148646A (en) * 2007-09-06 2008-03-26 武汉东方天琪生物工程有限公司 Method for producing high-activity chitosanase preparation
DE102007039490A1 (en) * 2006-09-11 2008-03-27 Zakrytoe Aktsionernoe Obschestvo "Bio Tekhnologii" Producing powdered chitosan oligomers comprises enzymatically hydrolyzing chitosan in an aqueous solution of an organic acid, removing the acid by ion exchange and filtering, concentrating and drying the hydrolysis product
CN101200711A (en) * 2006-12-12 2008-06-18 华东理工大学 Chitosan degradation complex enzyme preparation and method for preparing the same
CN103361374A (en) * 2013-07-23 2013-10-23 扬州日兴生物科技股份有限公司 Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene
CN104357361A (en) * 2014-11-14 2015-02-18 青岛农业大学 Bacillus subtilis capable of producing chitinase and application thereof
CN104911125A (en) * 2015-05-28 2015-09-16 威海斯瑞海洋生物科技有限公司 Chitosanase production strain and application thereof
CN106998695A (en) * 2014-09-17 2017-08-01 拜耳作物科学有限合伙公司 Include recombinated bacillus cell and insecticide composition
CN107105673A (en) * 2014-09-17 2017-08-29 拜耳作物科学有限合伙公司 Belong to the composition of (Bacillus) cell and fungicide comprising recombinated bacillus
WO2017161181A1 (en) * 2016-03-16 2017-09-21 Spogen Biotech Inc. Fusion proteins, recombinant bacteria, and exosporium fragments for animal health and aquaculture
CN107208042A (en) * 2014-09-17 2017-09-26 斯波根生物技术公司 Fusion protein, recombinant bacteria and the method using recombinant bacteria
CN107236721A (en) * 2017-07-28 2017-10-10 中科荣信（苏州）生物科技有限公司 A kind of bacillus subtilis chitosan enzyme and its preparation method and application
CN109640649A (en) * 2016-03-16 2019-04-16 斯波根生物技术公司 Promote the method for plant health using resolvase and the microorganism for being overexpressed enzyme

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001069975A (en) * 1999-09-03 2001-03-21 Hankyu Bio Ind Kk Chitosanase
DE102007039490A1 (en) * 2006-09-11 2008-03-27 Zakrytoe Aktsionernoe Obschestvo "Bio Tekhnologii" Producing powdered chitosan oligomers comprises enzymatically hydrolyzing chitosan in an aqueous solution of an organic acid, removing the acid by ion exchange and filtering, concentrating and drying the hydrolysis product
KR100803095B1 (en) * 2006-11-10 2008-02-18 제주대학교 산학협력단 Gene coding chitosanase from bacillus subtilis ch2 expression vector comprising the gene transformant transfected with the expression vector and method for purification of protein produced by thereof
CN101200711A (en) * 2006-12-12 2008-06-18 华东理工大学 Chitosan degradation complex enzyme preparation and method for preparing the same
CN101148646A (en) * 2007-09-06 2008-03-26 武汉东方天琪生物工程有限公司 Method for producing high-activity chitosanase preparation
CN103361374A (en) * 2013-07-23 2013-10-23 扬州日兴生物科技股份有限公司 Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene
CN107105673A (en) * 2014-09-17 2017-08-29 拜耳作物科学有限合伙公司 Belong to the composition of (Bacillus) cell and fungicide comprising recombinated bacillus
CN106998695A (en) * 2014-09-17 2017-08-01 拜耳作物科学有限合伙公司 Include recombinated bacillus cell and insecticide composition
CN107208042A (en) * 2014-09-17 2017-09-26 斯波根生物技术公司 Fusion protein, recombinant bacteria and the method using recombinant bacteria
CN104357361A (en) * 2014-11-14 2015-02-18 青岛农业大学 Bacillus subtilis capable of producing chitinase and application thereof
CN104911125A (en) * 2015-05-28 2015-09-16 威海斯瑞海洋生物科技有限公司 Chitosanase production strain and application thereof
WO2017161181A1 (en) * 2016-03-16 2017-09-21 Spogen Biotech Inc. Fusion proteins, recombinant bacteria, and exosporium fragments for animal health and aquaculture
CN109640649A (en) * 2016-03-16 2019-04-16 斯波根生物技术公司 Promote the method for plant health using resolvase and the microorganism for being overexpressed enzyme
CN107236721A (en) * 2017-07-28 2017-10-10 中科荣信（苏州）生物科技有限公司 A kind of bacillus subtilis chitosan enzyme and its preparation method and application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GENEBANK: "chitosanase [Bacillus subtilis] NCBI Reference Sequence: WP_129137795.1", 《GENEBANK》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112175921A (en) * 2020-10-23 2021-01-05 中国科学院成都生物研究所 Chitosanase mutant G21R and application thereof
CN112175921B (en) * 2020-10-23 2022-06-07 中国科学院成都生物研究所 Chitosanase mutant G21R and application thereof
CN113278546A (en) * 2021-05-13 2021-08-20 青岛海洋生物医药研究院股份有限公司 Bacillus subtilis LC1-1 capable of efficiently producing enzyme and enzyme production method and application thereof
CN113913450A (en) * 2021-10-29 2022-01-11 湖南省植物保护研究所 Method for expressing chitosanase by using rhodopseudomonas palustris, chitosanase, recombinant plasmid, recombinant bacterium, fermentation inoculant and application
CN113913450B (en) * 2021-10-29 2024-02-02 湖南省植物保护研究所 Method for expressing chitosanase by rhodopseudomonas palustris, chitosanase, recombinant plasmid, recombinant bacteria, fermentation bacteria and application
CN114480537A (en) * 2021-12-21 2022-05-13 中国海洋大学 Method for preparing chitosan oligosaccharide with high polymerization degree
CN114480537B (en) * 2021-12-21 2023-06-20 中国海洋大学 Method for preparing chitosan oligosaccharide with high polymerization degree

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