CN106434475B - One streptomycete category polysaccharide degradation bacteria and its cultural method and application - Google Patents
One streptomycete category polysaccharide degradation bacteria and its cultural method and application Download PDFInfo
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Abstract
The present invention relates to a streptomycete category polysaccharide degradation bacteria and its cultural method and applications.One streptomycete (Streptomyces sp.) CB16 bacterial strain, on April 11st, 2016 are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number: CGMCC No.12351.The invention further relates to the strain culturing methods and application.Bacterial strain of the present invention is one plant of multi-functional polysaccharide degradation bacteria, compound polysaccharide degrading enzyme preparation degradable plant, seaweed, animal and the microbial polysaccharide prepared using the bacterial strain, especially to significant from the hyaluronic acid of higher mammal connective tissue, chondroitin sulfate A (CSA), chondroitin sulfate C and chondroitin sulfate E degrading activity, it has potential application, it is completely not identical as existing known streptomycete (Streptomyces sp.) function.
Description
Technical field
The present invention relates to a streptomycete category polysaccharide degradation bacteria and its cultural method and applications, belong to field of biotechnology.
Background technique
Polysaccharide is as duplicate single sugar unit or complicated sugar unit by polymer made of glucosides key connection, both wired
Property molecule, also there is branched molecule or network shaped polymer.The type of polysaccharide is more, quantity is big, is widely present in nature,
Such as: cellulose, xylan, mannosan in higher plant, agar-agar, algin, carragheen in seaweed, in crustacean
Chitin and microbe-derived peptide glycan, xanthan gum etc..These polysaccharide do not form the structural material of organism still,
Or the medium for storing energy, is important one of living resources[1].Moreover, polysaccharide and its catabolite have a variety of important lifes
Reason activity, medicine, cosmetics, food industry and in terms of with important application value.It is separated from microorganism
The fracture that polysaccharide degrading enzyme can be catalyzed glycosidic bond by the various ways such as hydrolyzing, cracking, generating has novel bioactivity
Oligomeric saccharic composition, to generate great economic benefit and social benefit.
Polysaccharide degradation bacteria[2,3]Refer to the bacterial strain that can be grown using a plurality of types of polysaccharide as sole carbon source.Polysaccharide degradation bacteria institute
The type multiplicity for producing polysaccharide degrading enzyme, has important Development volue.Currently, reported polysaccharide degradation bacteria is mainly isolated from sea
Ocean.The marine bacteria Persicobacter sp.JZB09 that Korean monarch etc. screens in ooze[4]、Flammeovirga
sp.MY04[5]It can be sole carbon source growth using 10 kinds or more of polysaccharide.International 2 plants of famous polysaccharide degradation bacterias
Zobellia galactanivorans[6]、Saccharophagus degradans 2-40[7]And it is isolated from marine environment.
In contrast, it is rarely reported from the polysaccharide degradation bacteria of terrestrial soil.
Streptomycete is a category of most species in actinomyces door, is widely present in soil, is Filamentous gram sun
Property bacterium, mainly by sporogenesis, most streptomycetes are harmless[8,9].Streptomycete is to generate various antibiotic
Main source, the antibiotic derived from streptomycete include erythromycin, tetracycline, streptomysin, chloramphenicol, neomycin, nystatin, card
That mycin, cycloheximide and anphotericin etc.[10], therefore, it is anti-that bacterial strain generation is focused primarily upon for the existing research of streptomycete
Ability and size of bacterium active material etc..The research for producing polysaccharide degrading enzyme about streptomycete focuses primarily upon the produced list of bacterial strain
One polysaccharide degrading enzyme, such as chitinase, zytase and cellulase[11-13], rare single streptomycete can generate a variety of
The report of type polysaccharide degrading enzyme.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides one plant of can drop in vegetable drug bulbus fritillariae cirrhosae root soil
Solution utilizes the streptomycete of a variety of different type polysaccharide and its cultural method and application.
The present invention is achieved by the following technical solutions:
One streptomycete (Streptomyces sp.) CB16 bacterial strain, on April 11st, 2016 are preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, address: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro-
Biological study institute, deposit number: CGMCC No.12351.
Streptomycete (Streptomyces sp.) CB16 bacterial strain, Gram-positive are observed on solid medium, bacterium
It falls in flower shape, intermediate meat-like protrusion, relatively wet, edge is radial and dry, can generate uranidin, as shown in Figure 1A；It adopts
Luxuriant with ordinary optical microscope and the growth of scanning electron microscopic observation aerial hyphae, branch is more, is in dendroid, spore is in oval
Shape, as shown in Figure 1B.Solid medium for thalli morphology observation is TSB solid medium, and component is as follows: tryptone
17g/L, phytone 3g/L, sodium chloride 5g/L, potassium dihydrogen phosphate 2.5g/L, glucose 2.5g/L, agar 20g/L, surplus
Water, pH7.2.
Streptomycete (Streptomyces sp.) CB16 bacterial strain, after measured, the gene order length of 16S rRNA is
1448bp, as shown in SEQ ID NO.1.By using U.S. Biotechnology Information center (National Center for
Biotechnology Information, NCBI) comparison of BLASTN program, the gene sequence of the 16S rRNA of bacterial strain of the invention
The gene order homology with higher of column and streptomycete reference culture (Streptomyces) the 16S rRNA of NCBI registration.
Through comparing, with reference culture Streptomyces cyaneofuscatus ATCC 19746, Streptomyces griseus
23345 affiliation of ATCC is nearest, and similarity is respectively 99.2%, 99.5%, but above-mentioned standard bacterial strain has no and can degrade
The report of a variety of polysaccharide.
Streptomycete (Streptomyces sp.) CB16 bacterial strain, using 16S rRNA Phylogenetic Tree construction method:
Using the Clustal W in MEGA6.0 software package to the similar sequences in the 16S rRNA gene order and gene pool measured,
Multiple Sequence Alignment is carried out together, with Neighbor-Joining method phylogenetic tree construction, and carries out 1000 Bootstraps
It examines, obtains statistics tree, as a result as shown in Figure 3.The result shows that CB16 bacterial strain and the type strain of streptomyces cluster, and position
In the branch internal.Therefore, CB16 bacterial strain is identified to streptomyces.
The basic skills of above-mentioned 16S rRNA gene sequencing is: preparing bacterial strain with bacterial genomes extracts kit (Tiangeng)
Genomic DNA expanded using the genomic DNA of bacterial strain as template using the universal primer of bacterial 16 S rRNA gene, use
Plastic recovery kit (Tiangeng) purifies pcr amplification product and is connected to pEASY-Blunt Simple Cloning after electrophoresis verifying
Vector, Transformed E .coli DH5 α competent cell.Through ammonia benzyl resistance screening, positive colony is obtained.16S rRNA gene sequencing
It is completed by Sangon Biotech (Shanghai) Co., Ltd., gained sequence and American National Bioinformatics Institute (NCBI) is received
The 16S rRNA gene order of the reference culture of record is compared.
The cultural method of above-mentioned streptomycete (Streptomyces sp.) CB16 bacterial strain, steps are as follows:
(1) streptomycete (Streptomyces sp.) CB16 bacterial strain is taken to line on solid medium, 25~30 DEG C of inversions
Activation culture 3~7 days, bacterial strain after activation is made；
(2) strain inoculated is 25~30 DEG C, revolution in temperature into fluid nutrient medium after taking step (1) activation obtained
Under conditions of 150~220 revs/min, shaking table culture 3~7 days, seed liquor is made；
(3) seed liquor made from step (2) is taken, is inoculated in fluid nutrient medium by 1~10% percent by volume, in temperature
Under conditions of degree is 25~30 DEG C, revolution is 150~220 revs/min, expand culture 3~7 days, streptomycete is made
(Streptomyces sp.) CB16 bacterium solution.
According to the present invention preferably, the solid medium in the step (1), every liter of component are as follows:
Tryptone 17g, phytone 3g, sodium chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, agar 20g,
Water is settled to 1L, pH7.2.
According to the present invention preferably, the fluid nutrient medium in the step (2) and (3), every liter of component are as follows:
1~20g of carbon source, NaCl 0.5g, K2HPO4 0.5g、KNO3 1g、FeSO4 0.01g、MgSO4·7H2O 0.5g,
Water is settled to 1L, pH value 6.5~7.5.
According to the present invention it is further preferred that the carbon source is selected from: cellulose, carboxymethyl cellulose, xylan, sweet dew
Glycan, chitin, chitosan, starch, pectin, sodium alginate, hyaluronic acid, chondroitin sulfate A (CSA), chondroitin sulfate C, sulfuric acid are soft
One of ossein E or xanthan gum.
Preferred according to the present invention, every liter of component of the fluid nutrient medium is as follows:
Tryptone 17g, phytone 3g, sodium chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, water are settled to
1L, pH 7.2.
Application of above-mentioned streptomycete (Streptomyces sp.) the CB16 bacterial strain in compound polysaccharide of degrading.
A kind of preparation method of compound polysaccharide degrading enzyme preparation, steps are as follows:
(i) above-mentioned streptomycete (Streptomyces sp.) CB16 bacterium solution is taken, is inoculated in by 1~10% percent by volume
In fermentation medium, under conditions of temperature is 25~30 DEG C, revolving speed is 150~220 revs/min, expand culture 3~7 days, system
Obtain streptomycete CB16 bacterial strain fermentation liquor；
(ii) fermentation liquid of streptomycete CB16 bacterial strain made from step (i) is taken, is separated by solid-liquid separation, liquid is taken, ammonium sulfate is added,
Make saturation degree 80%, gained precipitating under 80% ammonium sulfate saturation degree is collected after centrifugation, and buffered with the TGE of 20~50 times of volumes
Precipitating is resuspended in liquid, and compound polysaccharide degrading enzyme preparation is made in dialysis removal ammonium sulfate.
Preferred according to the present invention, the fermentation medium in the step (i) is No. II culture medium of Gao Shi, and every liter of component is such as
Under:
Soluble starch 20g, glucose 20g, beef extract 3g, yeast extract 10g, CaCO30.5g, NaCl 0.5g,
MgSO4·7H2O 0.5g, water are settled to 1L, pH7.0.
It is preferred according to the present invention, in the step (ii), be separated by solid-liquid separation as centrifugation, condition are as follows: 12000 × g, 4 DEG C from
5~20min of the heart.
It is preferred according to the present invention, in the step (ii), centrifugation are as follows: 15,000 × g, 4 DEG C of 5~20min of centrifugation.
Preferred according to the present invention, in the step (ii), TGE buffer composition is as follows:
50mM Tris, 50mM NaCl, 5mM EDTA, 5mM dithiothreitol (DTT) (DTT), the glycerol of 5.0% percent by volume
(Glycerol), excess water, pH 7.9.
It is preferred according to the present invention, in the step (ii), dialyse as using molecule interception 10, the bag filter of 000Da,
It is stirred dialysis.
Beneficial effect
Separation obtains a streptomycete (Streptomyces sp.) to the present invention from high mountain bulbus fritillariae cirrhosae root soil for the first time
CB16 bacterial strain, the bacterial strain can be using land plant, seaweed, animal and microbe-derived a plurality of types of polysaccharide as sole carbon source
It grows (Fig. 2), it is one plant of multi-functional polysaccharide degradation bacteria that the type of produced polysaccharide degrading enzyme is abundant, utilizes answering for bacterial strain preparation
Mould assembly polysaccharide degrading enzyme preparation degradable plant, seaweed, animal and microbial polysaccharide, especially to from higher mammal connective group
Hyaluronic acid, chondroitin sulfate A (CSA), chondroitin sulfate C and the chondroitin sulfate E degrading activity knitted are significant (Fig. 4), have potential
Application value, this is not completely identical as existing known streptomycete (Streptomyces sp.) function.
Detailed description of the invention
The grown form feature photo of Fig. 1, streptomycete CB16 bacterial strain；
Wherein, A, colonial morphology of the bacterial strain on TSB culture medium after culture 3 days, 6 days and 10 days；
B, left figure is optical microscope photograph, and middle figure and right figure are electromicroscopic photograph；
The highest cell concentration histogram that Fig. 2, streptomycete CB16 bacterial strain are grown in sole carbon source culture medium；
The chadogram that Fig. 3, streptomycete CB16 bacterial strain are drawn based on 16S rRNA gene order；
Analysis histogram of the compound polysaccharide degrading enzyme preparation of Fig. 4, streptomycete CB16 bacterial strain to polysaccharide degradation capability；
The HPLC analysis graph of Fig. 5, compound polysaccharide degrading enzyme preparation degradation hyaluronic acid products therefrom.
Specific embodiment
Below with reference to embodiment, technical scheme is described further, but institute's protection scope of the present invention is not limited to
This.
Embodiment 1, bulbus fritillariae cirrhosae root soil microorganism isolation and purification
Bulbus fritillariae cirrhosae root soil leachate is taken, supernatant 1mL is added in 9mL sterile water, and being diluted to concentration respectively is 10-1、10-2、10-3、10-4、10-55 concentration gradients.Bacteria suspension after dilution is filled with 50 DEG C or so No. I culture medium of Gao Shi
Divide and mix, each concentration does two in parallel, and 28 DEG C are cultivated 7 days.The more typical actinomyces bacterium colony of form is chosen, is then passed through
It after 3 times plate streaking isolates and purifies, chooses single bacterium and falls in TSB fluid nutrient medium, 28 DEG C, 200 revs/min are cultivated 3 days, and culture is taken
400 μ l glycerol are added in object 1.6ml, in -80 DEG C of refrigerator long-term preservations after mixing.
Above-mentioned TSB fluid nutrient medium, every liter of component are as follows:
Tryptone 17g, phytone 3g, sodium chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, water are settled to
1L, pH7.2.
No. I culture medium of Gao Shi, every liter of component are as follows:
Soluble starch 20g, KNO310g, NaCl 0.5g, FeSO40.01g, MgSO40.5g, agar 15g, dichromic acid
Potassium 0.1g, water are settled to 1L, pH7.2~7.4.
The screening of embodiment 2, polysaccharide degradation bacteria
It bacterial strain obtained in embodiment 1, is inoculated on sole carbon source fluid nutrient medium respectively, 200 revs/min, 30
DEG C culture 72h, observe bacterium solution cloudiness, while take culture supernatant carry out carbazole reaction detection carbon source Expenditure Levels.Foundation
Above-mentioned two index selects bacterium producing multi enzyme preparation.Will in each sole carbon source culture medium the muddy and lesser bacterium of carbazole reaction numerical value
It is cultivated on strain picking to TSB solid medium, and conservation is denoted as CB16.
The preparation method of above-mentioned sole carbon source culture medium is:
Polysaccharide substrate is added respectively into ion culture medium to final concentration of 0.10% (w/v)；
Above-mentioned polysaccharide substrate is selected from: cellulose, carboxymethyl cellulose, xylan, mannosan, chitin, chitosan, is formed sediment
Powder, pectin, agarose, sodium alginate, hyaluronic acid, chondroitin sulfate A (CSA), chondroitin sulfate C, chondroitin sulfate E, acetyl sulfate
Heparin, dermatan sulfate or xanthan gum；In 115 DEG C of high pressure sterilization 20min.
Above-mentioned ion culture medium, every liter of component are as follows:
NaCl 0.5g、K2HPO4 0.5g、KNO3 1g、FeSO4 0.01g、MgSO4·7H2O 0.5g, distilled water are settled to
1L adjusts pH value 7.2~7.4.
Such as Fig. 1, the streptomycete (Streptomyces sp.) CB16 bacterial strain, Gram-positive is trained in solid
Supporting the bacterium colony on base is in flower shape, and intermediate meat-like protrusion is relatively wet, and edge is radial and dry, energy chromogenesis, the raw bacterium of gas
Silk growth is luxuriant, and branch is more, is in dendroid, and spore is in oblong.
As shown in Fig. 2, above-mentioned streptomycete (Streptomyces sp.) CB16 bacterial strain, can not only utilize terrestrial higher plant
The polysaccharide in source, such as: microcrystalline cellulose, carboxymethyl cellulose, xylan, mannosan, starch and pectin；Also seaweed can be utilized
The polysaccharide in source, such as: algin；The polysaccharide that animal origin can also be utilized, especially from higher mammal connective tissue
Hyaluronic acid, chondroitin sulfate A (CSA), chondroitin sulfate C and chondroitin sulfate E, and chitin and shell from crustacean
Glycan；In addition, degradation Utilization ability is significant for the xanthan gum for deriving from microorganism.By to the more of streptomycete CB16 bacterial strain
Sugared Utilization ability analysis, CB16 bacterial strain can be grown at least 14 kinds of sole carbon source culture mediums, illustrate that the bacterial strain is more than one plant
Sugared degradation bacteria.
Streptomyces (Streptomyces sp.) CB16 bacterial strain that above-mentioned separation is obtained, preservation on April 11 in 2016
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number Institute of Microorganism, Academia Sinica, deposit number: CGMCC No.12351.
Embodiment 3, Molecular Identification of streptomycete (Streptomyces sp.) the CB16 bacterial strain based on 16S rRNA gene
Strain gene group DNA is prepared using bacterial genomes extracts kit (Tiangeng), using strain gene group DNA as mould
Plate is expanded using the universal primer of bacterial 16 S rRNA gene, is produced with plastic recovery kit (Tiangeng) purifying PCR amplification
Object is connected to pEASY-Blunt Simple Cloning Vector, is transformed into E.coli DH5 α after electrophoresis verifying.Through ammonia
Benzyl resistance screening obtains positive colony.16S rRNA gene sequencing is completed by Sangon Biotech (Shanghai) Co., Ltd.,
The 16S rRNA gene order for the reference culture that sequence and American National Bioinformatics Institute (NCBI) are included is compared, is answered
With MEGA6.0 phylogenetic tree construction.
The above-mentioned universal primer for bacterial strain 16S rRNA gene magnification are as follows:
Forward primer is 27f:5 '-AGAGTTTGATCCTG GCT CAG-3 '；
Reverse primer is 1492r:5 '-GGTTACCTTGTTACGACTT-3 '.
The above-mentioned reaction system for bacterial strain 16S rRNA gene magnification is as follows, 30 μ L of total volume:
Gene amplification reagent used is purchased from Dalian treasured Bioisystech Co., Ltd.
The above-mentioned program for bacterial strain 16S rRNA gene magnification are as follows:
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30S, 55 DEG C of annealing 30S, 72 DEG C of extension 90S, totally 35 recycle；72 DEG C are prolonged
Stretch 15min；It is cooled to 4 DEG C and keeps the temperature 15min.
It is above-mentioned to be based on 16S rRNA Phylogenetic Tree construction method:
Using the Clustal W in MEGA6.0 software package in the 16S rRNA gene order and gene pool measured
Similar sequences carry out Multiple Sequence Alignment together, with Neighbor-Joining method phylogenetic tree construction, and carry out 1000 times
Bootstraps is examined, and obtains statistics tree.
As a result as shown in figure 3, CB16 bacterial strain and reference culture Streptomyces that the present invention screens
Cyaneofuscatus ATCC 19746, Streptomyces griseus 23345 affiliations of ATCC are nearest, 16S
Similarity between rRNA gene is respectively 99.2%, 99.5%.And it is based on the gene constructed resulting statistics tree table of 16S rRNA
Bright, the type strain cluster of CB16 bacterial strain and streptomyces is located at the branch internal.Therefore, CB16 bacterial strain is accredited to strepto-
Pseudomonas.
The preparation of embodiment 4, the compound polysaccharide degrading enzyme preparation of streptomycete (Streptomyces sp.) CB16 bacterial strain
(1) from -80 DEG C of refrigerator scribing line streptomycete CB16 bacterial strains on TSB solid medium, 28 DEG C of inversions are cultivated 4 days；
(2) picking CB16 single bacterium drops down onto TSB fluid nutrient medium, in the item that temperature is 28 DEG C, revolving speed is 200 revs/min
Under part, shaking table culture 4 days, seed liquor is made；
(3) it by seed liquor made from step (2), is inoculated in Gao Shi II fluid nutrient medium by 1% percent by volume,
Under conditions of temperature is 28 DEG C, revolving speed is 220 revs/min, expand culture 6 days, streptomycete CB16 bacterial strain fermentation liquor is made；
(4) fermentation liquid for taking streptomycete CB16 bacterial strain made from step (3) is filtered with sterile gauze and removes most of bacterium
Body is separated by solid-liquid separation, and liquid is taken, and ammonium sulfate is added, makes saturation degree 80%, collects after 15000 × g, 4 DEG C of centrifugation 20min
Gained precipitating under 80% ammonium sulfate saturation degree is resuspended precipitating with the TGE buffer of 20 times of volumes, and is using molecule interception
Extracellular enzyme preparation is made to remove ammonium sulfate in 10,000Da bag filter dialysis.
TSB culture medium in the step (1), (2) is pancreas peptone soybean broth culture medium, and every liter of component is as follows:
Tryptone 17g, phytone 3g, sodium chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, water are settled to
1L, pH7.2；The agar that mass concentration is 2% is also added into solid medium.
The above-mentioned culture medium for strain fermentation is No. II culture medium of Gao Shi, and every liter of component is as follows:
Soluble starch 20g/L, glucose 20g/L, beef extract 3g/L, yeast extract 10g/L, CaCO30.5g/L,
NaCl 0.5g/L, MgSO4·7H2O 0.5g/L, water are settled to 1L, pH7.0.
The solid-liquid separating method is centrifugation, condition are as follows: 12,000 × g, 4 DEG C of centrifugation 10min.
The component of above-mentioned TGE buffer is as follows:
50mM Tris, 50mM NaCl, 5mM EDTA, 5mM dithiothreitol (DTT) (DTT), the glycerol of 5.0% percent by volume
(Glycerol), excess water, pH 7.9.
The dialysis, the bag filter for the use of molecule interception being 10,000Da, to 20~50 times of bodies in low temperature environment
Long-pending TGE buffer is stirred dialysis.
The analysis of embodiment 5, compound polysaccharide degrading enzyme preparation to not homopolysaccharide degradation capability
The polysaccharide substrate of concentration 3mg/mL, compound polysaccharide degrading enzyme preparation, PBS buffer solution and water are pressed into 2:1:2:1
After the ratio mixing of (volume ratio), 12h is reacted at 30 DEG C, pH7.4, incubating 10min in boiling water bath inactivates enzyme, 12,000 ×
G, 4 DEG C of centrifugation 10min, takes supernatant, as the enzymolysis product of compound polysaccharide degrading enzyme preparation, is gone back with what the detection of DNS method generated
Raw sugar.
As a result the terrestrial as shown in figure 4, the compound polysaccharide degrading enzyme preparation of streptomycete CB16 bacterial strain preparation can not only degrade
The polysaccharide in higher plant source, such as: microcrystalline cellulose, carboxymethyl cellulose, xylan, mannosan, starch and pectin；Also can
The polysaccharide in degradation seaweed source, such as: algin；Can also degrade the polysaccharide of animal origin, especially to deriving from higher mammal
Hyaluronic acid, chondroitin sulfate A (CSA), chondroitin sulfate C and the chondroitin sulfate E of connective tissue have higher degrading activity；In addition,
For deriving from the xanthan gum of microorganism, it may have certain degradation capability.Therefore, answering using the preparation of streptomycete CB16 bacterial strain
The degradable a variety of polysaccharide of mould assembly polysaccharide degrading enzyme preparation, to hyaluronic acid, the chondroitin sulfate for deriving from higher mammal connective tissue
Plain A, chondroitin sulfate C and chondroitin sulfate E degrading activity are significant, have potential application.
The HPLC analysis of embodiment 6, compound polysaccharide degrading enzyme preparation degradation hyaluronic acid products therefrom
The hyaluronic acid of concentration 3mg/mL, compound polysaccharide degrading enzyme preparation, PBS buffer solution and water are pressed into 2:1:2:1
After the ratio mixing of (volume ratio), 12h is reacted under conditions of 30 DEG C, pH7.4, incubating 10min in boiling water bath inactivates enzyme,
12,000 × g, 4 DEG C of centrifugation 10min, takes supernatant, the enzymolysis product as compound polysaccharide degrading enzyme preparation.It is preparatory with boiling water bath
The enzyme of inactivation carries out control group experiment.
Above-mentioned 20 μ L of enzymolysis product is taken to carry out efficient liquid phase (HPLC) analysis, gel column used is Superdex
Peptide10/300GL (GE), mobile phase are 0.2M ammonium hydrogen carbonate, flow velocity 0.4mL/min；Testing conditions are UV232nm.
As a result as shown in figure 5, in the reaction product of compound polysaccharide degrading enzyme preparation and hyaluronic acid, the appearance of principal product
Time is about 42min, and prompt is unsaturated hyaluronic acid disaccharides.It can't detect in the product of control group with characteristic absorption
Ingredient.Therefore, the compound polysaccharide degrading enzyme preparation prepared with streptomycete (Streptomyces sp.) CB16 bacterial strain, can be used for
Produce hyaluronic acid disaccharides.
Bibliography:
[1] Qingdao research [D] of the ocean Korean monarch colloidal sol bacterium Flammeovirga sp.MY04 agarase (being): in
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[2]Andrykovitch G,Marx I.Isolation of a new polysaccharide-digesting
bacterium from a salt marsh.Applied and Environmental Microbiology,1988,54
(4):1061-1062.
[3]Ekborg NA,Gonzalez JM,Howard MB,et al.Saccharophagus degradans
gen.nov.,sp.Nov.,a versatile marine degrader of complex
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Microbiology,2005,55(4):1545-1549.
[4] Korean monarch, separation, identification and agarose degradation capability [J] of mono- plant of polysaccharide degradation bacteria of such as Zhao Shuai, Liu Huihui
Microorganism journal, 2012,52 (6): 776-783.
[5]Wenjun Han,Jingyan Gu,Qiujie Yan,et al.A polysaccharide-degrading
marine bacterium,Flammeovirga sp.MY04 and its extracellular agarase
system.Journal of Ocean University of China.2012,11(3):375-382.
[6]Barbeyron,T,L’Haridon,S,Corre,E,et al.Zobellia galactanovorans
gen.nov.,sp.nov.,a marine species of Flavobacteriaceae isolated from a red
alga,and classification of[Cytophaga]uliginosa(ZoBell and Upham 1944)
Reichenbach 1989 as Zobellia uliginosagen.nov.,comb.nov..International
Journal of Systematic and Evolutionary Microbiology,51,985-997.
[7]Andrykovitch G,Marx I.Isolation of a new polysaccharide-digesting
bacterium from a salt marsh.Applied and Environmental Microbiolology,1988,54
(4):1061-1062.
[8]Dees M W,Somervuo P,Lysoe E,et al.Species identification and
microarray-based comparative genome analysis of Streptomyces species isolated
from potato scab lesions in Norway.Molecular Plant Pathology,2012,13(2):174-
186.
[9]WangYong,ZhangWei,ZhangWenge,et al.Study on identification and
separation of the antifungal antibiotic from its fermentation broth of
Streptomyces hygroscopicus BOS-013 strain.Asian Journal of Chemistry,2012,24
(9):3821-3824.
[10]Ayari A,Morakchi H,Djamila K G.Identification and antifungal
activity of Streptomyces sp.S72 isolated from Lake Oubeira sediments in
North-East of Algeria.African Journal of Biotechnology,2012,11(2):305-311.
[11]Mitsutomi M,Hata T,Kuwahara T.Purification and characterization
of novel chitinases from Streptomyces griseus HUT6037.Journal of Fermentation
and Bioengineering,1995,80:153-158.
[12]Suchita N,Mukesh K,Ramesh C K.Purification and characterization
of extracellular xylanase from Streptomyces cyaneus SN32.Bioresource
Technology,2008,99(2008):1252-1258.
[13]Katsuhiko F,Masataka S,Youhei F,et al.Streptomyces abietis
sp.nov.,a cellulolytic bacterium isolated from soil of a pine
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2013,63:4754-4759.
SEQUENCE LISTING
<110>Wutong Aroma Chemicals Co., Ltd.
<120>one streptomycete category polysaccharide degradation bacterias and its cultural method and application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1448
<212> DNA
<213> Streptomyces sp.
<400> 1
cgtcccaatc gccagtccca ccttcgacag ctccctccca caaggggttg ggccaccggc 60
ttcgggtgtt accgactttc gtgacgtgac gggcggtgtg tacaaggccc gggaacgtat 120
tcaccgcagc aatgctgatc tgcgattact agcaactccg acttcatggg gtcgagttgc 180
agaccccaat ccgaactgag accggctttt tgagattcgc tccgcctcgc ggcatcgcag 240
ctcattgtac cggccattgt agcacgtgtg cagcccaaga cataaggggc atgatgactt 300
gacgtcgtcc ccaccttcct ccgagttgac cccggcagtc tcctgtgagt ccccatcacc 360
ccgaagggca tgctggcaac acagaacaag ggttgcgctc gttgcgggac ttaacccaac 420
atctcacgac acgagctgac gacagccatg caccacctgt ataccgacca caaggggggc 480
accatctctg atgctttccg gtatatgtca agccttggta aggttcttcg cgttgcgtcg 540
aattaagcca catgctccgc tgcttgtgcg ggcccccgtc aattcctttg agttttagcc 600
ttgcggccgt actccccagg cggggaactt aatgcgttag ctgcggcacc gacgacgtgg 660
aatgtcgcca acatctagtt cccaacgttt acggcgtgga ctaccagggt atctaatcct 720
gttcgctccc cacgctttcg ctcctcagcg tcagtaatgg cccagagatc cgccttcgcc 780
accggtgttc ctcctgatat ctgcgcattt caccgctaca ccaggaattc cgatctcccc 840
taccacactc tagctagccc gtatcgaatg cagacccggg gttaagcccc gggctttcac 900
atccgacgtg acaagccgcc tacgagctct ttacgcccaa taattccgga caacgcttgc 960
gccctacgta ttaccgcggc tgctggcacg tagttagccg gcgcttcttc tgcaggtacc 1020
gtcactttcg cttcttccct gctgaaagag gtttacaacc cgaaggccgt catccctcac 1080
gcggcgtcgc tgcatcaggc tttcgcccat tgtgcaatat tccccactgc tgcctcccgt 1140
aggagtctgg gccgtgtccc agtcccagtg tggccggtcg ccctctcagg ccggctaccc 1200
gtcgtcgcct tggtaggcca ttaccccacc aacaagctga taggccgcgg gctcatcctt 1260
caccgccgga gcttttaacc ccgtcccaag cgggacagag tgttatccgg tattagaccc 1320
cgtttccagg gcttgtccca gagtgaaggg cagattgccc acgagttact cacccgttcg 1380
ccactaatcc accccgaaag gcttcatcgt tcgacttgca tgtgttaagc acgccgccag 1440
cgttcgtc 1448
Claims (13)
1. a streptomycete (StreptomycesSp.) CB16 bacterial strain, on April 11st, 2016 are preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center, address: the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Object research institute, deposit number: CGMCC No.12351.
2. streptomycete described in claim 1 (StreptomycesSp.) the cultural method of CB16 bacterial strain, which is characterized in that step
It is as follows:
(1) take streptomycete (StreptomycesSp.) CB16 bacterial strain lines on solid medium, 25~30 DEG C of inversion activation
Bacterial strain after activation is made in culture 3 ~ 7 days；
(2) strain inoculated is into fluid nutrient medium after taking step (1) activation obtained, temperature be 25~30 DEG C, revolution 150
Under conditions of~220 revs/min, shaking table culture 3~7 days, seed liquor is made；
(3) seed liquor made from step (2) is taken, is inoculated in fluid nutrient medium by 1~10% percent by volume, is in temperature
25~30 DEG C, revolution be 150~220 revs/min under conditions of, expand culture 3~7 days, be made streptomycete
(StreptomycesSp.) CB16 bacterium solution.
3. cultural method as claimed in claim 2, which is characterized in that the solid medium in the step (1), every liter of component
It is as follows:
Tryptone 17g, phytone 3g, sodium chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, agar 20g, water are fixed
Hold to 1L, pH7.2.
4. cultural method as claimed in claim 2, which is characterized in that the fluid nutrient medium in the step (2) and (3), often
It is as follows to rise component:
1~20g of carbon source, NaCl 0.5g, K2HPO4 0.5g、KNO3 1g、FeSO4 0.01g、MgSO4·7 H20.5 g of O, water are fixed
Hold to 1L, pH value 6.5~7.5.
5. cultural method as claimed in claim 4, which is characterized in that the carbon source is selected from: cellulose, carboxymethyl cellulose
Element, xylan, mannosan, chitin, chitosan, starch, pectin, sodium alginate, hyaluronic acid, chondroitin sulfate A (CSA), sulfuric acid
One of chondroitin C, chondroitin sulfate E or xanthan gum.
6. cultural method as claimed in claim 2, which is characterized in that every liter of component of the fluid nutrient medium is as follows:
Tryptone 17g, phytone 3g, sodium chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, water are settled to 1L, pH
7.2。
7. streptomycete described in claim 1 (StreptomycesSp.) application of the CB16 bacterial strain in compound polysaccharide of degrading；
The compound polysaccharide is selected from microcrystalline cellulose, carboxymethyl cellulose, xylan, mannosan, starch, pectin, brown alga
Glue, hyaluronic acid, chondroitin sulfate A (CSA), chondroitin sulfate C, chondroitin sulfate E, chitin, chitosan and/or xanthan gum.
8. a kind of preparation method of compound polysaccharide degrading enzyme preparation, which is characterized in that steps are as follows:
(i) take streptomycete described in claim 2 (Streptomyces Sp.) CB16 bacterium solution is connect by 1~10% percent by volume
Kind is in fermentation medium, under conditions of temperature is 25~30 DEG C, revolving speed is 150~220 revs/min, expands culture 3~7
It, is made streptomycete CB16 bacterial strain fermentation liquor；
(ii) the fermentation liquid of streptomycete CB16 bacterial strain made from step (i) is taken, is separated by solid-liquid separation, liquid is taken, ammonium sulfate is added, makes to satisfy
It is 80% with degree, gained precipitating under 80% ammonium sulfate saturation degree is collected after centrifugation, and be resuspended with the TGE buffer of 20~50 times of volumes
Precipitating, dialysis removal ammonium sulfate, is made compound polysaccharide degrading enzyme preparation；
The compound polysaccharide is selected from microcrystalline cellulose, carboxymethyl cellulose, xylan, mannosan, starch, pectin, brown alga
Glue, hyaluronic acid, chondroitin sulfate A (CSA), chondroitin sulfate C, chondroitin sulfate E, chitin, chitosan and/or xanthan gum.
9. preparation method as claimed in claim 8, which is characterized in that the fermentation medium in the step (i) is Gao Shi II
Number culture medium, every liter of component are as follows:
Soluble starch 20g, glucose 20g, beef extract 3g, yeast extract 10g, CaCO30.5g, NaCl 0.5g,
MgSO4·7H2O 0.5g, water are settled to 1L, pH7.0.
10. preparation method as claimed in claim 8, which is characterized in that in the step (ii), be separated by solid-liquid separation as centrifugation, item
Part are as follows: 12000 × g, 4 DEG C of 5~20min of centrifugation.
11. preparation method as claimed in claim 8, which is characterized in that in the step (ii), centrifugation are as follows: 15,000 × g, 4
DEG C centrifugation 5 ~ 20min.
12. preparation method as claimed in claim 8, which is characterized in that in the step (ii), TGE buffer composition is as follows:
50mM Tris, 50mM NaCl, 5mM EDTA, 5mM dithiothreitol (DTT), the glycerol of 5.0% percent by volume, excess water, pH
7.9。
13. preparation method as claimed in claim 8, which is characterized in that in the step (ii), dialyse to be retained using molecule
The bag filter for measuring 10,000Da, is stirred dialysis.
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