CN106434475A - Streptomyces polysaccharide degradation bacterium as well as culture method and application thereof - Google Patents
Streptomyces polysaccharide degradation bacterium as well as culture method and application thereof Download PDFInfo
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- CN106434475A CN106434475A CN201610937102.5A CN201610937102A CN106434475A CN 106434475 A CN106434475 A CN 106434475A CN 201610937102 A CN201610937102 A CN 201610937102A CN 106434475 A CN106434475 A CN 106434475A
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- streptomyces
- bacterial strain
- streptomycete
- polysaccharide
- culture
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Classifications
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention relates to a streptomyces polysaccharide degradation bacterium as well as a culture method and application thereof. A streptomyces sp. CB16 strain is preserved in China General Microbiological Culture Collection Center on April 11, 2016 and the preservation number is CGMCC No.12351. The invention further relates to the culture method and the application of the strain. The strain provided by the invention is a multifunctional polysaccharide degradation bacterium; a compound type polysaccharide degradation enzyme preparation prepared from the strain can be used for degrading plant, seaweed, animal and microorganism polysaccharides and particularly has remarkable degradation activity on hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C and chondroitin sulfate E from connective tissues of higher animals; and the streptomyces polysaccharide degradation bacterium has a potential application value and has functions which are completely different from functions of existing streptomyces sp.
Description
Technical field
The present invention relates to a streptomycete belongs to polysaccharide degradation bacteria and its cultural method and application, belong to biological technical field.
Background technology
Polysaccharide is the polymer being formed by connecting by glycosidic bond by the single sugar unit for repeating or complicated sugar unit, both wired
Property molecule, also has branched molecule, or network shaped polymer.The species of polysaccharide is many, quantity is big, is widely present in nature,
Such as：Cellulose, xylan, mannan in higher plant etc., the agar in Sargassum, Algin, carrageenan, in crustacean
Chitin, and microbe-derived Peptidoglycan, xanthan gum etc..These polysaccharide still do not constitute the structural material of organism,
Or the medium of energy is stored, is one of important living resources[1].And, polysaccharide and its catabolite have multiple important lifes
Reason activity, at the aspect such as medicine, cosmetics, food industry and agricultural with important using value.Detached from microorganism
Polysaccharide degrading enzyme can be catalyzed the fracture of glycosidic bond by various ways such as hydrolysis, cracking, generate with novel biological activity
Oligomeric saccharic composition, so as to produce great economic benefit and social benefit.
Polysaccharide degradation bacteria[2,3]The bacterial strain that can grow as sole carbon source is referred to polytype polysaccharide.Polysaccharide degradation bacteria institute
The type for producing polysaccharide degrading enzyme is various, with important Development volue.At present, the polysaccharide degradation bacteria that has reported is mainly isolatable from sea
Ocean.The marine bacteria Persicobacter sp.JZB09 that Korean monarch etc. is screened in sea mud[4]、Flammeovirga
sp.MY04[5]More than 10 kinds of polysaccharide can be all utilized to grow for sole carbon source.International in repute 2 plants of polysaccharide degradation bacteria
Zobellia galactanivorans[6]、Saccharophagus degradans 2-40[7]And it is isolatable from marine environment.
By contrast, the polysaccharide degradation bacteria from terrestrial soil is rarely reported.
Streptomycete is a category of most species in actinomycetes door, is widely present in soil, is thread gram sun
Property antibacterial, mainly pass through sporogenesis, most streptomycetes are harmless[8,9].Streptomycete is to produce various antibiotic
Main source, the antibiotic for coming from streptomycete includes erythromycin, tetracycline, streptomycin, chloromycetin, neomycin, Nystatin, card
That mycin, cycloheximide and amphotericin etc.[10], therefore, for streptomycete research focus primarily upon bacterial strain produce anti-
The aspects such as the ability and size of bacterium active substance.The research for polysaccharide degrading enzyme being produced with regard to streptomycete focuses primarily upon the produced list of bacterial strain
One polysaccharide degrading enzyme, such as chitinase, xylanase and cellulase etc.[11-13], rare single streptomycete can produce multiple
The report of type polysaccharide degrading enzyme.
Content of the invention
The present invention is directed to the deficiencies in the prior art, provides one plant of can drop in vegetable drug Bulbus Fritillariae Cirrhosae root soil
Solution, the streptomycete using number of different types 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 is preserved in China Microbiological bacterium
Plant 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, observe on solid medium, bacterium
Fall in flower shape, middle meat-like projection, relatively moisten, edge is radial and dries, and can produce flavochrome, as shown in Figure 1A；Adopt
Luxuriant with ordinary optical microscope and the growth of scanning electron microscopic observation aerial hyphae, branch is more, and in dendroid, spore is in oval
Shape, as shown in Figure 1B.It is TSB solid medium for the solid medium of thalli morphology observation, 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 its 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 the bacterial strain of the present invention
Row have higher homology with the gene order of streptomycete reference culture (Streptomyces) the 16S rRNA of NCBI registration.
Through comparing, with reference culture Streptomyces cyaneofuscatus ATCC 19746, Streptomyces griseus
23345 sibship of ATCC is nearest, and similarity is respectively 99.2%, 99.5%, but above-mentioned standard bacterial strain has no can degrade
The report of multiple polysaccharide.
Streptomycete (Streptomyces sp.) CB16 bacterial strain, using 16S rRNA Phylogenetic Tree construction method：
Clustal W in application MEGA6.0 software kit to the similar sequences in the 16S rRNA gene order that measures and gene bank,
Carry out Multiple Sequence Alignment together, with Neighbor-Joining method phylogenetic tree construction, and carry out 1000 Bootstraps
Inspection, obtains statistics tree, as a result as shown in Figure 3.As a result show, CB16 bacterial strain is clustered with the type strain of streptomyces, and position
In the branch internal.Therefore, by CB16 identification of strains to streptomyces.
The basic skills of above-mentioned 16S rRNA gene sequencing is：Bacterial strain is prepared with bacterial genomes extracts kit (Tiangeng)
Genomic DNA, with the genomic DNA of bacterial strain as template, expanded using the universal primer of bacterial 16 S rRNA gene, use
Glue reclaim test kit (Tiangeng) purification pcr amplification product, after electrophoresis checking, is connected to pEASY-Blunt Simple Cloning
Vector, Transformed E .coli DH5 α competent cell.Through ammonia benzyl resistance screening, positive colony is obtained.16S rRNA gene sequencing
Completed by Sangon Biotech (Shanghai) Co., Ltd., gained sequence is received with American National Bioinformatics Institute (NCBI)
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, step is as follows：
(1) take streptomycete (Streptomyces sp.) CB16 bacterial strain to line on solid medium, 25～30 DEG C of inversions
Activation culture 3～7 days, is obtained bacterial strain after activation；
(2) after taking the obtained activation of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution of step (1), inoculation, in fluid medium, is 25～30 DEG C, revolution
Under conditions of 150～220 revs/min, shaking table culture 3～7 days, prepared seed liquor；
(3) seed liquor obtained in step (2) is taken, is inoculated in fluid medium by 1～10% percent by volume, in temperature
Spend under conditions of being 150～220 revs/min for 25～30 DEG C, revolution, amplification culture 3～7 days, prepared streptomycete
(Streptomyces sp.) CB16 bacterium solution.
According to currently preferred, in step (1) solid medium, per liter of component is 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 currently preferred, in step (2) and (3) fluid medium, per liter of component is as follows：
1～20g of carbon source, NaCl 0.5g, K2HPO40.5g、KNO31g、FeSO40.01g、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 described carbon source is selected from：Cellulose, carboxymethyl cellulose, xylan, manna
Polysaccharide, chitin, shitosan, starch, pectin, sodium alginate, hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, sulphuric acid are soft
One of ossein E or xanthan gum.
According to currently preferred, per liter of component of the fluid medium is as follows：
Tryptone 17g, phytone 3g, Sodium Chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, water is 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, step is as follows：
I () takes above-mentioned streptomycete (Streptomyces sp.) CB16 bacterium solution, be inoculated in by 1～10% percent by volume
In fermentation medium, temperature be 25～30 DEG C, rotating speed be 150～220 revs/min under conditions of, amplification culture 3～7 days, system
Obtain streptomycete CB16 bacterial strain fermentation liquor；
(ii) fermentation liquid of streptomycete CB16 bacterial strain obtained in step (i) taken, solid-liquid separation, liquid is taken, ammonium sulfate is added,
Saturation is made for 80%, gained precipitation under 80% ammonium sulfate saturation of collected after centrifugation, and buffered with the TGE of 20～50 times of volumes
The resuspended precipitation of liquid, dialysis removes ammonium sulfate, and compound polysaccharide degrading enzyme preparation is obtained.
According to currently preferred, the fermentation medium in the step (i) is No. II culture medium of Gao Shi, and per 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 is settled to 1L, pH7.0.
According to currently preferred, in step (ii), solid-liquid separation for centrifugation, condition is：12000 × g, 4 DEG C from
5～20min of the heart.
According to currently preferred, in step (ii), it is centrifuged and is：15,000 × g, 4 DEG C of 5～20min of centrifugation.
According to currently preferred, in 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.
According to currently preferred, in step (ii), dialysis is the bag filter using molecular retention amount 10,000Da,
It is stirred dialysis.
Beneficial effect
The present invention is separated from high mountain Bulbus Fritillariae Cirrhosae root soil first and obtains a streptomycete (Streptomyces sp.)
CB16 bacterial strain, the bacterial strain can be with land plant, Sargassum, animal and microbe-derived polytype polysaccharide as sole carbon source
Growth (Fig. 2), the abundant species of produced polysaccharide degrading enzyme, are one plant of multi-functional polysaccharide degradation bacteria, using prepared by the bacterial strain answering
Mould assembly polysaccharide degrading enzyme preparation degradable plant, Sargassum, animal and microbial polysaccharide, especially to deriving from higher mammal connective group
Hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C and the chondroitin sulfate E degrading activity that knits is notable (Fig. 4), with potential
Using value, this is differed completely with existing known streptomycete (Streptomyces sp.) function.
Description of the drawings
Fig. 1, the grown form feature photo of streptomycete CB16 bacterial strain；
Wherein, A, culture 3 days, 6 days and 10 days after colonial morphology of the bacterial strain in TSB culture medium；
B, left figure are optical microscope photograph, and middle figure and right figure are electromicroscopic photograph；
The highest cell concentration block diagram that Fig. 2, streptomycete CB16 bacterial strain are grown in sole carbon source culture medium；
The cladogram that Fig. 3, streptomycete CB16 bacterial strain are drawn based on 16S rRNA gene order；
Analysis block diagram of the compound polysaccharide degrading enzyme preparation of Fig. 4, streptomycete CB16 bacterial strain to polysaccharide degradation capability；
Fig. 5, the HPLC analysis graph of compound polysaccharide degrading enzyme preparation degraded hyaluronic acid products therefrom.
Specific embodiment
With reference to embodiment, technical scheme is described further, but institute's protection domain of the present invention is not limited to
This.
Embodiment 1, the isolation and purification of Bulbus Fritillariae Cirrhosae root soil microorganism
Bulbus Fritillariae Cirrhosae root soil leachate being taken, supernatant 1mL is added in 9mL sterilized water, concentration is diluted to respectively for 10-1、10-2、10-3、10-4、10-55 Concentraton gradient.No. I culture medium of Gao Shi of bacteria suspension after dilution with 50 DEG C or so is filled
Point mix, each concentration do two parallel, 28 DEG C cultivate 7 days.Form more typical actinomycetes bacterium colony is chosen, is then passed through
After 3 times plate streaking is isolated and purified, single bacterium colony is chosen in TSB fluid medium, 28 DEG C, 200 revs/min are cultivated 3 days, take culture
Thing 1.6ml adds 400 μ l glycerol, mixes and is preserved after -80 DEG C of refrigerators for a long time.
Above-mentioned TSB fluid medium, per liter of component is as follows：
Tryptone 17g, phytone 3g, Sodium Chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, water is settled to
1L, pH7.2.
No. I culture medium of Gao Shi, per liter of component is as follows：
Soluble starch 20g, KNO310g, NaCl 0.5g, FeSO40.01g, MgSO40.5g, agar 15g, dichromic acid
Potassium 0.1g, water is settled to 1L, pH7.2～7.4.
Embodiment 2, the screening of polysaccharide degradation bacteria
The bacterial strain obtained by embodiment 1, it is inoculated on sole carbon source fluid medium respectively, 200 revs/min, 30
DEG C culture 72h, observe bacterium solution cloudiness, while take culture supernatant carry out carbazole reaction detect carbon source Expenditure Levels.Foundation
Above-mentioned two index selects bacterium producing multi enzyme preparation.Will in each sole carbon source culture medium equal muddy and less bacterium of carbazole reaction numerical value
Strain picking is cultivated on TSB solid medium, and conservation is designated as CB16.
The compound method of above-mentioned sole carbon source culture medium is：
Add polysaccharide substrate in ion culture medium respectively to final concentration of 0.10% (w/v)；
Above-mentioned polysaccharide substrate is selected from：Cellulose, carboxymethyl cellulose, xylan, mannan, chitin, shitosan, forms sediment
Powder, pectin, agarose, sodium alginate, hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate E, acetyl sulfate
Heparin, dermatan sulfate or xanthan gum；In 115 DEG C of autoclaving 20min.
Above-mentioned ion culture medium, per liter of component is as follows：
NaCl 0.5g、K2HPO40.5g、KNO31g、FeSO40.01g、MgSO4·7H2O 0.5g, distilled water are settled to
1L, adjusts pH value 7.2～7.4.
As Fig. 1, described streptomycete (Streptomyces sp.) CB16 bacterial strain, Gram-positive, train in solid
Bacterium colony on foster base is in flower shape, and middle meat-like projection is relatively moistened, and edge is radial and dries, energy chromogenesises, the raw bacterium of gas
Silk growth is luxuriant, and branch is more, and in dendroid, spore is in Long Circle.
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, mannan, starch and pectin；Sargassum can also be utilized
The polysaccharide in source, such as：Algin；The polysaccharide of animal origin can also be utilized, especially from higher mammal connective tissue
Hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C and chondroitin sulfate E, and the chitin from crustacean and shell
Polysaccharide；In addition, for the xanthan gum from microorganism, degraded Utilization ability is notable.By to many of streptomycete CB16 bacterial strain
The analysis of sugared Utilization ability, CB16 bacterial strain can be grown at least 14 kinds sole carbon source culture medium, illustrate that the bacterial strain is more than one plant
Sugared degradation bacteria.
By above-mentioned streptomyces (Streptomyces sp.) the CB16 bacterial strain for separating and obtaining, the preservation of 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, streptomycete (Streptomyces sp.) Molecular Identification of the CB16 bacterial strain based on 16S rRNA gene
Strain gene group DNA is prepared using bacterial genomes extracts kit (Tiangeng), with strain gene group DNA as mould
Plate, is expanded using the universal primer of bacterial 16 S rRNA gene, is produced with glue reclaim test kit (Tiangeng) purification PCR amplification
Thing, after electrophoresis checking, is connected to pEASY-Blunt Simple Cloning Vector, is transformed into E.coli DH5 α.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 of the reference culture that sequence is included with American National Bioinformatics Institute (NCBI) is compared, should
Use MEGA6.0 phylogenetic tree construction.
The above-mentioned universal primer for bacterial strain 16S rRNA gene amplification is：
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 amplification is as follows, 30 μ L of cumulative volume：
Gene amplification reagent used is purchased from Dalian treasured Bioisystech Co., Ltd.
The above-mentioned program for bacterial strain 16S rRNA gene amplification is：
94 DEG C of denaturations 5min；94 DEG C of degeneration 30S, 55 DEG C of annealing 30S, 72 DEG C of extension 90S, totally 35 circulations；72 DEG C are prolonged
Stretch 15min；It is cooled to 4 DEG C and is incubated 15min.
Above-mentioned based on 16S rRNA Phylogenetic Tree construction method：
Clustal W in application MEGA6.0 software kit is in the 16S rRNA gene order that measures and gene bank
Similar sequences, carry out Multiple Sequence Alignment together, with Neighbor-Joining method phylogenetic tree construction, and carry out 1000 times
Bootstraps is checked, and obtains statistics tree.
As a result as shown in figure 3, the CB16 bacterial strain that screens of the present invention and reference culture Streptomyces
23345 sibship of cyaneofuscatus ATCC 19746, Streptomyces griseus ATCC is nearest, 16S
Similarity between rRNA gene is respectively 99.2%, 99.5%.And the statistics tree table based on the gene constructed gained of 16S rRNA
Bright, CB16 bacterial strain is clustered with the type strain of streptomyces, positioned at the branch internal.Therefore, CB16 bacterial strain is identified to strepto-
Pseudomonas.
The preparation of the compound polysaccharide degrading enzyme preparation of embodiment 4, streptomycete (Streptomyces sp.) CB16 bacterial strain
(1) streptomycete CB16 bacterial strain is rule in TSB solid medium from -80 DEG C of refrigerators, 28 DEG C are inverted culture 4 days；
(2) picking CB16 single bacterium colony is to TSB fluid medium, temperature be 28 DEG C, rotating speed be 200 revs/min of bar
Under part, shaking table culture 4 days, prepared seed liquor；
(3) by seed liquor obtained in step (2), it is inoculated in Gao Shi II fluid medium by 1% percent by volume,
Temperature be 28 DEG C, rotating speed be 220 revs/min under conditions of, amplification culture 6 days, prepared streptomycete CB16 bacterial strain fermentation liquor；
(4) fermentation liquid of streptomycete CB16 bacterial strain obtained in step (3) is taken, is filtered with sterile gauze and remove most of bacterium
Body, through solid-liquid separation, takes liquid, adds ammonium sulfate, makes saturation for 80%, collects after 15000 × g, 4 DEG C of centrifugation 20min
Gained precipitation under 80% ammonium sulfate saturation, with the resuspended precipitation of the TGE buffer of 20 times of volumes, and using molecular retention amount be
10,000Da bag filter dialysis, to remove ammonium sulfate, prepared extracellular enzyme preparation.
TSB culture medium in step (1), (2) is pancreas peptone soybean broth culture medium, and per liter of component is as follows：
Tryptone 17g, phytone 3g, Sodium Chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, water is settled to
1L, pH7.2；The agar that mass concentration is 2% is also added in solid medium.
The above-mentioned culture medium for strain fermentation is No. II culture medium of Gao Shi, and per 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 is settled to 1L, pH7.0.
Described solid-liquid separating method is for being centrifuged, and condition is：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.
Described dialysis, is the bag filter of 10,000Da using molecular retention amount, to 20～50 times of bodies in low temperature environment
The TGE buffer of product 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 and water are pressed 2：1：2：1
After the ratio mixing of (volume ratio), under 30 DEG C, pH7.4,12h is reacted, 10min is incubated in boiling water bath inactivates enzyme, 12,000 ×
G, 4 DEG C of centrifugation 10min, take supernatant, as the enzymatic hydrolysate of compound polysaccharide degrading enzyme preparation, detect that with DNS method generated goes back
Raw sugar.
As a result as shown in figure 4, compound polysaccharide degrading enzyme preparation prepared by streptomycete CB16 bacterial strain can not only be degraded terrestrial
The polysaccharide in higher plant source, such as：Microcrystalline Cellulose, carboxymethyl cellulose, xylan, mannan, starch and pectin；Also can
The polysaccharide of degraded algal source, such as：Algin；Can also degrade the polysaccharide of animal origin, especially to deriving from higher mammal
The hyaluronic acid of connective tissue, chondroitin sulfate A, chondroitin sulfate C and chondroitin sulfate E have higher degrading activity；In addition,
For the xanthan gum from microorganism, it may have certain degradation capability.Therefore, using prepared by streptomycete CB16 bacterial strain answering
The multiple polysaccharide of mould assembly polysaccharide degrading enzyme preparation degradable, to the hyaluronic acid from higher mammal connective tissue, chondroitin sulfate
Plain A, chondroitin sulfate C and chondroitin sulfate E degrading activity are notable, with potential using value.
Embodiment 6, the HPLC analysis of compound polysaccharide degrading enzyme preparation degraded hyaluronic acid products therefrom
The hyaluronic acid of concentration 3mg/mL, compound polysaccharide degrading enzyme preparation, PBS and water are pressed 2：1：2：1
After the ratio mixing of (volume ratio), under conditions of 30 DEG C, pH7.4,12h is reacted, 10min is incubated in boiling water bath inactivates enzyme,
12,000 × g, 4 DEG C of centrifugation 10min, take supernatant, as the enzymatic hydrolysate of compound polysaccharide degrading enzyme preparation.Advance with boiling water bath
The enzyme of inactivation, carries out matched group experiment.
Taking 20 μ L of above-mentioned enzymatic hydrolysate carries out efficient liquid phase (HPLC) analysis, and gel column used is Superdex
Peptide10/300GL (GE), it is 0.4mL/min that mobile phase is 0.2M ammonium hydrogen carbonate, flow velocity；Testing conditions are UV232nm.
As a result as shown in figure 5, in compound polysaccharide degrading enzyme preparation and the product of hyaluronic acid, the appearance of principal product
Time is about 42min, and prompting is undersaturated hyaluronic acid disaccharide.Can't detect with characteristic absorption in the product of matched group
Composition.Therefore, the compound polysaccharide degrading enzyme preparation for being prepared with streptomycete (Streptomyces sp.) CB16 bacterial strain, can be used for
Production hyaluronic acid disaccharide.
List of references：
[1] Korean monarch. the research [D] of ocean colloidal sol antibacterial Flammeovirga sp.MY04 agarase (being). Qingdao:In
State ocean University Ph.D. Dissertation, 2012.
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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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[4] Korean monarch, Zhao Shuai, Liu Huihui etc. the separation of one plant of polysaccharide degradation bacteria, identification and agarose degradation capability [J].
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[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
forest.International Journal of Systematic and Evolutionary Microbiology,
2013,63:4754-4759.
SEQUENCE LISTING
<110>Wutong Aroma Chemicals Co., Ltd.
<120>One streptomycete belongs to polysaccharide degradation bacteria 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 (10)
1. a streptomycete (Streptomyces sp.) CB16 bacterial strain, on April 11st, 2016 is 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
Thing institute, deposit number：CGMCC No.12351.
2. the cultural method of streptomycete described in claim 1 (Streptomyces sp.) CB16 bacterial strain, it is characterised in that step
As follows：
(1) take streptomycete (Streptomyces sp.) CB16 bacterial strain to line on solid medium, 25～30 DEG C are inverted activation
Culture 3～7 days, is obtained bacterial strain after activation；
(2) after taking the obtained activation of step (1), inoculation is to fluid medium, temperature be 25～30 DEG C, revolution be
Under conditions of～220 revs/min, shaking table culture 3～7 days, prepared seed liquor；
(3) seed liquor obtained in step (2) is taken, is inoculated in fluid medium by 1～10% percent by volume, in temperature is
25～30 DEG C, revolution be 150～220 revs/min under conditions of, amplification culture 3～7 days, prepared streptomycete (Streptomyces
Sp.) CB16 bacterium solution.
3. cultural method as claimed in claim 2, it is characterised in that the solid medium in step (1), per liter of component
As follows：
Tryptone 17g, phytone 3g, Sodium Chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, agar 20g, water is fixed
Hold to 1L, pH7.2.
4. cultural method as claimed in claim 2, it is characterised in that the fluid medium in step (2) and (3), per
Rise component as follows：
1～20g of carbon source, NaCl 0.5g, K2HPO40.5g、KNO31g、FeSO40.01g、MgSO4·7H2O 0.5g, water is fixed
Hold to 1L, pH value 6.5～7.5.
5. cultural method as claimed in claim 4, it is characterised in that described carbon source is selected from：Cellulose, carboxymethyl cellulose
Element, xylan, mannan, chitin, shitosan, starch, pectin, sodium alginate, hyaluronic acid, chondroitin sulfate A, sulphuric acid
One of chrondroitin C, chondroitin sulfate E or xanthan gum.
6. cultural method as claimed in claim 2, it is characterised in that per liter of component of the fluid medium is as follows：
Tryptone 17g, phytone 3g, Sodium Chloride 5g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, water is settled to 1L, pH
7.2.
7. application of streptomycete described in claim 1 (Streptomyces sp.) the CB16 bacterial strain in compound polysaccharide of degrading.
8. a kind of preparation method of compound polysaccharide degrading enzyme preparation, it is characterised in that step is as follows：
I () takes above-mentioned streptomycete (Streptomyces sp.) CB16 bacterium solution, be inoculated in fermentation by 1～10% percent by volume
In culture medium, temperature be 25～30 DEG C, rotating speed be 150～220 revs/min under conditions of, amplification culture 3～7 days, prepared chain
Mycete CB16 bacterial strain fermentation liquor；
(ii) fermentation liquid of streptomycete CB16 bacterial strain obtained in step (i) taken, solid-liquid separation, liquid is taken, ammonium sulfate is added, makes to satisfy
It is gained precipitation 80%, under 80% ammonium sulfate saturation of collected after centrifugation with degree, and the TGE buffer weight with 20～50 times of volumes
Outstanding precipitation, dialysis removes ammonium sulfate, and compound polysaccharide degrading enzyme preparation is obtained.
9. preparation method as claimed in claim 8, it is characterised in that the fermentation medium in the step (i) be
Number culture medium, per liter of component is as follows：
Soluble starch 20g, glucose 20g, beef extract 3g, yeast extract 10g, CaCO30.5g, NaCl 0.5g,
MgSO4·7H2O 0.5g, water is settled to 1L, pH7.0.
10. preparation method as claimed in claim 8, it is characterised in that in step (ii), solid-liquid separation is centrifugation, bar
Part is：12000 × g, 4 DEG C of 5～20min of centrifugation；
Preferably, in described step (ii), it is centrifuged and is：15,000 × g, 4 DEG C of 5～20min of centrifugation；
Preferably, in described 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；
Preferably, in described step (ii), dialysis is the bag filter using molecular retention amount 10,000Da, is stirred dialysis.
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