CN106635920A - High-yield fucoidanase ocean alternating pseudomonas and application thereof - Google Patents

High-yield fucoidanase ocean alternating pseudomonas and application thereof Download PDF

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CN106635920A
CN106635920A CN201710030991.1A CN201710030991A CN106635920A CN 106635920 A CN106635920 A CN 106635920A CN 201710030991 A CN201710030991 A CN 201710030991A CN 106635920 A CN106635920 A CN 106635920A
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fucoidin
bacterial strain
ocean
culture medium
fucoidanase
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CN106635920B (en
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刘伟治
于广利
张柯柯
律倩倩
赵小亮
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Ocean University of China
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)

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Abstract

The invention provides a high-yield fucoidanase degrading enzyme strain. The preservation number of the strain is CGMCC No.13477. The microbiological strain is used for producing fucoidanase, the fucoidanase produced by the screened strain can degrade fucoidan, and oligomerization fucoidanase with potential biological activity is generated. The microorganism has the advantages that the fermentation period is short, the enzyme activity is high, and most activity is intracellular activity, collection and preservation of samples are promoted, and a foundation is laid for subsequent industrial application.

Description

A kind of ocean pseudoalteromonas of high yield fucoidanase and its application
Technical field
The invention belongs to marine microorganism triage techniques field, and in particular to a kind of ocean of high yield fucoidanase replaces Pseudomonad and its application.
Background technology
Fucoidin sulfuric ester (Fucoidan) is a kind of water-soluble polysaccharide that brown alga cell membrane contains, mainly by L- rock algaes The L-fucose composition of sugar and different Sulfations.Additionally, also containing a certain amount of D- galactolipins, D-MANNOSE, D-Glucose Aldehydic acid, a small amount of D- wood sugars and L-arabinose, are a kind of acidity of all sufficiently complex HMW of chemical composition and structure Compound of polysaccharide.Research finds that there is such polysaccharide anti-oxidant, reducing blood lipid, anticoagulation, antiviral, antitumor, enhancing body to exempt from The various BAs such as epidemic disease power, and its physiologically active has important with polysaccharide molecular weight, the content of sulfate radical and its position of substitution Relation.Therefore, the saccharoidal structure-activity relationship is furtherd investigate, it is necessary to which polysaccharide is degraded, and this is saccharoidal fixed The acquisition of specific digestive enzyme is depended on to degraded.Series of oligosaccharides is obtained using the polysaccharide degrading enzyme, and is explained from oligosaccharide structure aspect Bright its action target spot, this is the important channel of original new drug and the exploitation of series function oligosaccharide preparation.
In order to realize above-mentioned target, the acquisition of fucoidin digestive enzyme is most important.But due to the fermentoid exist vigor compared with Low problem, commercially producing for the enzyme is not carried out all the time, therefore it is still a weight to screen high vigor microbes producing cellulase bacterial strain The problem wanted.
The content of the invention
It is an object of the invention to provide a plant height produces fucoidin digestive enzyme bacterial strain, so as to promote the system of fucose Standby and application, to make up the deficiencies in the prior art.
First purpose of the present invention is the pseudoalteromonas for providing one plant of product fucoidanase (Pseudoalteromonas sp.) OU03 strains, the microbial strains are stored in positioned at the institute of Haidian District, Beijing City North Star West Road 1 The China Committee for Culture Collection of Microorganisms's common micro-organisms center of No. 3, deposit number be CGMCC No.13477, preservation Date is on December 22nd, 2016.
The microbial strains of the present invention are used to produce fucoidin digestive enzyme；
Present invention also offers a kind of culture for inducing above-mentioned marine microorganism expression to produce fucoidin digestive enzyme Base, the basic carbon source of culture medium is fucoidin sulfuric ester；Most of enzyme activity that the bacterial strain inducing is produced all concentrates on intracellular, has Beneficial to the enrichment of consequent activities enzyme.
Used as the concrete record of embodiment, the formula of described culture medium is as follows：Fucoidin sulfuric ester 2g/L, peptone 1g/L, it is 7.6-7.8 that pH is adjusted after being prepared with seawater.
Another aspect of the invention is to provide a kind of method for preparing fucoidin digestive enzyme, is entered using the bacterial strain of screening Row fermentation prepares fucoidin digestive enzyme.
Fucoidin digestive enzyme produced by the bacterial strain of present invention screening can degrade fucoidin sulfuric ester, and generation has The oligomerization fucose sulfate of potential source biomolecule activity.The microorganism has fermentation period short, and enzymatic activity is high, and most activity are Intracellular enzyme activity, is conducive to the collection and preservation of subsequent sample, is that its industrial applications is laid a good foundation.
Description of the drawings
Fig. 1：The growth curve chart of OU03 bacterium,
Fig. 2：OU03 different parts fucoidanase degrading activity figure (DNS methods) after induction,
Fig. 3：The C-PAGE analysis charts of the fucoidanase that Fiber differentiation OU03 is produced, wherein A is endocellular enzyme；B is fermentation Liquid.
Specific embodiment
It is single-minded that present invention screening from the symbiotic microorganism in oceanic invertebrate glandula digestive source obtains plant height product Property fucoidanase bacterial strain, and most activity is intracellular enzyme activity, is conducive to the enzyme that the later use bacterial strain is produced to enter professional etiquette Modelling prepares fucose sulfuric ester.
The present invention is described in detail with reference to embodiment
Embodiment 1：The screening and identification of microorganism
1st, the screening of microorganism
Aseptically scallop living, sea urchin and abalone are dissected, their glandula digestive is isolated, in the sea extracted SPSS is added in foreign bio-digestion gland and rotten sea-tangle, scallop, sea urchin, abalone glandula digestive and rotten are obtained after extraction The tissue suspension of sea-tangle.Take tissue suspension 1-2mL respectively to access in fucoidin sulfuric ester liquid selective medium A, and Cultivated in 25 DEG C, 150rpm shaken cultivation casees.The product fucoidin enzymatic activity of each bacterial strain is measured after 24h, is selected The bacterium solution 100-200 μ L for selecting enzymatic activity are applied on fucoidin sulfuric ester solid selection medium B.Culture plate is placed in into 25 Cultivated in DEG C incubator.The good colony inoculation of picking growth conditions is in fucoidin sulfuric ester liquid selective from flat board In culture medium D, the shaken cultivation in shaking table.The product fucoidin enzymatic activity of each bacterial strain is measured after 24h, selects enzymatic activity High bacterial strain is subsequently screened.
Culture medium prescription is as follows：
Liquid selective medium A：Fucoidin 2g/L, peptone 1g/L, cross the configuration of film seawater, pH 7.6-7.8.
Solid selection medium B：On the basis of liquid medium within D, agar is added.Liquid fermentation medium：Rock algae is more Sugared 1g/L, peptone 5g/L, dusty yeast 2g/L, K2HPO4 0.2g/L, MgSO40.05g/L, crosses the configuration of film seawater, pH 7.6- 7.8。
The product of the degraded fucoidin sulfuric ester of the bacterial strain high to enzymatic activity is analyzed, and finally filters out product rock algae many Anase activity highest, the high bacterial strain of enzymolysis product unicity.
The microorganism is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.13477, preservation date is on December 22nd, 2016.
2nd, amplification, the determination and analysis of sequence of the 16S rRNA genes of bacterial strain OU03
The genome of the high activity bacterial strain for having screened is extracted using bacterial genomes extracts kit (Tiangen), with This is template, expands the 16S rRNA genes of the bacterial strain.Using following primer：
27F:5’-AGAGTTTGATCCTGGCTCAG-3’
1492R:5’-GGTTACCTTGTTACGACTT-3’
PCR reactions are carried out according to following condition：95 DEG C of denaturations 3min；95 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 68 DEG C are prolonged Stretching 90s carries out altogether 35 circulations, afterwards 68 DEG C of extension 10min.RCR products Jing glue reclaim kits (Omega) purifying of acquisition Afterwards, it is connected with pMD19-T carriers (Takara), the competent cells of Transformed E .coli Top 10, picking positive colony carries out DNA Sequencing.
The phylogenetic analysis of bacterial strain 16S rRNA show that it is nearest with the affiliation of Pseudoalteromonassp.
Embodiment 2：The enzymatic production of microorganism
1st, fermented and cultured
The high activity bacterial strain that screening is obtained is carried out into fermented and cultured, the microorganism is obtained in fucoidin sulfuric ester culture medium In growth curve.
By the inoculum concentration of 1-3%, seed liquor is accessed in fucoidin sulfuric ester liquid fermentation medium, 25-37 DEG C, Cultivate in 150rpm shaking tables, in different time zymotic fluid is collected, not connect the culture medium of bacterium as blank, determine bacterium solution Light absorption value at 600nm, draws the growth curve (Fig. 1) of the bacterium.As a result the bacterium is found in fucoidin sulfuric ester culture medium, greatly The plateau of growth is about reached in 18-24 hours, now OD600=5.3 or so.This as microorganism collection in future it is optimal when Between.
2nd, the extraction and determination of activity (operate is carried out below under the conditions of 4 DEG C) of different parts enzyme
1. ectoenzyme：Zymotic fluid is centrifuged under the conditions of 20,000g 30min.Supernatant after centrifugation is carried out dialysis In 50mMTris-HClpH7.5,200mMNaCl buffer solution.
2. endocellular enzyme：Thalline 50mMTris-HClpH7.5 in previous step after zymotic fluid centrifugation, 200mMNaCl buffering Liquid is resuspended, and ultrasonication (ultrasonic 3s is spaced 5s, total time 15min) is carried out afterwards, and the liquid after ultrasonication is 20,000g Under the conditions of be centrifuged 20min, collect the supernatant after centrifugation and be the endocellular enzyme for extracting.
Detect that the method for reduced sugar carries out determination of activity to the enzyme component for extracting using DNS.Respectively with glucose and rock algae Polysaccharide sulfate is that carbon source induces OU03 strain enzyme-producings, by the endocellular enzyme for extracting and each 100 μ L of ectoenzyme (zymotic fluid freeze-dried powder) In being separately added into the fucoidin sulphate solution of 200 μ L 0.2%, 25 DEG C are reacted 12h or so, and the buffer solution of enzyme digestion reaction is 50mMTris-HCl pH7.5 200mMNaCl.After reaction terminates, take 200 μ L reactant liquors and add equal-volume DNS, 100 DEG C of heating 5min develops the color, 12000rpm centrifugation 5min, takes the light absorption value at the μ L of supernatant 200 detection 520nm, as a result as shown in Figure 2.Determine knot Fruit shows that the fucoidanase of the bacterium is produced by the induction of fucoidin sulfuric ester, and cell is located at fucoidanase more It is interior.
3rd, the electrophoretic analysis of enzymolysis product
Fucoidin sulfuric ester is degraded with the intracellular and extracellular thick enzyme extracted, to add same volume inactivator to make For control, react and after terminating catabolite is analyzed using PAGE methods.Sample and sample-loading buffer (10% sugarcane after degraded Sugar, 0.01% is phenol red) mixing, from 6% concentration glue and 27% separation gel, electrode buffer is 50mMTris-HCl 2mM EDTA pH8.7,200V electrophoresis 2 hours, electrophoresis is dyeed with Alcian blue dyes after terminating with reference to silver staining.
As a result as shown in figure 3, enzymolysis product has obvious band to occur in electrophoretogram, illustrated that compound sugar is produced, table It is bright to have obvious fucoidin enzymatic activity in the intracellular extract of the bacterium.
4th, the mass spectral analysis of enzymolysis product
The preparation of enzymolysis product
The buffer solution of enzyme digestion reaction is 50mMTris-HCl (pH7.5200mMNaCl), by the endocellular enzyme 15mL for extracting and born of the same parents Exoenzyme 20mL (zymotic fluid freeze-dried powder) is separately added into the fucoidin sulfuric acid aqueous solution of ester of 35mL0.2%, 25 DEG C of reaction 48h. After reaction terminates, 100 DEG C of heating 10min are inactivated to enzyme, and 12000rpm room temperatures centrifugation 20min collects supernatant and is enzyme Solution product.
Enzymolysis mixed liquor collects saccharic composition through G10 gel column desalinations, and appropriate second is used in freeze-drying after eluent concentration Mass spectral analysis is carried out after nitrile/water dissolves.Then mass spectrometric measurement determination sample molecular weight is utilized.Measurement result is proved in enzymolysis product Containing molecular weight be 324Da, the component of 550Da, 696Da and 776Da, show after calculating its it is corresponding be respectively 1 Fuc2S, 2 Fuc3S, 3 Fuc3S and 3 Fuc4S, the enzyme that this further demonstrates the microorganism secretion has degraded rock algae The activity of polysaccharide sulfate.

Claims (6)

1. one plant of ocean pseudoalteromonas belongs to bacterial strain, it is characterised in that described ocean pseudoalteromonas belong to bacterial strain preservation In China Committee for Culture Collection of Microorganisms's common micro-organisms of the institute 3 of Haidian District, Beijing City North Star West Road 1 The heart, deposit number is CGMCC No.13477, and preservation date is on December 22nd, 2016.
2. the ocean pseudoalteromonas described in claim 1 belong to application of the bacterial strain in production fucoidin digestive enzyme.
3. it is a kind of to produce fucoidin digestive enzyme for inducing the ocean pseudoalteromonas described in claim 1 to belong to bacterial strain expression Culture medium, it is characterised in that the basic carbon source of described culture medium select fucoidin sulfuric ester.
4. culture medium as claimed in claim 4, it is characterised in that the formula of described culture medium is as follows：Fucoidin sulfuric acid Ester 2g/L, peptone 1g/L, it is 7.6-7.8 that pH is adjusted after being prepared with seawater.
5. a kind of method for preparing fucoidin digestive enzyme, it is characterised in that described method is that usage right is required described in 1 Ocean pseudoalteromonas belong to bacterial strain to carry out fermentation to prepare fucoidin digestive enzyme.
6. method as claimed in claim 5, it is characterised in that described method is to replace the ocean described in claim 1 Pseudomonas strain is inoculated into claim 3 or the culture medium described in claim 4 and is fermented.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109055460A (en) * 2018-09-13 2018-12-21 青岛高迈生物科技有限公司 A kind of low weight molecular fucoidan and its preparing the application in cosmetics
CN109593672A (en) * 2018-12-13 2019-04-09 山东大学 One plant of Pseudoalteromonas polysaccharide degradation bacteria and its cultural method and application
CN112695029A (en) * 2020-12-25 2021-04-23 北京雷力海洋生物新产业股份有限公司 Genetic engineering strain for high yield of fucoidin degrading enzyme and preparation method thereof
CN112708575A (en) * 2020-12-09 2021-04-27 汪秋宽 Chinese ocean micro-bacterium and application thereof in preparing fucoidan sulfate degrading enzyme
CN115088719A (en) * 2022-07-20 2022-09-23 北京雷力海洋生物新产业股份有限公司 Application of fucooligosaccharide

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109055460A (en) * 2018-09-13 2018-12-21 青岛高迈生物科技有限公司 A kind of low weight molecular fucoidan and its preparing the application in cosmetics
CN109055460B (en) * 2018-09-13 2021-06-29 青岛创通生物科技有限公司 Low molecular weight fucoidin and application thereof in preparing cosmetics
CN109593672A (en) * 2018-12-13 2019-04-09 山东大学 One plant of Pseudoalteromonas polysaccharide degradation bacteria and its cultural method and application
CN112708575A (en) * 2020-12-09 2021-04-27 汪秋宽 Chinese ocean micro-bacterium and application thereof in preparing fucoidan sulfate degrading enzyme
CN112708575B (en) * 2020-12-09 2024-02-02 汪秋宽 Oceanic Chinese microbe and its application in preparing fucoidan sulfate degrading enzyme
CN112695029A (en) * 2020-12-25 2021-04-23 北京雷力海洋生物新产业股份有限公司 Genetic engineering strain for high yield of fucoidin degrading enzyme and preparation method thereof
CN112695029B (en) * 2020-12-25 2022-05-31 北京雷力海洋生物新产业股份有限公司 Genetic engineering strain for high yield of fucoidin degrading enzyme and preparation method thereof
CN115088719A (en) * 2022-07-20 2022-09-23 北京雷力海洋生物新产业股份有限公司 Application of fucooligosaccharide
CN115088719B (en) * 2022-07-20 2024-05-24 北京雷力海洋生物新产业股份有限公司 Use of fucoidin

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