CN106635920B

CN106635920B - Marine alternans for high yield of fucosidase and application thereof

Info

Publication number: CN106635920B
Application number: CN201710030991.1A
Authority: CN
Inventors: 刘伟治; 于广利; 张柯柯; 律倩倩; 赵小亮
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2017-01-17
Filing date: 2017-01-17
Publication date: 2020-07-03
Anticipated expiration: 2037-01-17
Also published as: CN106635920A

Abstract

The invention provides a high-yield fucoidin degrading enzyme strain, and the preservation number of the strain is CGMCC No. 13477. The microbial strain is used for producing fucoidin degrading enzyme, and the fucoidin degrading enzyme produced by the strain screened by the invention can degrade fucoidin sulfate to produce oligomeric fucoidin sulfate with potential biological activity. The microorganism has the excellent characteristics of short fermentation period, high enzyme activity, intracellular enzyme activity of most of the activities and the like, is favorable for collecting and storing samples, and lays a foundation for subsequent industrial application.

Description

Marine alternans for high yield of fucosidase and application thereof

Technical Field

The invention belongs to the technical field of marine microorganism screening, and particularly relates to marine alternan pseudomonas for high yield of fucosidase and application thereof.

Background

Fucoidan (Fucoidan) is a water-soluble polysaccharide contained in the cell wall of brown algae, and mainly comprises L-fucose and different sulfated L-fucose. In addition, the polysaccharide also contains a certain amount of D-galactose, D-mannose and D-glucuronic acid, and a small amount of D-xylose and L-arabinose, and is an acidic polysaccharide compound with quite complex chemical composition and structure and high molecular weight. Researches show that the polysaccharide has various biological activities of resisting oxidation, reducing blood fat, resisting coagulation, resisting virus and tumor, enhancing immunity of organisms and the like, and the physiological activity of the polysaccharide has important relation with the molecular weight of the polysaccharide, the content of sulfate radical and the substitution position of the sulfate radical. Therefore, the structure-activity relationship of the polysaccharide is deeply studied, and the polysaccharide is degraded directionally depending on the availability of specific degrading enzymes. The polysaccharide degrading enzyme is used to obtain a series of oligosaccharides, and the action targets of the oligosaccharides are clarified from the oligosaccharide structure level, which is an important way for developing innovative drugs and a series of functional oligosaccharide products.

In order to achieve the above object, the acquisition of the fucoidan-degrading enzyme is crucial. However, since the enzymes have problems such as low activity and commercial production of the enzymes has not been achieved, screening of microbial strains producing enzymes with high activity is still an important issue.

Disclosure of Invention

The invention aims to provide a strain for highly producing fucoidin degrading enzyme, thereby promoting the preparation and application of fucoidin and making up the defects of the prior art.

The first objective of the invention is to provide an alternating pseudomonad (Pseudoalteromonas sp.) OU03 strain for producing fucosidase, which is preserved in China general microbiological culture collection center of culture Collection of microorganisms No. 3 of West Lu No.1, North Cheng, Haisheng district, Beijing, with the preservation number of CGMCC No.13477 and the preservation date of 2016, 12 and 22 days.

The microbial strain is used for producing the fucoidin degrading enzyme;

the invention also provides a culture medium for inducing the marine microorganisms to express and produce the fucoidin degradation enzyme, wherein the basic carbon source of the culture medium is fucoidin sulfate; most of enzyme activity induced by the strain is concentrated in cells, which is beneficial to the enrichment of subsequent active enzymes.

As a concrete description of the examples, the formulation of the culture medium is as follows: 2g/L fucoidan sulfate and 1g/L peptone, and adjusting pH to 7.6-7.8 after being prepared with seawater.

In another aspect of the present invention, there is provided a method for preparing a fucoidan-degrading enzyme by fermenting using the selected strain.

The fucoidan degrading enzyme produced by the strain screened by the invention can degrade fucoidan sulfate and produce oligomeric fucose sulfate with potential biological activity. The microorganism has short fermentation period and high enzyme activity, most of the activity is intracellular enzyme activity, and the microorganism is beneficial to the collection and storage of subsequent samples and lays a foundation for the industrial application of the microorganism.

Drawings

FIG. 1: a growth curve chart of the OU03 strain,

FIG. 2: graph of fucosidase degradation activity at different positions of OU03 after induction (DNS method),

FIG. 3: C-PAGE analysis of fucosidase produced by induced culture OU03, wherein A is an intracellular enzyme; b is fermentation liquor.

Detailed Description

The invention screens and obtains a strain of fucosidase with high yield and specificity from symbiotic microorganisms from digestive gland sources of marine invertebrates, and most of the activities are intracellular enzyme activities, thereby being beneficial to the subsequent large-scale preparation of fucooligosaccharide sulfate by using the enzyme produced by the strain.

The present invention will now be described in detail with reference to examples

Example 1: screening and identification of microorganisms

1. Screening of microorganisms

Dissecting live scallop, sea urchin and abalone under aseptic condition, separating digestive glands of the live scallop, sea urchin and abalone, adding aseptic normal saline into the extracted marine organism digestive gland and rotten kelp, and extracting to obtain tissue suspension of the scallop, sea urchin, abalone digestive glands and rotten kelp. Respectively taking 1-2mL of the tissue suspension, inoculating into fucoidan sulfate liquid selection culture medium A, and culturing in a shaking incubator at 25 ℃ and 150 rpm. After 24h, the fucoidan enzyme activity of each strain is measured, and 200 mu L of the enzyme-activity bacterium liquid is selected and coated on the fucoidan sulfate solid selection medium B. The plates were incubated in an incubator at 25 ℃. Colonies with good growth status were picked from the plate and inoculated into fucoidan sulfate liquid selection medium D, followed by shake culture in a shaker. And measuring the fucoidan enzyme activity of each strain after 24h, and selecting the strain with high enzyme activity for subsequent screening.

The formula of the culture medium is as follows:

liquid selection medium a: 2g/L fucoidin, 1g/L peptone and 7.6-7.8 pH value, which are prepared from membrane-passing seawater.

Solid selection medium B: on the basis of the liquid medium D, agar was supplemented. Liquid fermentation medium: fucoidan 1g/L, peptone5g/L, 2g/L yeast powder, K₂HPO4 0.2g/L，MgSO₄0.05g/L, prepared by passing through membrane seawater, and has pH of 7.6-7.8.

And analyzing the products of the strains with high enzyme activity for degrading the fucoidan sulfate, and finally screening the strains with highest fucoidan enzyme activity and high enzymolysis product unicity.

The microorganism is preserved in the China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.13477, and the preservation date is 2016, 12 and 22 days.

2. Amplification, sequencing and analysis of 16S rRNA Gene of Strain OU03

Extracting the genome of the screened high-activity strain by using a bacterial genome extraction kit (Tiangen), and amplifying the 16S rRNA gene of the strain by using the extracted genome as a template. The following primers were used:

27F:5’-AGAGTTTGATCCTGGCTCAG-3’

1492R:5’-GGTTACCTTGTTACGACTT-3’

the PCR reaction was performed under the following conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 68 ℃ for 90s for 35 cycles, followed by extension at 68 ℃ for 10 min. The obtained RCR product was purified by gel recovery kit (Omega), ligated with pMD19-T vector (Takara), E.coli Top 10 competent cells were transformed, and positive clones were selected for DNA sequencing.

Phylogenetic tree analysis of strain 16S rRNA showed its closest relationship to Pseudomonas.

Example 2: production of enzymes by fermentation of microorganisms

1. Fermentation culture

And (3) carrying out fermentation culture on the screened high-activity strain to obtain a growth curve of the microorganism in the fucoidin sulfate culture medium.

Inoculating the seed liquid into fucoidan sulfate liquid fermentation medium according to the inoculation amount of 1-3%, culturing in a shaking table at 25-37 deg.C and 150rpm, collecting the fermentation liquid at different time, taking the culture medium without inoculated strain as blank control, measuring the absorbance at 600nm of the strain liquid, and drawing the growth curve of the strain (figure 1). As a result, it was found that the bacterium is useful for fucoidanIn sulfate medium, a plateau of growth is reached within about 18-24 hours, at which time the OD is₆₀₀About 5.3. This is the optimum time for collecting the bacterial cells in the future.

2. Extraction and Activity measurement of enzymes at different sites (all performed at 4 ℃ C.)

① extracellular enzyme the fermentation broth was centrifuged at 20,000g for 30min and the centrifuged supernatant was dialyzed into 50mM Tris-HClpH7.5, 200mM NaCl buffer.

② intracellular enzyme, the thallus after the fermentation broth centrifugation in the previous step is resuspended in 50mM Tris-HClpH7.5, 200mM NaCl buffer solution, then is subjected to ultrasonication (ultrasonication for 3s, interval for 5s, total time for 15min), the liquid after ultrasonication is centrifuged for 20min under the condition of 20,000g, and the centrifuged supernatant is the extracted intracellular enzyme.

And (3) performing activity determination on the extracted enzyme component by adopting a DNS (domain name system) reducing sugar detection method. Respectively using glucose and fucoidin sulfate as carbon sources to induce the OU03 strain to produce enzyme, respectively adding 100 μ L of the extracted intracellular enzyme and extracellular enzyme (fermentation liquid freeze-dried powder) into 200 μ L of 0.2% fucoidin sulfate solution, and reacting at 25 deg.C for about 12h, wherein the buffer solution for enzymolysis is 50mM Tris-HCl pH7.5200mM NaCl. After the reaction, 200. mu.L of the reaction solution was added to DNS of equal volume, heated at 100 ℃ for 5min for color development, centrifuged at 12000rpm for 5min, and 200. mu.L of the supernatant was collected to detect the absorbance at 520nm, the results are shown in FIG. 2. The measurement results show that the fucosidase of the strain is generated by the induction of fucoidan sulfate, and the fucosidase is more in cells.

3. Electrophoretic analysis of enzymatic products

Degrading fucoidan sulfate with crude enzyme extracted from intracellular and extracellular parts, adding inactivated enzyme with the same volume as the crude enzyme, and analyzing the degradation product by PAGE method after the reaction is finished. And mixing the degraded sample with a loading buffer solution (10% of sucrose and 0.01% of phenol red), selecting 6% concentrated gel and 27% separation gel, carrying out electrophoresis at 200V for 2 hours by using an electrode buffer solution of 50mM Tris-HCl 2mM EDTA (Methedita) pH8.7, and dyeing by combining alisnew blue dye with silver dye after the electrophoresis is finished.

The results are shown in FIG. 3, and the enzymatic products showed distinct bands in the electrophoretogram, indicating that there was oligosaccharide production, indicating that there was a distinct fucosidase activity in the intracellular extracts of this strain.

4. Mass spectrometric analysis of enzymatic products

Preparation of enzymatic hydrolysate

The buffer solution for enzymolysis reaction is 50mM Tris-HCl (pH7.5200mM NaCl), and 15mL of the extracted intracellular enzyme and 20mL of the extracted extracellular enzyme (lyophilized powder of fermentation broth) are respectively added into 35mL of 0.2% fucoidan sulfate aqueous solution, and reacted for 48h at 25 ℃. After the reaction is finished, heating at 100 ℃ for 10min to inactivate the enzyme, centrifuging at 12000rpm for 20min at room temperature, and collecting supernatant, namely an enzymolysis product.

Desalting the enzymolysis mixed solution through a G10 gel column, collecting sugar components, concentrating eluent, freeze-drying, dissolving with appropriate amount of acetonitrile/water, and performing mass spectrometry. The sample molecular weight is then determined using mass spectrometry. The determination result proves that the enzymolysis product contains components with the molecular weight of 324Da, 550Da, 696Da and 776Da, and the calculation shows that the components correspond to 1 Fuc2S, 2 Fuc3S, 3 Fuc3S and 3 Fuc4S respectively, which further proves that the enzyme secreted by the microorganism has the activity of degrading fucoidan sulfate.

Claims

1. A strain of marine alteromonas is characterized in that the strain of marine alteromonas (Pseudomonas sp.) is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.13477 and the preservation date of 2016, 12 and 22 days of No. 3 of China Committee for culture Collection of microorganisms in Xilu No.1 of Beijing Haizi lake.

2. Use of the alteromonas ocean strain of claim 1 for the production of a fucoidan-degrading enzyme.

3. A method for producing a fucoidan-degrading enzyme by fermentation using the strain of Pseudomonas marini according to claim 1.