CN103911315B - Bacterial strain and the application thereof of algin catenase are produced in one strain - Google Patents
Bacterial strain and the application thereof of algin catenase are produced in one strain Download PDFInfo
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- CN103911315B CN103911315B CN201410008138.6A CN201410008138A CN103911315B CN 103911315 B CN103911315 B CN 103911315B CN 201410008138 A CN201410008138 A CN 201410008138A CN 103911315 B CN103911315 B CN 103911315B
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Abstract
The invention discloses bacterial strain and application thereof that algin catenase is produced in a strain, it is characterized in that, does is described bacterial strain at the preserving number at China Microbiological bacterial strain preservation management committee's common micro-organisms center: CGMCC? No.8312, preservation date is: on October 9th, 2013, described strain classification called after: ocean is addicted to Halomonas SH-19 (Halomonas? elongata), the 16S of described bacterial strain? does is the polynucleotide sequence of rDNA SEQ? ID? polynucleotide sequence shown in No.1.Bacterial strain SH-19 of the present invention can high expression algin catenase, can be applicable to High-efficient Production algin catenase, the fermention medium Easy dosing of bacterial strain SH-19 of the present invention, it is all very general easily acquisition in laboratory, ferment effect is remarkable, the production cost of algin catenase is reduced greatly, can be applicable to scale operation.
Description
Technical field
The invention belongs to field of marine biotechnology, be specifically related to a kind of bacterial strain and application thereof of producing algin catenase.
Background technology
Algae is described as " energy rising star ", and compared with other terrestrial plants, algae can obtain higher biomass under unit cultured area, and also easier commerial growing and mechanize are gathered.Therefore algae becomes modern bioenergy material gradually, and output, production capacity and total cost all have significant advantage.
Containing a large amount of algins in brown alga, the complex structure of phaeophyta itself, its cell walls moiety algin is difficult to degraded, and various polysaccharide is tightly combined into an organic whole, and complicated structure causes the difficulty that algae utilizes.This adds the cost utilizing algae bio Energy production virtually.In addition, can prepare the oligosaccharides of different lengths after algin is cleaved, this oligosaccharides has multiple biological activity, has potential industrial value.Therefore, the cell wall polysaccharides lyase being devoted to find economical and efficient becomes one of study hotspot.
Current, conventional lyase exists that hydrolysis efficiency is low, the problem such as yield poorly.So, by existing technique means, the Microbial resources in nature and animal intestinal are excavated, with expect to obtain a kind of in degraded algin on the outstanding bacterial strain of ability.For degraded strategy in follow-up employing systems approach research nature, for developing the theory and technology basis that effective brown alga processing technology provides important.
Summary of the invention
One of the object of the invention is to provide a strain to produce the bacterial strain of algin catenase, and bacterial strain of the present invention screens first to obtain from sea urchin enteron aisle abrasive material, can synthesize algin catenase efficiently under optimum condition.
Two of the object of the invention is to provide a strain to produce the application of the bacterial strain of algin catenase, be about to the preparation for algin catenase of the HalomonaselongataSH-19 bacterial strain that filters out, thus produce algin catenase efficiently, to make up the deficiency of existing algin hydrolysis effect difference.
For achieving the above object, the technical solution used in the present invention is:
The bacterial strain of algin catenase is produced in one strain, it is characterized in that, described bacterial strain at the preserving number at China Microbiological bacterial strain preservation management committee's common micro-organisms center is: CGMCCNo.8312, preservation date is: on October 9th, 2013, described strain classification called after: ocean is addicted to Halomonas SH-19 (Halomonaselongata).
Preferably, described bacterial strain screening is from sea urchin enteron aisle.
Produce a method for algin catenase, it is characterized in that, apply bacterial strain as claimed in claim 1 or 2 and produce algin catenase.
Preferably, described method specifically comprises the following steps: in the algin catenase production phase, the carbon source of producing fermention medium is sodium alginate, and sodium alginate is 1 ~ 3% producing the mass percent concentration in fermention medium, and nitrogenous source is ammonium sulfate or ammonium chloride.
Preferably, described method specifically comprises the following steps:
1) seed liquor preparatory phase: utilize each seed liquor of bacterial strain system as claimed in claim 1 or 2;
2) in the culture propagation stage: after described seed liquor is inoculated in one time fermentation substratum, described one time fermentation substratum continuously ferments under pH is 4.5 ~ 9.5 and temperature is the condition of 26 ~ 30 DEG C;
3) the algin catenase production phase: add sodium alginate and sodium-chlor in described one time fermentation substratum, be mixed with production fermention medium, sodium alginate in production fermention medium and the concentration of sodium-chlor are respectively 1 ~ 3%, the pH of described production fermention medium is 4.5 ~ 9.5, continuously ferments 8 ~ 14 days under 26 ~ 30 DEG C of conditions.
Preferably, described one time fermentation medium component is: carbon source, nitrogenous source and Chen Haishui, and wherein, carbon source is extractum carnis or/yeast extractive substance, and described nitrogenous source is ammonium sulfate or ammonium chloride.
Preferably, in described one time fermentation substratum, the mass percent concentration of extractum carnis and yeast extractive substance is respectively 0.1 ~ 1.5%, and the mass percent concentration of ammonium sulfate or ammonium chloride is 0.1-2%.
Preferably, described step 3) in, sodium alginate is 1% producing the mass percent concentration in fermention medium, and sodium-chlor is 1% producing the mass percent concentration in fermention medium.
Preferably, described one time fermentation substratum and described production fermention medium pH are 7.5 ~ 8.5.
Preferably, described one time fermentation substratum and described production fermention medium pH are 7.5.
Beneficial effect of the present invention bacterial strain SH-19 of the present invention screens first from sea urchin enteron aisle abrasive material, can high expression algin catenase under relatively suitable growth conditions, can be applicable to High-efficient Production algin catenase, the present invention also explores the living condition of described bacterial strain SH-19 high expression algin catenase, obtain described bacterial strain and express optimum carbon source in the fermentation culture of algin catenase, optimum nitrogenous source, the living conditions such as optimum sodium chloride concentration, the fermention medium Easy dosing of bacterial strain SH-19 of the present invention, it is all very general easily acquisition in laboratory, ferment effect is remarkable, the production cost of algin catenase is reduced greatly, can be applicable to scale operation.
Accompanying drawing explanation
Fig. 1 is the phylogenetic tree of the 16srRNA sequence construct of the bacterium that bacterial strain of the present invention is close with blast result.
Fig. 2 be bacterial strain of the present invention in the fermention medium of different carbon source time enzyme activity and biomass comparison diagram.
Fig. 3 be bacterial strain of the present invention in the fermention medium of different nitrogen sources time enzyme activity and biomass comparison diagram.
Fig. 4 be bacterial strain of the present invention in the fermention medium of different concns sodium-chlor time enzyme activity and biomass comparison diagram.
Fig. 5 be bacterial strain of the present invention in the fermention medium of different pH value time enzyme activity and biomass comparison diagram.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
The HalomonaselongataSH-19 bacterial strain of the present invention's screening at the preserving number at China Microbiological bacterial strain preservation management committee's common micro-organisms center is: CGMCCNo.8312, preservation date is: on October 9th, 2013, described strain classification called after: ocean is addicted to Halomonas SH-19 (Halomonaselongata), and the polynucleotide sequence of the 16SrDNA of described bacterial strain is the polynucleotide sequence shown in SEQIDNo.1.
The morphological specificity of HalomonaselongataSH-19:
Bacterium colony is circular, smooth, regular, glossy, opaque, brown bacterium colony.Diameter is at 2-3mm.Thalline width 0.5-0.8 μm, length 1-2 μm, gramstaining is positive.
The genomics qualification of bacterial classification:
By the 16SrDNA sequencing to bacterial strain SH-19, sequence in this bacterium 16srDNA fragment and Genbank compared thus determine the kind of this bacterium, obtaining this bacterial strain is a strain extreme halotolerant Pseudomonas bacterium.
Below in conjunction with specific embodiment, detailed description is done to the invention process process:
The selection systems of embodiment 1 bacterial strain
1, the screening of bacterium producing multi enzyme preparation
The present invention be with grinding after sea urchin enteron aisle screen the production bacterial strain of algin catenase, to be cultivate in sole carbon source screening culture medium in sodium alginate after sea urchin enteron aisle ground sample doubling dilution, the bacterial strain that can survive in screening culture medium of picking for the fermentative production of algin catenase, bacterial strain after qualification freezen protective in glycerine pipe.Concrete steps are as follows:
By after the sea urchin intestinal samples doubling dilution after grinding, to coat sodium alginate be in the Selective agar medium of sole carbon source, cultivate 1 ~ 7 day at being placed in 25 DEG C ~ 30 DEG C. choose the bacterium colony that can grow with this understanding, the fermentation strain obtained is selected the highest strain alive of product enzyme and is accredited as Halomonassp. through 16srDNA sequential analysis, called after HalomonaselongataSH-19.
Screening culture medium consists of (w/v): sodium alginate 1 ~ 2%, ammonium sulfate 0.5 ~ 2%, iron 0.1 ~ 1%, agar 1.5 ~ 2.5%, adds the old seawater of 1000ml, pH=7.6 ~ 8.5.
2, the morphological specificity of HalomonaselongataSH-19
Its bacterium colony of bacterial strain that the present invention obtains is circular, smooth, regular, glossy, opaque, white colony edge slightly black ring.Diameter is at 2-3mm.Thalline width 0.5-0.8 μm, length 1-2 μm, gramstaining is positive.
3, the genomics qualification of bacterial strain
Utilize colony polymerase chain reaction (PCR) method, sequence in bacterial strain 16srDNA fragment and Genbank is compared thus determines the kind of this bacterium.
1) bacterium colony PCR:
The bacterial strain screened is rule in screening culture medium single bacterium colony to be chosen after pure culture three generations and carry out bacterium colony 16srDNAPCR, expect to obtain the phylogenetic feature that this bacterial strain is determined in the 16srDNA characteristic fragment of this bacterium and Genbank comparison.
16srDNA primer is: 1492R:5'-TACCTTGTTACGACTT-3 '
27F：5'-AGAGTTTGATCCTGGCTCAG-3’
Amplification system:
Amplified reaction program:
Denaturation: 95 DEG C, 5min
Sex change: 95 DEG C, 45s;
Annealing: 65 ~ 56 DEG C, 60s;
Extend: 72 DEG C, 90s;
From denaturation, be denatured, annealed to the circulation of extension coreaction 25.
2) sequence alignment analysis:
16SrDNA sequence alignment and Phylogenetic Analysis: the 16SrRNA full length gene sequence 1445nt altogether of bacterial strain HalomonaselongataSH-19.Compare of analysis result shows, the sequence similarity of HalomonaselongataSH-19 and reference culture HalomonaselongataDSM2581 and ChromohalobactersalexigensDSM3043 is the highest, is 95% and 93%.But golden section line between the kind lower than 97%, therefore thinks and which represent a new bacterium kind.Meanwhile, Phylogenetic Analysis result shows, HalomonaselongataSH-19 belongs to bacterial strain on phylogenetic tree sibship with HalomonaselongataDSM2581 is nearest, therefore thinks and which represent the novel species that Halomonas belongs to.
The phylogenetic tree of the 16srRNA sequence construct of the bacterium that HalomonaselongataSH-19 and blast result is close as shown in Figure 1.
Embodiment 2 carries out the measuring of the suitableeest living condition of fermentation algin catenase to the bacterial strain SH-19 that screening obtains:
1) to the selection determination experiment of carbon source in fermention medium:
First, be set to five experimental group, wherein, in fermention medium except carbon source, all nitrogenous source be ammonium sulfate, pH value is 6.5, NaCl mass percent concentration be 3% and temperature be 30 DEG C of conditions under ferment, described carbon source respectively with mass percent concentration be 0.5% glucose, sucrose, starch, sodium alginate or wood sugar.
After above-mentioned five experimental group fermentation culture terminate, measure each experimental group fermentation broth enzyme vigor respectively, test different carbon source is on the impact of producing enzyme.As shown in Figure 2, result shows, add one group of sodium alginate and to live compared to other group enzymes and significantly improve, biomass also significantly increases, and illustrates that sodium alginate can play the effect of inducible strain growth and product algin catenase.
2) to the selection determination experiment of carbon source in fermention medium:
First, be set to five experimental group, wherein, in fermention medium except nitrogenous source, all carbon source be sodium alginate, pH value is 6.5, NaCl mass percent concentration be 3% and temperature be 30 DEG C of conditions under ferment, described nitrogenous source be respectively 0.5% ammonium sulfate, ammonium chloride, extractum carnis, peptone or yeast extractive substance.
After above-mentioned five experimental group fermentation culture terminate, measure the enzyme activity of each experimental group fermented liquid respectively, test nitrogenous source is on the impact of strain enzyme-producing.As shown in Figure 3, found that the group of adding ammonium sulfate and ammonium chloride, biomass is obviously few than the experimental group of adding peptone and yeast extractive substance, but enzyme activity is obviously very high than the experimental group of adding peptone and yeast extractive substance, illustrate that ammonium sulfate or ammonium chloride are the reasonable nitrogenous source of described bacterial strain, wherein ammonium sulfate is best.Add extractum carnis, the experimental group of yeast extractive substance, biomass is apparently higher than other groups, the basic disappearance but enzyme is lived, show that this bacterium abandons the decomposition to being difficult to the sodium alginate of degrading, and utilize carbon source in compound nitrogen source as source of nutrition, only have after with the addition of sodium alginate in the medium and just produce a large amount of algin catenase, further illustrate sodium alginate and can play the effect that enzyme is produced in induction in the fermentation medium, but to live as one group of biomass of nitrogenous source and enzyme with peptone and substantially do not have, illustrate that peptone is not the utilizable nitrogenous source of bacterial classification or carbon source.
3) NaCl concentration is on the impact of producing enzyme:
First, be set to seven experimental group, wherein, the fermention medium of described seven experimental group is except NaCl concentration and pH value, by mass percent concentration be 0.5% ammonium sulfate and sodium alginate carry out under 30 DEG C of conditions, in the fermention medium of described seven experimental group, NaCl concentration (w/v) measures fermentation broth enzyme work after being followed successively by 0%, 1.0%, 2.0%, 3.0%, 4%, 5%, 6% cultivation 48h, and test NaCI concentration is on the impact of strain enzyme-producing.As shown in Figure 4, it is the highest under 4%NaCl concentration that experimental result shows this bacteria biomass, but enzyme work is the highest under 1%NaCl concentration.Consider to choose the formula of 1%NaCl concentration as fermention medium in conjunction with two aspects.
4) substratum initial ph value is on the impact of producing enzyme:
First, be set to six experimental group, wherein, the fermention medium of described six experimental group is except pH value, by NaCl concentration be 1%, mass percent concentration be 0.5% ammonium sulfate and sodium alginate carry out under 30 DEG C of conditions, the initial ph value of the fermention medium of described six experimental group is respectively 4.5,5.5,6.5,7.5,8.5,9.5. measures fermentation broth enzyme vigor after cultivating 48h, test medium initial ph value is on the impact of strain enzyme-producing.As shown in Figure 5, result is that the product enzyme of this bacterium when pH7.5 is most effective.
Explanation, bacterial strain SH-19 carry out the fermenting the suitableeest living condition of algin catenase be nitrogenous source is ammonium sulfate or ammonium chloride, carbon source is extractum carnis and yeast extractive substance, be produce enzyme induction thing with sodium alginate, and interpolation is the NaCl of 1% to mass percent concentration in fermention medium or uses Chen Haishui to dissolve Carbon and nitrogen sources, the optimal pH of fermention medium is 7.5.
The bacterial strain producing algin catenase described in embodiment 3, for producing the method for algin catenase, comprises the following steps:
1) seed liquor is prepared: being inoculated in seed culture medium by screening the bacterial strain SH-19 obtained, within 24 ~ 48 hours, arriving logarithmic phase at 26 ~ 30 DEG C of bottom fermentations, stopping fermentation, obtaining seed liquor;
2) bacterial strain amount reproduction: after described seed liquor is inoculated in one time fermentation substratum, described one time fermentation substratum carries out the fermentation of continuous 36 ~ 60 hours under pH is 4.5 ~ 9.5 and temperature is the condition of 26 ~ 30 DEG C, thus make described bacterial strain carry out amount reproduction, for later stage algin catenase provides condition;
3) algin catenase is induced to synthesize in a large number: after bacterial strain SH-19 amount reproduction terminates, in described one time fermentation substratum, add mass percent concentration be respectively the sodium alginate of 1 ~ 3% and the sodium-chlor of 1 ~ 3% is mixed with Secondary Fermentation substratum, the pH of described Secondary Fermentation substratum is 4.5 ~ 9.5, continuously ferment under 26 ~ 30 DEG C of conditions 8 ~ 14 days, wherein, described sodium alginate synthesizes algin catenase for inducing described bacterial strain.
The enzyme activity determination of embodiment 4 algin catenase:
After fermentation ends, centrifugal for fermented liquid rear mistake 0.22 μm of filter membrane is removed remaining thalline and not tolerant.In order to the activity of not destructive enzyme, the crude enzyme liquid of acquisition with ultraviolet absorption method measure enzyme live after cryopreservation.The measuring method that enzyme is lived is (ultraviolet light absorption method): grip altogether in non-reducing end formation double bond and shuttle base according to enzymolysis algin, have the principle of uv-absorbing consumingly to carry out enzyme activity determination at 235nm place.
By 0.3mL enzyme liquid, 0.3mL sodium alginate (1%) and 2.4mLTris-HCl (0.05mol/L, pH7.0) damping fluid mixing, at 400 DEG C of reaction 30min, the change of light absorption value is surveyed under certain temperature and pH value under 235nm wavelength, the cracking of every milliliter of enzyme liquid per minute catalysis sodium alginate, making substrate light absorption value raise 0.01 is a Ge Meihuo unit.(△ OD is the change of 235nm light absorption value to enzyme activity (u/mL)=Δ oD/T/0.3/0.01xn, T is the reaction times, 0.3 for adding enzyme liquid measure, and 0.01 increases by 0.01 suitable 1 enzyme activity unit for the actual absorbancy of per minute, and n is the extension rate of enzyme liquid).
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and shown here embodiment.
Claims (9)
1. the bacterial strain of algin catenase is produced in a strain, it is characterized in that, described bacterial strain at the preserving number at China Microbiological bacterial strain preservation management committee's common micro-organisms center is: CGMCCNo.8312, and preservation date is: on October 9th, 2013, described strain classification called after: ocean addicted to Halomonas (
halomonaselongata) SH-19.
2. produce a method for algin catenase, it is characterized in that, apply bacterial strain as claimed in claim 1 and produce algin catenase.
3. method as claimed in claim 2, it is characterized in that, specifically comprise the following steps: in the algin catenase production phase, the carbon source of producing fermention medium is sodium alginate, sodium alginate is 1 ~ 3% producing the mass percent concentration in fermention medium, and nitrogenous source is ammonium sulfate or ammonium chloride.
4. method as claimed in claim 3, is characterized in that, specifically comprise the following steps:
1) seed liquor preparatory phase: utilize bacterial strain as claimed in claim 1 to prepare seed liquor;
2) in the culture propagation stage: after described seed liquor is inoculated in one time fermentation substratum, be continuously ferment under the condition of 26 ~ 30 DEG C in temperature, described one time fermentation medium pH is 4.5 ~ 9.5;
3) the algin catenase production phase: add sodium alginate and sodium-chlor in described one time fermentation substratum, be mixed with production fermention medium, sodium alginate in production fermention medium and the concentration of sodium-chlor are respectively 1 ~ 3%, the pH of described production fermention medium is 4.5 ~ 9.5, continuously ferments 8 ~ 14 days under 26 ~ 30 DEG C of conditions.
5. method as claimed in claim 4, it is characterized in that, described one time fermentation medium component is: carbon source, nitrogenous source and Chen Haishui, and wherein, carbon source is extractum carnis and yeast extractive substance, and described nitrogenous source is ammonium sulfate or ammonium chloride.
6. method as claimed in claim 5, it is characterized in that, in described one time fermentation substratum, the mass percent concentration of extractum carnis and yeast extractive substance is respectively 0.1 ~ 1.5%, and the mass percent concentration of ammonium sulfate or ammonium chloride is 0.1-2%.
7. method as claimed in claim 4, is characterized in that, described 3) in, sodium alginate is 1% producing the mass percent concentration in fermention medium, and sodium-chlor is 1% producing the mass percent concentration in fermention medium.
8. method as claimed in claim 4, it is characterized in that, described one time fermentation substratum and described production fermention medium pH are 7.5 ~ 8.5.
9. method as claimed in claim 8, it is characterized in that, described one time fermentation substratum and described production fermention medium pH are 7.5.
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Families Citing this family (5)
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| CN104195080B (en) * | 2014-08-23 | 2017-01-18 | 中国科学院天津工业生物技术研究所 | Bacillus sp capable of producing alginate lyase and application thereof |
| CN106701627B (en) * | 2017-01-05 | 2020-07-03 | 中国海洋大学 | Marine vibrio with high yield of alginate lyase and application thereof |
| CN108018234B (en) * | 2017-12-14 | 2020-10-30 | 华侨大学 | Bacterial strain for producing alginate lyase and application thereof |
| CN110338374A (en) * | 2019-07-30 | 2019-10-18 | 威海长青海洋科技股份有限公司 | A method of brown alga extracting solution is prepared using the enzyme-linked fermentation method of microorganism |
| CN112551692B (en) * | 2020-11-23 | 2022-09-20 | 自然资源部第三海洋研究所 | Halomonas with aerobic denitrification and heterotrophic sulfur oxidation functions and application thereof |
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