CN103911315A - Strain for producing alginate lyase and use thereof - Google Patents
Strain for producing alginate lyase and use thereof Download PDFInfo
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Abstract
The invention discloses a strain for producing alginate lyase and a use thereof. The strain is preserved in the China general microbiological culture collection center on October 9, 2013, has an accession number of CGMCC No.8312 and is named as Halomonas elongata SH-19. A 16S rDNA polynucleotide sequence of the strain is shown in the formula of SEQ ID No.1. The strain SH-19 can efficiently express alginate lyase and can be used for high-efficiency production of alginate lyase. A strain SH-19 fermentation medium comprises simple ingredients commonly used in a laboratory, has substantial fermentation effects, greatly reduces an alginate lyase production cost and can be used for large-scale production.
Description
Technical field
The invention belongs to marine biotechnology field, 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, all has significant advantage in output, production capacity and total cost.
In brown alga, contain a large amount of algins, the complex structure of phaeophyta itself, its cell wall components composition algin hard degradation, various polysaccharide are tightly combined into an organic whole, the difficulty that complicated structure has caused algae to utilize.This has increased the cost that utilizes 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 of, 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 on aspect degraded algin the outstanding bacterial strain of ability.For degraded strategy in follow-up employing systems approach research nature, provide important theory and technology basis for developing effective brown alga processing technology.
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 is from sea urchin enteron aisle abrasive material, to screen and obtain first, can under optimum condition, synthesize algin catenase efficiently.
Two of the object of the invention is to provide a strain to produce the application of the bacterial strain of algin catenase, the Halomonas elongata SH-19 bacterial strain that is about to filter out is for the preparation of algin catenase, thereby produce efficiently algin catenase, to make up the poor deficiency of existing algin hydrolysis effect.
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 Chinese microorganism strains preservation management committee's common micro-organisms center is: CGMCC No.8312, preservation date is: on October 9th, 2013, described strain classification called after: Halomonas SH-19 (Halomonas elongata) is had a liking in ocean.
Preferably, described bacterial strain screening is from sea urchin enteron aisle.
A method of producing algin catenase, is characterized in that, applies bacterial strain as claimed in claim 1 or 2 and produces 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% at the mass percent concentration of producing 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 the each seed liquor of bacterial strain system as claimed in claim 1 or 2;
2) the culture propagation stage: described seed liquor is inoculated in after one time fermentation substratum, described one time fermentation substratum pH be 4.5～9.5 and temperature be to continuously ferment under the condition of 26～30 ℃;
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, under 26～30 ℃ of conditions, continuously ferments 8～14 days.
Preferably, described one time fermentation medium component is: carbon source, nitrogenous source and Chen Haishui, wherein, carbon source be extractum carnis or/yeast extractive substance, 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% at the mass percent concentration of producing in fermention medium, sodium-chlor is 1% at the mass percent concentration of producing 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 efficient expression algin catenase under more suitable growth conditions, can be applicable to High-efficient Production algin catenase, the present invention also explores the living condition of the high efficient expression algin catenase of described bacterial strain SH-19, obtain described bacterial strain and expressed 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 is enzyme activity and the biomass comparison diagram of bacterial strain of the present invention in the fermention medium of different carbon sources time.
Fig. 3 is enzyme activity and the biomass comparison diagram of bacterial strain of the present invention in the fermention medium of different nitrogen sources time.
Fig. 4 is enzyme activity and the biomass comparison diagram of bacterial strain of the present invention in the fermention medium of different concns sodium-chlor time.
Fig. 5 is enzyme activity and the biomass comparison diagram of bacterial strain of the present invention in the fermention medium of different pH values time.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, to make those skilled in the art can implement according to this with reference to specification sheets word.
The Halomonas elongata SH-19 bacterial strain of the present invention's screening at the preserving number at Chinese microorganism strains preservation management committee's common micro-organisms center is: CGMCC No.8312, preservation date is: on October 9th, 2013, described strain classification called after: Halomonas SH-19 (Halomonas elongata) is had a liking in ocean, the polynucleotide sequence of the 16S rDNA of described bacterial strain is the polynucleotide sequence shown in SEQ ID No.1.
The morphological specificity of Halomonas elongata SH-19:
Bacterium colony is circular, smooth, regular, glossy, opaque, brown bacterium colony.Diameter is at 2-3mm.Thalline width 0.5-0.8 μ m, length 1-2 μ m, gramstaining is positive.
The genomics of bacterial classification is identified:
By the 16SrDNA sequencing to bacterial strain SH-19, thereby sequence in this bacterium 16s rDNA fragment and Genbank is compared and determined the kind of this bacterium, obtaining this bacterial strain is a strain extreme halotolerant Pseudomonas bacterium.
Below in conjunction with specific embodiment, the invention process process is done to detailed description:
Screening and the evaluation of embodiment 1 bacterial strain
1, the screening of bacterium producing multi enzyme preparation
The present invention is the production bacterial strain that screens algin catenase with the sea urchin enteron aisle after grinding, to after sea urchin enteron aisle ground sample doubling dilution, be to cultivate in sole carbon source screening culture medium in sodium alginate, the bacterial strain that picking can be survived in screening culture medium is for the fermentative production of algin catenase, and bacterial strain is freezing being stored in glycerine pipe after identifying.Concrete steps are as follows:
To after the sea urchin enteron aisle sample doubling dilution after grinding, coat in the selection substratum that sodium alginate is sole carbon source, be placed at 25 ℃～30 ℃ and cultivate 1～7 day. choose the bacterium colony that can grow with this understanding, the fermentation strain obtaining is selected the highest strain alive of product enzyme and is accredited as Halomonas sp. through 16s rDNA sequential analysis, called after Halomonas elongata SH-19.
Screening culture medium consists of (w/v): sodium alginate 1～2%, and 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 Halomonas elongata SH-19
Its bacterium colony of bacterial strain that the present invention obtains is circular, smooth, regular, glossy, opaque, slightly black ring of white colony edge.Diameter is at 2-3mm.Thalline width 0.5-0.8 μ m, length 1-2 μ m, gramstaining is positive.
3, the genomics of bacterial strain is identified
Utilize colony polymerase chain reaction (PCR) method, thereby sequence in bacterial strain 16s rDNA fragment and Genbank is compared and determined the kind of this bacterium.
1) bacterium colony PCR:
The bacterial strain screening is rule after pure culture three generations single bacterium colony is chosen and carried out bacterium colony 16srDNA PCR in screening culture medium, and the 16s rDNA characteristic fragment of expecting to obtain this bacterium and Genbank compare the phylogenetic feature of definite this bacterial strain.
16s rDNA primer is: 1492R:5'-TACCTTGTTACGACTT-3 '
27F：5'-AGAGTTTGATCCTGGCTCAG-3’
Amplification system:
Amplified reaction program:
Denaturation: 95 ℃, 5min
Sex change: 95 ℃, 45s;
Annealing: 65～56 ℃, 60s;
Extend: 72 ℃, 90s;
From denaturation, be denatured, annealed to and extend coreaction 25 circulations.
2) sequence alignment analysis:
16S rDNA sequence alignment and Phylogenetic Analysis: the 16S rRNA full length gene sequence of bacterial strain Halomonas elongata SH-19 is 1445nt altogether.Compare of analysis result shows, the sequence similarity of Halomonas elongata SH-19 and reference culture Halomonas elongata DSM2581 and Chromohalobacter salexigens DSM3043 is the highest, is 95% and 93%.But lower than golden section line between 97% kind, therefore think that it has represented a new bacterium kind.Meanwhile, Phylogenetic Analysis result shows, to belong to the sibship of bacterial strain nearest with Halomonas elongata DSM2581 on phylogenetic tree for Halomonas elongata SH-19, therefore thinks that it has represented the novel species that Halomonas belongs to.
The phylogenetic tree of the 16srRNA sequence construct of the bacterium that Halomonas elongata SH-19 is close with blast result as shown in Figure 1.
The ferment measuring of the suitableeest living condition of algin catenase of the bacterial strain SH-19 that embodiment 2 obtains screening:
1) the Selective determination experiment to carbon source in fermention medium:
First, be set to five experimental group, wherein, in fermention medium except carbon source, all be at nitrogenous source that ammonium sulfate, pH value are 6.5, NaCl mass percent concentration be 3% and temperature be to ferment under 30 ℃ of conditions, glucose, sucrose, starch, sodium alginate or wood sugar that described carbon source is 0.5% with mass percent concentration respectively.
After above-mentioned five experimental group fermentation culture finish, measure respectively each experimental group fermentation broth enzyme vigor, test different carbon sources to producing the impact of enzyme.As shown in Figure 2, result shows, organizes enzyme work for one group of interpolation sodium alginate significantly improve than other, and biomass also significantly increases, and the effect that sodium alginate can play inducible strain growth and produce algin catenase is described.
2) the Selective determination experiment to carbon source in fermention medium:
First, be set to five experimental group, wherein, in fermention medium except nitrogenous source, all be in carbon source that sodium alginate, pH value are 6.5, NaCl mass percent concentration be 3% and temperature be to ferment under 30 ℃ of conditions, described nitrogenous source is respectively 0.5% ammonium sulfate, ammonium chloride, extractum carnis, peptone or yeast extractive substance.
After above-mentioned five experimental group fermentation culture finish, measure respectively the enzyme activity of each experimental group fermented liquid, the impact of test nitrogenous source on 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 abandoned the decomposition of the sodium alginate to hard degradation, and utilize carbon source in compound nitrogen source as source of nutrition, only added sodium alginate in substratum after, just produce a large amount of algin catenases, further illustrate the effect that sodium alginate can play induction product enzyme in fermention medium, but live and substantially do not have as one group of biomass of nitrogenous source and enzyme with peptone, illustrate that peptone is not the utilizable nitrogenous source of bacterial classification or carbon source.
3) NaCl concentration is on producing the impact of enzyme:
First, be set to seven experimental group, wherein, the fermention medium of described seven experimental group is except NaCl concentration and pH value, the ammonium sulfate that is 0.5% by mass percent concentration and sodium alginate carry out under 30 ℃ of conditions, in the fermention medium of described seven experimental group, NaCl concentration (w/v) is measured fermentation broth enzyme work after being followed successively by 0%, 1.0%, 2.0%, 3.0%, 4%, 5%, 6% cultivation 48h, the impact of test NaCI concentration on strain enzyme-producing.As shown in Figure 4, experimental result shows that this bacteria biomass is the highest under 4%NaCl concentration, 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 is on producing the impact of enzyme:
First, be set to six experimental group, wherein, the fermention medium of described six experimental group is except pH value, carry out under 30 ℃ of conditions by NaCl concentration is 1%, mass percent concentration is 0.5% ammonium sulfate and sodium alginate, the Initial pH 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, the impact of test medium Initial pH on strain enzyme-producing after cultivating 48h.As shown in Figure 5, result is that the product enzyme of this bacterium in the time of pH7.5 is most effective.
Explanation, the ferment the suitableeest living condition of algin catenase of bacterial strain SH-19 is that nitrogenous source is ammonium sulfate or ammonium chloride, carbon source is extractum carnis and yeast extractive substance, take sodium alginate as producing enzyme induction thing, and the NaCl that to add to mass percent concentration in fermention medium be 1% or use Chen Haishui to dissolve Carbon and nitrogen sources, the optimal pH of fermention medium is 7.5.
Described in embodiment 3, produce the bacterial strain of algin catenase for producing the method for algin catenase, comprise the following steps:
1) prepare seed liquor: the bacterial strain SH-19 that screening is obtained is inoculated in seed culture medium, within 24～48 hours, arrive logarithmic phase at 26～30 ℃ of bottom fermentations, stop fermentation, obtain seed liquor;
2) bacterial strain amount reproduction: described seed liquor is inoculated in after one time fermentation substratum, described one time fermentation substratum pH be 4.5～9.5 and temperature be to carry out the fermentation of continuous 36～60 hours under the condition of 26～30 ℃, thereby make described bacterial strain carry out amount reproduction, for later stage algin catenase provides condition;
3) induction algin catenase is synthetic in a large number: after bacterial strain SH-19 amount reproduction finishes, to adding in described one time fermentation substratum, mass percent concentration is respectively 1～3% sodium alginate and 1～3% sodium-chlor is mixed with Secondary Fermentation substratum, the pH of described Secondary Fermentation substratum is 4.5～9.5, under 26～30 ℃ of conditions, continuously ferment 8～14 days, wherein, described sodium alginate is used for inducing described bacterial strain to synthesize algin catenase.
The enzyme activity determination of embodiment 4 algin catenases:
After fermentation ends, centrifugal fermented liquid rear mistake 0.22 μ m filter membrane is removed to remaining thalline and not tolerant.For the activity of destructive enzyme not, for the crude enzyme liquid of acquisition ultraviolet absorption method measure enzyme and live after cryopreservation.The measuring method that enzyme is lived be (ultraviolet light absorption method): form pair keys and shuttle base is gripped altogether according to enzymolysis algin at non-reducing end, 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 mixes, at 400 ℃ of reaction 30min, under 235nm wavelength, survey the variation of light absorption value under certain temperature and pH value, the cracking of every milliliter of enzyme liquid per minute catalysis sodium alginate, making substrate light absorption value rising 0.01 is a Ge Meihuo unit.(△ OD is the variation 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 utilization in specification sheets and embodiment, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other modification, 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 (10)
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 Chinese microorganism strains preservation management committee's common micro-organisms center is: CGMCC No.8312, preservation date is: on October 9th, 2013, described strain classification called after: Halomonas SH-19 (Halomonas elongata) is had a liking in ocean.
2. the bacterial strain that produces as claimed in claim 1 algin catenase, is characterized in that, described bacterial strain screening is from sea urchin enteron aisle.
3. a method of producing algin catenase, is characterized in that, applies bacterial strain as claimed in claim 1 or 2 and produces algin catenase.
4. method as claimed in claim 3, 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% at the mass percent concentration of producing in fermention medium, and nitrogenous source is ammonium sulfate or ammonium chloride.
5. method as claimed in claim 4, is characterized in that, specifically comprises the following steps:
1) seed liquor preparatory phase: utilize the each seed liquor of bacterial strain system as claimed in claim 1 or 2;
2) the culture propagation stage: described seed liquor is inoculated in after one time fermentation substratum, described one time fermentation substratum pH be 4.5～9.5 and temperature be to continuously ferment under the condition of 26～30 ℃;
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, under 26～30 ℃ of conditions, continuously ferments 8～14 days.
6. method as claimed in claim 5, 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, described nitrogenous source is ammonium sulfate or ammonium chloride.
7. method as claimed in claim 6, 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%.
8. method as claimed in claim 4, is characterized in that, described step 3) in, sodium alginate is 1% at the mass percent concentration of producing in fermention medium, sodium-chlor is 1% at the mass percent concentration of producing in fermention medium.
9. method as claimed in claim 5, is characterized in that, described one time fermentation substratum and described production fermention medium pH are 7.5～8.5.
10. method as claimed in claim 9, is characterized in that, described one time fermentation substratum and described production fermention medium pH are 7.5.
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| CN104195080A (en) * | 2014-08-23 | 2014-12-10 | 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 |
| CN106701627A (en) * | 2017-01-05 | 2017-05-24 | 中国海洋大学 | Vibiro splendidus highly yielding alginate lyase and application thereof |
| CN108018234A (en) * | 2017-12-14 | 2018-05-11 | 华侨大学 | One plant of bacterial strain for producing algin catenase and its application |
| 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 |
| CN112551692A (en) * | 2020-11-23 | 2021-03-26 | 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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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104195080A (en) * | 2014-08-23 | 2014-12-10 | 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 |
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| 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 |
| CN108018234A (en) * | 2017-12-14 | 2018-05-11 | 华侨大学 | One plant of bacterial strain for producing algin catenase and its application |
| 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 |
| CN112551692A (en) * | 2020-11-23 | 2021-03-26 | 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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| CN103911315B (en) | 2016-03-02 |
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