CN103923853A - Paenibacillus sp. and method for preparing k-carrageenanase by using same - Google Patents
Paenibacillus sp. and method for preparing k-carrageenanase by using same Download PDFInfo
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- CN103923853A CN103923853A CN201410093127.2A CN201410093127A CN103923853A CN 103923853 A CN103923853 A CN 103923853A CN 201410093127 A CN201410093127 A CN 201410093127A CN 103923853 A CN103923853 A CN 103923853A
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Abstract
The invention relates to Paenibacillus sp. fjfst-333 and a method for preparing k-carrageenanase by using the same. Paenibacillus sp. fjfst-333 is registered and preserved in China Center for Type Culture Collection on December, 25th, 2013, with an accession number of CCTCC M2013707. According to the invention, Paenibacillus sp. is used as a culture, a fermentation medium composed of a carbon source, a nitrogen source and an inorganic salt is employed, and under optimized culture conditions, activity of k-carrageenanase reaches 67 U/ml. Dry k-carrageenanase powder can be obtained by subjecting fermentation broth to salting-out with ammonium sulfate, carrying out centrifugation to collect a precipitate, then carrying out dialysis, allowing dialysate to pass through an ion exchange column and a gel column and finally, carrying out freeze drying.
Description
Technical field
The invention belongs to biological chemical field, relate to a strain series bacillus and the preparation method for kappa-carrageenan enzyme thereof.
Background technology
Carrageenin be by 1,3-β-D-galactopyranose and Isosorbide-5-Nitrae-α-D-galactopyranose as basic framework, the sulfuric acid linear polysaccharide being alternately formed by connecting, is mainly present in the cell walls of Eucheuma, Chondrus, China fir Trentepohlia and husky Lepidium etc. in Rhodophyceae.According to having or not of the quantity of sulfate group contained in its disaccharide unit and interior ether ring, carrageenin can be divided into κ-, ι-, γ-, λ-, ξ-, ω-and type such as ν-carrageenin, industrial production and use be mainly 3 kinds of kappa-carrageenan, ι-carrageenin and lambda-carrageenans.
Carrageenin is the even polysaccharide of a kind of marine alga sulfuric acid gala, after chemistry or biological means degraded and modifying, the galactooligosacchariwith sulfuric acid vinegar obtaining and derivative thereof have multiple biological activity as antiviral, antitumor, anticoagulation etc., and the cellular immunization to human body and humoral immune function also have significant enhancement.Studies show that, the biologic activity of carrageenin and oligose thereof and molecular size range and sulfate radical content are closely related, it is generally acknowledged, polysaccharide molecular weight has obvious biological activity in 5000-60000 scope, and the carraoligose sulfuric acid therefore obtaining after degraded extremely likely becomes the important sources of novel sea medicine.
Carrageenase is mainly derived from pseudomonas
paenibacillus, Cytophaga
cytophaga, vibrios
vibrio, other Zymomonas mobilis
alteromonasdeng.The method of utilizing at present carrageenin to prepare galactooligosacchariwith sulfuric acid vinegar mainly depends on chemical method.Because chemical degradation method exists the shortcoming that reaction conditions is wayward, degraded productive rate is low, object product is not easily separated etc. cannot overcome; and enzymolysis process farthest the active group of protective reaction substrate can in degradation process, not be damaged, thereby have laid a good foundation for medicine source exploitation.Therefore, become scientific worker's both domestic and external main direction by the bacterium that screening can produce carrageenase from marine alga material or marine organisms.
The research that obtains carrageenase with microbial method has had the research history of more than 30 years, but up to now, only has several microorganisms few in number to be found to have the function of Biological preparation carrageenase, comprises
pseudomonas carrageenovora(MeLean M W et.
journal of Biotechnology, 1979),
cytophaga sp.lk mono-C783(Sarwar G et.
journal of Biotechnology, 1987),
delesseria sanguinea(Potin P et.
biotechnology and Bioengineering, 1991),
pseudoalteromonas carrageenovora(Miehel G et.
journal of Fermentation and Bioengineering, 1999),
pseudoalteromonas porphyrae(gene clone and the high efficient expression of Liu Guang et. marine microorganism polysaccharide hydrolase of heap of stone, 2012) etc.Therefore screening new microbial strains carries out enzymolysis process degraded carrageenan and becomes investigator's new task.
Summary of the invention
The object of this invention is to provide the bacterial strain of a strain energy efficient degradation kappa-carrageenan and prepare the method for kappa-carrageenan enzyme with it.
Technical scheme of the present invention: a strain screening, from the bacterium of carrageen, can be used for producing kappa-carrageenan degrading enzyme, its called after series bacillus (
paenibacillussp.) fjfst-333, has registered preservation at Chinese Typical Representative culture collection center on December 25th, 2013, its preserving number is CCTCC NO:M 2013707, and preservation address is Wuhan University.
The screening of microorganism strains and qualification:
The present invention carries out the screening of carrageenin degradation bacteria from carrageen.Carrageen is shredded with seawater (3% sea salt configuration) immersion 2d, get supernatant liquor and add in enrichment medium 5 DEG C, 160r/min shaking table is cultivated after 3d, repetitive operation 3 times, get 0.1ml enrichment culture and be also coated with 28 DEG C of inversion cultivation 48h of primary dcreening operation isolation medium, picking forms the bacterium colony line primary dcreening operation substratum of obvious transparent circle and pit around, cultivates after 48h, picking list bacterium colony moves and connects the medium slant that goes down to posterity, and obtains the pure culture of carrageenin degradation bacteria.Purebred 32 DEG C of the fermentation culture kinds of receiving, 160r/min cultivates the vigor that detects kappa-carrageenan enzyme in nutrient solution after 2d, finishing screen is selected aimed strain B-333 it is carried out to Physiology and biochemistry qualification and 16s rDNA sequential analysis, determine that it is series bacillus (
paenibacillussp.), intend called after series bacillus (
paenibacillussp.) fjfst-333.
With described series bacillus (
paenibacillussp.) method steps of fjfst-333 production kappa-carrageenan enzyme is as follows:
1. by series bacillus (
paenibacillussp.) fjfst-333 is inoculated in culture medium, the bottled 30ml culture medium of 250ml triangle, sterilizing according to a conventional method, cooling and inoculate bacterium colony, 32 DEG C, 160r/min shaking table are cultivated after 24h, get 1ml nutrient solution and again inoculate culture medium, 32 DEG C, 160r/min shaking table cultivation 56h, the centrifugal (5000r/min of fermented liquid, 10min) remove precipitation, get supernatant liquor;
2. be only the substratum of 0.5% kappa-carrageenin substrate containing massfraction with the bottled 20ml of 250ml triangle, sterilizing according to a conventional method, cooling and inoculate 1ml supernatant fermenting enzyme liquid, in 32 DEG C of reaction 1h, obtains the fermented liquid containing kappa-carrageenan enzyme;
3. by fermention medium through 4 DEG C, after 8000-10000g frozen centrifugation 10-20min, get supernatant liquor;
4. supernatant liquor being added to massfraction is the saturated ammonium sulphate of 60%-80%, adds rear continuation to stir 1h, places refrigerator sedimentation and spends the night, and 4 DEG C, 8000-10000g is centrifugal, and 20-40min gets precipitation;
5. precipitation is placed in the distilled water 48h that dialyses, and obtains thick enzyme powder ,-20 DEG C of preservations after lyophilize;
6. thick enzyme dissolves with the Tris-HCI damping fluid of 0.02mo/L pH7.5, filter paper filtering is removed insoluble impurities, separate through anion-exchange chromatography Q-sepharose Fast Flow, carry out continuous gradient wash-out with the Tris-HCI damping fluid of the 0.05mol/L pH7.2 that contains 0.1 ~ 0.6mol/L NaCI, flow velocity is 1.2ml/min, detects the protein content in elutriant in 280nm, distributes and collects elutriant, 4.5ml/ pipe, measures the enzyme of peak position and lives;
7. activated part in test tube is merged and carries out lyophilize, kappa-carrageenan enzyme work in-process.
8. by damping fluid (0.02mol/L for Sephcryl S-100 gel permeation chromatography post (φ 1.6cm*80cm), the Tris-HCl of pH7.5) after balance, weigh kappa-carrageenan enzyme work in-process 0.05g and be dissolved in 4mL level pad, after 6000g low-temperature centrifugation 20min, get chromatography column on supernatant liquor.Carry out wash-out with damping fluid (0.02mol/L, the Tris-HCl of pH7.5), coutroi velocity is 0.1ml/min.Elutriant is through ultraviolet detection fraction collection, and 4ml/ manages, and the enzyme that determines respectively peak part test tube is lived, and great-hearted part is merged, and after abundant dialysis desalting, ultra-filtration membrane is concentrated, to concentrated solution lyophilize, obtains kappa-carrageenan enzyme finished product.
Described culture medium: sodium-chlor 15g, kappa-carrageenan 2g, yeast extract paste 1g, inorganic salt mother liquor 100ml, water 900ml, pH7.5, wherein inorganic salt mother liquor: NaNO
320g, MgSO
4.7H
2o 5g, K
2hPO
410g, CaC1
2lg, distilled water 1L.
Step is described substratum 2.: kappa-carrageenan 5g, distilled water 1000ml, pH7.5.
The detection method of the kappa-carrageenan enzyme of producing by the method:
Get step and 3. obtain the fermented liquid supernatant liquid 1mL that contains kappa-carrageenan enzyme, add the carrageenin substrate (wherein containing 0.5% carrageenin) of 20mL, be placed in 32 DEG C of enzymolysis 1h, after add 1.0mLDNS, boiling water bath 5min.Be cooled to after room temperature, with distilled water polishing scale, use spectrophotometer to detect under 540nm.1U is defined as the value of 1min generation 1ug carrageenan oligosaccharide.
Beneficial effect of the present invention:
1. newly filter out the bacterial strain of a strain energy High-efficient Production kappa-carrageenan enzyme, Classification And Nomenclature be series bacillus (
paenibacillussp.) fjfst-333.
2. the present invention is using this bacterium as bacterial classification, with common carbon source, and nitrogenous source, the fermention medium of inorganic salt composition can reach maximum enzyme output in 48-60h, and enzyme is lived as 67U/ml.
Embodiment
Be below explanation specific embodiments of the invention, but the present invention is not limited to this.
Embodiment 1
Make sample with carrageen and carry out the screening of kappa-carrageenan degradation bacteria.Carrageen is shredded with seawater (3% sea salt configuration) immersion 2d, get supernatant liquor and add in enrichment medium 5 DEG C, 160r/min shaking table is cultivated after 3d, repetitive operation 3 times, get 0.1ml enrichment culture and be also coated with 28 DEG C of inversion cultivation 48h of primary dcreening operation isolation medium, picking forms the bacterium colony line primary dcreening operation substratum of obvious transparent circle and pit around, cultivates after 48h, picking list bacterium colony moves and connects the medium slant that goes down to posterity, and obtains the pure culture of kappa-carrageenan degradation bacteria.Purebred 32 DEG C of the fermentation culture kinds of receiving, 160r/min cultivates the vigor that detects kappa-carrageenan enzyme in nutrient solution after 2d, and finishing screen is selected aimed strain B-333.
Embodiment 2
Through morphology and physio-biochemical characteristics research, the feature of B-333 bacterial strain is as follows:
Colonial morphology: the micro-protuberance in bacterium colony surface, be milk yellow, adhesion, neat in edge.
Cellular form: this is Gram-positive, feminine gender, form is shaft-like, flagellum.
Physiological and biochemical property: be facultative anaerobe, oxydase reaction is variable, V-P reaction (acetyl methyl carbinol generation) is variable, and the pH 4～6 of V-P liquid, does not produce hydrogen sulfide.
16S rRNA gene to this bacterial strain carries out pcr amplification and sequencing, and the Gene Partial fragment length of finding its 16S rRNA is 1539bp, compares through NCBI and rrna database, be accredited as series bacillus (
paenibacillussp.), intend called after series bacillus (
paenibacillussp.) fjfst-333, the concrete visible sequence table of 16S rRNA sequence of bacterial strain.
Embodiment 3
1. by series bacillus (
paenibacillussp.) fjfst-333 is inoculated in culture medium, the bottled 30ml culture medium of 250ml triangle, sterilizing according to a conventional method, cooling and inoculate bacterium colony, 32 DEG C, 160r/min shaking table are cultivated after 24h, get 1ml nutrient solution and again inoculate culture medium, 32 DEG C, 160r/min shaking table cultivation 56h.Fermented liquid centrifugal (5000r/min, 10min) is removed precipitation, gets supernatant liquor.Described culture medium: sodium-chlor 15g, kappa-carrageenan 2g, yeast extract paste 1g, inorganic salt mother liquor 100ml, water 900ml, pH7.5.
Inorganic salt mother liquor: NaNO
320g, MgSO
4.7H
2o 5g, K
2hPO
410g, CaC1
2lg, distilled water 1L.
2. be only the substratum of 0.5% kappa-carrageenin substrate containing massfraction with the bottled 20ml of 250ml triangle, sterilizing according to a conventional method, cooling and inoculate 1ml supernatant fermenting enzyme liquid.In 32 DEG C of reaction 1h, obtain containing the fermented liquid of kappa-carrageenan enzyme and survey its enzyme and live.Described substratum: kappa-carrageenan 5g, distilled water 1000ml, pH7.5.
The vigor of the agarase of this embodiment gained is 67U/mL.
Embodiment 4
1.. by series bacillus (
paenibacillussp.) fjfst-333 is inoculated in culture medium, the bottled 30ml culture medium of 250ml triangle, sterilizing according to a conventional method, cooling and inoculate bacterium colony, 32 DEG C, 160r/min shaking table are cultivated after 24h, get 1ml nutrient solution and again inoculate culture medium, 32 DEG C, 160r/min shaking table cultivation 56h, the centrifugal (5000r/min of fermented liquid, 10min) remove precipitation, get supernatant liquor.Described culture medium: sodium-chlor 15g, kappa-carrageenan 2g, yeast extract paste 1g, inorganic salt mother liquor 100ml, water 900ml, pH7.5.
Inorganic salt mother liquor: NaNO
320g, MgSO
4.7H
2o 5g, K
2hPO
410g, CaC1
2lg, distilled water 1L.
2.. only containing massfraction with the bottled 20ml of 250ml triangle is the substratum of 0.5% kappa-carrageenin substrate, sterilizing according to a conventional method, cooling and inoculate 1ml supernatant fermenting enzyme liquid, in 32 DEG C of reaction 1h, obtain the fermented liquid containing kappa-carrageenan enzyme, described substratum: kappa-carrageenan 5g, distilled water 1000ml, pH7.5.
3.. fermention medium, through 4 DEG C, after 8000-10000g frozen centrifugation 10-20min, is got to supernatant liquor.
4.. it is the saturated ammonium sulphate of 60%-80% that supernatant liquor is added to massfraction, adds rear continuation to stir 1h, places refrigerator sedimentation and spends the night, and 4 DEG C, 10000g is centrifugal, and 40min gets precipitation;
5.. precipitation is placed in the distilled water 48h that dialyses, and obtains thick enzyme powder ,-20 DEG C of preservations after lyophilize;
6.. thick enzyme dissolves with the Tris-HCI damping fluid of 0.02mo/L pH7.5, filter paper filtering is removed insoluble impurities, separate through anion-exchange chromatography Q-sepharose Fast Flow, carry out continuous gradient wash-out with the Tris-HCI damping fluid of the 0.05mol/L pH7.2 that contains 0.1 ~ 0.6mol/L NaCI, flow velocity is 1.2ml/min, detects the protein content in elutriant in 280nm, distributes and collects elutriant, 4.5ml/ pipe, measures the enzyme of peak position and lives;
7.. activated part in test tube is merged and carries out lyophilize, kappa-carrageenan enzyme work in-process;
8..by damping fluid (0.02mol/L for Sephcryl S-100 gel permeation chromatography post (φ 1.6cm*80cm), the Tris-HCl of pH7.5) after balance, weigh kappa-carrageenan enzyme work in-process 0.05g and be dissolved in 4mL level pad, after 6000g low-temperature centrifugation 20min, get chromatography column on supernatant liquor.Carry out wash-out with damping fluid (0.02mol/L, the Tris-HCl of pH7.5), coutroi velocity is 0.1ml/min.Elutriant is through ultraviolet detection fraction collection (4ml/ pipe), and the enzyme that determines respectively peak part test tube is lived, and great-hearted part is merged, and after abundant dialysis desalting, ultra-filtration membrane is concentrated, to concentrated solution lyophilize, obtains kappa-carrageenan enzyme finished product.
SEQUENCE LISTING
<110> University Of Agriculture and Forestry In Fujian
<120> mono-strain series bacillus and for the preparation method of kappa-carrageenan enzyme
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1539
<212> DNA
<213> 16s rDNA sequence
<400> 1
cagagtttga tcctggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag 60
cggagttatt ccttcgggga tagcttagcg gcggacgggt gagtaacacg taggtaacct 120
gcctgtaaga ctgggataac attcggaaac gaatgctaat accggatacg cgaattggtc 180
gcatggccga ttcgggaaag acggagcaat ctgtcgctta cagatggacc tgcggtgcat 240
tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt 300
gatcggccac actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa 360
tcttccgcaa tgggcgaaag cctgacggag caacgccgcg tgagtgatga aggttttcgg 420
atcgtaaagc tctgttgcca gggaagaacg cttgggagag taactgctct caaggtgacg 480
gtacctgaga agaaagcccc ggctaactac gtgccagcag ccgcggcaat acgtaggggg 540
caagcgttgt ccggaattat tgggcgtaaa gcgcgcgcag gcggtcaatt aagtctggtg 600
tttaaggctg gggctcaacc ccggttcgca ctggaaactg gttgacttga gtgcagaaga 660
ggaaagtgga attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg 720
cgaaggcgac tttctgggct gtaactgacg ctgaggcgcg aaagcgtggg gagcaaacag 780
gattagatac cctggtagtc cacgccgtaa acgatgaatg ctaggtgtta ggggtttcga 840
tacccttggt gccgaagtta acacattaag cattccgcct ggggagtacg gtcgcaagac 900
tgaaactcaa aggaattgac ggggacccgc acaagcagtg gagtatgtgg tttaattcga 960
agcaacgcga agaaccttac caggtcttga catccctctg accggattag agatagtcct 1020
tcccttcggg gcagaggaga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 1080
ttgggttaag tcccgcaacg agcgcaaccc ctaattttag ttgccagcac ttcgggtggg 1140
cactctaaag tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca 1200
tgccccttat gacctgggct acacacgtac tacaatggcc agtacaacgg gaagcgaagt 1260
cgcgagatgg agccaatcct atcaaagctg gtctcagttc ggattgcagg ctgcaactcg 1320
cctgcatgaa gtcggaattg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380
cgggccttgt acacaccgcc cgtcacacca cgagagttta caacacccga agtcggtggg 1440
gtaacccgca agggagccag ccgccgaagg tggggtagat gattggggtg aagtcgtaac 1500
aaggtagccg tatcggaagg tgcggctgga tcacctcct 1539
Claims (6)
1. a strain series bacillus, is characterized in that: described bacterial strain be series bacillus (
paenibacillussp.) fjfst-333, has registered preservation at Chinese Typical Representative culture collection center on December 25th, 2013, its preserving number is CCTCC M 2013707.
2. a strain series bacillus according to claim 1, is characterized in that: described series bacillus (
paenibacillussp.) the 16S rRNA sequence of fjfst-333 is as shown in SEQ ID NO.1.
3. the purposes of a strain strain series bacillus as claimed in claim 1, is characterized in that: for the preparation of kappa-carrageenan enzyme.
4. strain series bacillus as claimed in claim 1, for the preparation of the method for kappa-carrageenan enzyme, is characterized in that: comprise the steps:
1.. by series bacillus (
paenibacillussp.) fjfst-333 is inoculated in culture medium, the bottled 30ml culture medium of 250ml triangle, sterilizing according to a conventional method, cooling and inoculate bacterium colony, 32 DEG C, 160r/min shaking table are cultivated after 24h, get 1ml nutrient solution and again inoculate culture medium, 32 DEG C, 160r/min shaking table cultivation 56h, the centrifugal 10min of fermented liquid 5000r/min, remove precipitation, get supernatant liquor;
2.. only containing massfraction with the bottled 20ml of 250ml triangle is the substratum of 0.5% kappa-carrageenin substrate, sterilizing according to a conventional method, cooling and inoculate 1ml supernatant fermenting enzyme liquid, in 32 DEG C of reaction 1h, obtain the fermented liquid containing kappa-carrageenan enzyme, described substratum: kappa-carrageenan 5g, distilled water 1000ml, pH7.5;
3.. fermention medium, through 4 DEG C, after 8000-10000g frozen centrifugation 10-20min, is got to supernatant liquor;
4.. it is the saturated ammonium sulphate of 60%-80% that supernatant liquor is added to massfraction, adds rear continuation to stir 1h, places refrigerator sedimentation and spends the night, and 4 DEG C, 8000-10000g is centrifugal, and 20-40min gets precipitation;
5.. precipitation is placed in the distilled water 48h that dialyses, and obtains thick enzyme powder ,-20 DEG C of preservations after lyophilize;
6.. thick enzyme dissolves with the Tris-HCI damping fluid of 0.02mo/L pH7.5, filter paper filtering is removed insoluble impurities, separate through anion-exchange chromatography Q-sepharose Fast Flow, carry out continuous gradient wash-out with the Tris-HCI damping fluid of the 0.05mol/L pH7.2 that contains 0.1 ~ 0.6mol/L NaCI, flow velocity is 1.2ml/min, detects the protein content in elutriant in 280nm, distributes and collects elutriant, 4.5ml/ pipe, measures the enzyme of peak position and lives;
7.. activated part in test tube is merged and carries out lyophilize, kappa-carrageenan enzyme work in-process;
8.. by Sephcryl S-100 gel permeation chromatography post 0.02mol/L, after the Tris-HCl damping fluid balance of pH7.5, weighing kappa-carrageenan enzyme work in-process 0.05g is dissolved in 4mL level pad, after 6000g low-temperature centrifugation 20min, get chromatography column on supernatant liquor, with 0.02mol/L, the Tris-HCl damping fluid of pH7.5 carries out wash-out, coutroi velocity is 0.1ml/min, elutriant is through ultraviolet detection fraction collection, 4ml/ pipe, the enzyme that determines respectively peak part test tube is lived, great-hearted part is merged, after abundant dialysis desalting, ultra-filtration membrane is concentrated, to concentrated solution lyophilize, obtain kappa-carrageenan enzyme finished product.
5. a strain Pseudomonas aeruginosa according to claim 4 is for the preparation of the method for kappa-carrageenan enzyme, it is characterized in that: described culture medium: sodium-chlor 15g, kappa-carrageenan 2g, yeast extract paste 1g, inorganic salt mother liquor 100ml, water 900ml, pH7.5, wherein inorganic salt mother liquor: NaNO
320g, MgSO
4.7H
2o 5g, K
2hPO
410g, CaC1
2lg, distilled water 1L.
6. a strain Pseudomonas aeruginosa according to claim 4, for the preparation of the method for kappa-carrageenan enzyme, is characterized in that: step is described substratum 2.: kappa-carrageenan 5g, distilled water 1000ml, pH7.5.
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| CN105218670B (en) * | 2015-11-20 | 2019-01-22 | 宝鸡西姆现代生物技术工程有限公司 | A kind of tunning immune protein and its preparation process from bacillus firmus |
| CN109439709A (en) * | 2018-11-21 | 2019-03-08 | 青岛博智汇力生物科技有限公司 | A kind of preparation method of new Iota carrageenan oligosaccharide |
| CN112159774A (en) * | 2020-09-22 | 2021-01-01 | 自然资源部第一海洋研究所 | Deep-sea bacillus N1-1 for efficiently degrading selenocarrageenan and application thereof |
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