CN103923853B - One strain series bacillus and the preparation method for kappa-carrageenan enzyme thereof - Google Patents
One strain series bacillus and the preparation method for kappa-carrageenan enzyme thereof Download PDFInfo
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- CN103923853B CN103923853B CN201410093127.2A CN201410093127A CN103923853B CN 103923853 B CN103923853 B CN 103923853B CN 201410093127 A CN201410093127 A CN 201410093127A CN 103923853 B CN103923853 B CN 103923853B
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Abstract
Do you the present invention relates to a strain series bacillus (<i>Paenibacillus? </i>sp.) fjfst-333, register preservation on December 25th, 2013 in China typical culture collection center, does is its preserving number CCTCC? M2013707, and the method preparing kappa-carrageenan enzyme with this strain fermentation.The present invention is using this bacterial strain as bacterial classification, and adopt the fermention medium of carbon source, nitrogenous source, inorganic salt composition, under culture condition after optimization, the vigor of kappa-carrageenan enzyme reaches 67U/ml.Fermented liquid by ammonium sulfate precipitation, centrifugal collecting precipitation, after dialyse, get dialyzate and cross ion exchange column and gel column, lyophilize can obtain kappa-carrageenan enzyme dry powder.
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 is that the sulfuric acid linear polysaccharide be alternately formed by connecting, is mainly present in the cell walls of the Eucheuma in Rhodophyceae, Chondrus, China fir Trentepohlia and husky Lepidium etc. by 1,3-β-D-galactopyranose and Isosorbide-5-Nitrae-α-D-galactopyranose as basic framework.According to the quantity of sulfate group contained in its disaccharide unit and the presence or absence of interior ether ring, carrageenin can be divided into κ-, ι-, γ-, λ-, ξ-, the type such as ω-and ν-carrageenin, industrial production and use be mainly kappa-carrageenan, ι-carrageenin and lambda-carrageenan 3 kinds.
Carrageenin is the even polysaccharide of a kind of marine alga sulfuric acid gala, after chemistry or biological means degraded and modification, the galactooligosacchariwith sulfuric acid vinegar obtained and derivative thereof have multiple biological activity as antiviral, antitumor, anticoagulation etc., also have significant enhancement to the cellular immunization of human body and humoral immune function.Research display, the biologic activity of carrageenin and oligose thereof and molecular size range and sulfate radical content closely related, it is generally acknowledged, polysaccharide molecular weight has obvious biological activity in 5000-60000 scope, and the carraoligose sulfuric acid therefore obtained 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 utilizing carrageenin to prepare galactooligosacchariwith sulfuric acid vinegar at present 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 can the active group of farthest protective reaction substrate can not be damaged in degradation process, thus have laid a good foundation for the exploitation of medicine source.Therefore, by screening the Main way that the bacterium that can produce carrageenase becomes scientific worker both domestic and external from marine alga material or marine organisms.
There is the research history in more than 30 years with the research of microbial method acquisition carrageenase, but up to now, only had several microorganisms few in number to be found to have the function of Biological preparation carrageenase, comprise
pseudomonascarrageenovora(MeLeanMWet.
journalofBiotechnology, 1979),
cytophagasp.lk mono-C783(SarwarGet.
journalofBiotechnology, 1987),
delesseriasanguinea(PotinPet.
biotechnologyandBioengineering, 1991),
pseudoalteromonascarrageenovora(MiehelGet.
journalofFermentationandBioengineering, 1999),
pseudoalteromonasporphyrae(Liu Guang builds gene clone and the high expression of et. marine microorganism polysaccharide hydrolase, 2012) etc.Therefore screen new microbial strains and carry out the new task that enzymolysis process degraded carrageenan becomes investigator.
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, register preservation on December 25th, 2013 in China typical culture collection center, its preserving number is CCTCCNO:M2013707, 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 and soaks 2d with seawater (3% sea salt configuration), get supernatant liquor and to add in enrichment medium 5 DEG C, after 3d cultivated by 160r/min shaking table, repetitive operation 3 times, get 0.1ml enrichment culture and be also coated with primary dcreening operation isolation medium 28 DEG C inversion cultivation 48h, form the bacterium colony line primary dcreening operation substratum of obvious transparent circle and pit around picking, after cultivating 48h, picking list bacterium colony moves and connects Secondary Culture base inclined-plane, obtains the pure culture of carrageenin degradation bacteria.Purebredly receive fermentation culture kind 32 DEG C, 160r/min detects the vigor of kappa-carrageenan enzyme in nutrient solution after cultivating 2d, finishing screen is selected aimed strain B-333 and carried out Physiology and biochemistry qualification and 16srDNA sequential analysis to it, 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 inoculate bacterium colony, 32 DEG C, 160r/min shaking table cultivates after 24h, get 1ml nutrient solution and again inoculate culture medium, 32 DEG C, the cultivation of 160r/min shaking table 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 inoculate 1ml supernatant fermenting enzyme liquid, in 32 DEG C of reaction 1h, obtain 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 is added the saturated ammonium sulphate that massfraction is 60%-80%, add rear continuation and stir 1h, place refrigerator sedimentation and spend the night, 4 DEG C, 8000-10000g is centrifugal, and 20-40min gets precipitation;
5. precipitation is placed in distilled water and dialyses 48h, obtains thick enzyme powder ,-20 DEG C of preservations after lyophilize;
6. the Tris-HCI buffer solution of thick enzyme 0.02mo/LpH7.5, filter paper filtering removing insoluble impurities, be separated through anion-exchange chromatography Q-sepharoseFastFlow, continuous gradient wash-out is carried out with the Tris-HCI damping fluid of the 0.05mol/LpH7.2 containing 0.1 ~ 0.6mol/LNaCI, flow velocity is 1.2ml/min, detects the protein content in elutriant in 280nm, and elutriant is collected in distribution, 4.5ml/ manages, and the enzyme measuring peak position is lived;
7. activated part in test tube is merged and carries out lyophilize, kappa-carrageenan enzyme work in-process.
8. by SephcrylS-100 gel permeation chromatography post (φ 1.6cm*80cm) damping fluid (0.02mol/L, the Tris-HCl of pH7.5) balance after, weighing kappa-carrageenan enzyme work in-process 0.05g is dissolved in 4mL level pad, gets chromatography column on supernatant liquor after 6000g low-temperature centrifugation 20min.Carry out wash-out with damping fluid (Tris-HCl of 0.02mol/L, pH7.5), coutroi velocity is 0.1ml/min.Elutriant is through ultraviolet detection and fraction collection, and 4ml/ manages, and the enzyme determining peak part test tube is respectively lived, and great-hearted part is merged, and after enough hemodialysis desalination, ultra-filtration membrane concentrates, and 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
2o5g, 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 produced by the method:
Get the fermented liquid supernatant liquid 1mL that 3. step obtains containing 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.After being cooled to room temperature, with distilled water polishing scale, spectrophotometer is used to detect under 540nm.1U is defined as the value that 1min produces 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, and with common carbon source, 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 that specific embodiments of the invention are described, but the present invention is not limited to this.
Embodiment 1
The screening that sample carries out kappa-carrageenan degradation bacteria is done with carrageen.Carrageen is shredded and soaks 2d with seawater (3% sea salt configuration), get supernatant liquor and to add in enrichment medium 5 DEG C, after 3d cultivated by 160r/min shaking table, repetitive operation 3 times, get 0.1ml enrichment culture and be also coated with primary dcreening operation isolation medium 28 DEG C inversion cultivation 48h, form the bacterium colony line primary dcreening operation substratum of obvious transparent circle and pit around picking, after cultivating 48h, picking list bacterium colony moves and connects Secondary Culture base inclined-plane, obtains the pure culture of kappa-carrageenan degradation bacteria.Purebredly receive fermentation culture kind 32 DEG C, 160r/min detects the vigor of kappa-carrageenan enzyme in nutrient solution after cultivating 2d, finishing screen selects 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: bacterium colony surface micro-protuberance, in 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 pH4 ~ 6 of V-P liquid, do not produce hydrogen sulfide.
Pcr amplification and sequencing are carried out to the 16SrRNA gene of this bacterial strain, find that the Gene Partial fragment length of its 16SrRNA is 1539bp, compare 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 16SrRNA 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 inoculate bacterium colony, 32 DEG C, after 160r/min shaking table cultivates 24h, get 1ml nutrient solution and again inoculate culture medium, 32 DEG C, 160r/min shaking table cultivation 56h.Precipitation removed by fermented liquid centrifugal (5000r/min, 10min), 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
2o5g, 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 inoculate 1ml supernatant fermenting enzyme liquid.In 32 DEG C of reaction 1h, obtain the fermented liquid containing kappa-carrageenan enzyme and survey its enzyme living.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 inoculate bacterium colony, 32 DEG C, 160r/min shaking table cultivates after 24h, get 1ml nutrient solution and again inoculate culture medium, 32 DEG C, the cultivation of 160r/min shaking table 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
2o5g, 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 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.. by fermention medium through 4 DEG C, after 8000-10000g frozen centrifugation 10-20min, get supernatant liquor.
4.. supernatant liquor is added the saturated ammonium sulphate that massfraction is 60%-80%, add rear continuation and stir 1h, place refrigerator sedimentation and spend the night, 4 DEG C, 10000g is centrifugal, and 40min gets precipitation;
5.. precipitation is placed in distilled water and dialyses 48h, obtains thick enzyme powder ,-20 DEG C of preservations after lyophilize;
6.. the Tris-HCI buffer solution of thick enzyme 0.02mo/LpH7.5, filter paper filtering removing insoluble impurities, be separated through anion-exchange chromatography Q-sepharoseFastFlow, continuous gradient wash-out is carried out with the Tris-HCI damping fluid of the 0.05mol/LpH7.2 containing 0.1 ~ 0.6mol/LNaCI, flow velocity is 1.2ml/min, detects the protein content in elutriant in 280nm, and elutriant is collected in distribution, 4.5ml/ manages, and the enzyme measuring peak position is lived;
7.. activated part in test tube is merged and carries out lyophilize, kappa-carrageenan enzyme work in-process;
8..by SephcrylS-100 gel permeation chromatography post (φ 1.6cm*80cm) damping fluid (0.02mol/L, the Tris-HCl of pH7.5) balance after, weighing kappa-carrageenan enzyme work in-process 0.05g is dissolved in 4mL level pad, gets chromatography column on supernatant liquor after 6000g low-temperature centrifugation 20min.Carry out wash-out with damping fluid (Tris-HCl of 0.02mol/L, pH7.5), coutroi velocity is 0.1ml/min.Elutriant is through ultraviolet detection and fraction collection (4ml/ pipe), and the enzyme determining peak part test tube is respectively lived, and great-hearted part is merged, and after enough hemodialysis desalination, ultra-filtration membrane concentrates, and to concentrated solution lyophilize, obtains kappa-carrageenan enzyme finished product.
SEQUENCELISTING
<110> University Of Agriculture and Forestry In Fujian
<120> mono-strain series bacillus and the preparation method for kappa-carrageenan enzyme thereof
<130>1
<160>1
<170>PatentInversion3.3
<210>1
<211>1539
<212>DNA
<213>16srDNA sequence
<400>1
cagagtttgatcctggctcaggacgaacgctggcggcgtgcctaatacatgcaagtcgag60
cggagttattccttcggggatagcttagcggcggacgggtgagtaacacgtaggtaacct120
gcctgtaagactgggataacattcggaaacgaatgctaataccggatacgcgaattggtc180
gcatggccgattcgggaaagacggagcaatctgtcgcttacagatggacctgcggtgcat240
tagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggt300
gatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaa360
tcttccgcaatgggcgaaagcctgacggagcaacgccgcgtgagtgatgaaggttttcgg420
atcgtaaagctctgttgccagggaagaacgcttgggagagtaactgctctcaaggtgacg480
gtacctgagaagaaagccccggctaactacgtgccagcagccgcggcaatacgtaggggg540
caagcgttgtccggaattattgggcgtaaagcgcgcgcaggcggtcaattaagtctggtg600
tttaaggctggggctcaaccccggttcgcactggaaactggttgacttgagtgcagaaga660
ggaaagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtgg720
cgaaggcgactttctgggctgtaactgacgctgaggcgcgaaagcgtggggagcaaacag780
gattagataccctggtagtccacgccgtaaacgatgaatgctaggtgttaggggtttcga840
tacccttggtgccgaagttaacacattaagcattccgcctggggagtacggtcgcaagac900
tgaaactcaaaggaattgacggggacccgcacaagcagtggagtatgtggtttaattcga960
agcaacgcgaagaaccttaccaggtcttgacatccctctgaccggattagagatagtcct1020
tcccttcggggcagaggagacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatg1080
ttgggttaagtcccgcaacgagcgcaacccctaattttagttgccagcacttcgggtggg1140
cactctaaagtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatca1200
tgccccttatgacctgggctacacacgtactacaatggccagtacaacgggaagcgaagt1260
cgcgagatggagccaatcctatcaaagctggtctcagttcggattgcaggctgcaactcg1320
cctgcatgaagtcggaattgctagtaatcgcggatcagcatgccgcggtgaatacgttcc1380
cgggccttgtacacaccgcccgtcacaccacgagagtttacaacacccgaagtcggtggg1440
gtaacccgcaagggagccagccgccgaaggtggggtagatgattggggtgaagtcgtaac1500
aaggtagccgtatcggaaggtgcggctggatcacctcct1539
Claims (6)
1. a strain series bacillus, is characterized in that: described bacterial strain be series bacillus (
paenibacillussp.) fjfst-333, register preservation on December 25th, 2013 in China typical culture collection center, its preserving number is CCTCCNO:M2013707.
2. a strain series bacillus according to claim 1, is characterized in that: described series bacillus (
paenibacillussp.) the 16SrRNA sequence of fjfst-333 is as shown in SEQIDNO.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 is for the preparation of the method for kappa-carrageenan enzyme, it 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 inoculate bacterium colony, 32 DEG C, 160r/min shaking table cultivates after 24h, get 1ml nutrient solution and again inoculate culture medium, 32 DEG C, the centrifugal 10min of 160r/min shaking table cultivation 56h, fermented liquid 5000r/min, 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 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. by fermention medium through 4 DEG C, after 8000-10000g frozen centrifugation 10-20min, get supernatant liquor;
4. supernatant liquor is added the saturated ammonium sulphate that massfraction is 60%-80%, add rear continuation and stir 1h, place refrigerator sedimentation and spend the night, 4 DEG C, 8000-10000g is centrifugal, and 20-40min gets precipitation;
5. precipitation is placed in distilled water and dialyses 48h, obtains thick enzyme powder ,-20 DEG C of preservations after lyophilize;
6. the Tris-HCI buffer solution of thick enzyme 0.02mo/LpH7.5, filter paper filtering removing insoluble impurities, be separated through anion-exchange chromatography Q-sepharoseFastFlow, continuous gradient wash-out is carried out with the Tris-HCI damping fluid of the 0.05mol/LpH7.2 containing 0.1 ~ 0.6mol/LNaCI, flow velocity is 1.2ml/min, detects the protein content in elutriant in 280nm, and elutriant is collected in distribution, 4.5ml/ manages, and the enzyme measuring peak position is lived;
7. activated part in test tube is merged and carries out lyophilize, kappa-carrageenan enzyme work in-process;
8. by SephcrylS-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, chromatography column on supernatant liquor is got after 6000g low-temperature centrifugation 20min, 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 and fraction collection, 4ml/ manages, the enzyme determining peak part test tube is respectively lived, great-hearted part is merged, after enough hemodialysis desalination, ultra-filtration membrane concentrates, to concentrated solution lyophilize, obtain kappa-carrageenan enzyme finished product.
5. a strain series bacillus 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
2o5g, K
2hPO
410g, CaC1
2lg, distilled water 1L.
6. a strain series bacillus according to claim 4 is for the preparation of the method for kappa-carrageenan enzyme, it is characterized in that: step is described substratum 2.: kappa-carrageenan 5g, distilled water 1000ml, pH7.5.
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