CN105567606A - Arthrobacter globiformis and hyaluronidase generated by arthrobacter globiformis - Google Patents
Arthrobacter globiformis and hyaluronidase generated by arthrobacter globiformis Download PDFInfo
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Abstract
The invention provides arthrobacter globiformis and hyaluronidase generated by the arthrobacter globiformis, and relates to arthrobacter globiformis A152 from intertidal zone sludge. The bacterial strain has the characteristics that nutritional conditions are simple, and the culture is easy. The hyaluronidase generated by the bacterial strain provided by the invention has the characteristics that the enzyme production vitality is high; the stability is high; the cost is low; the large-scale culture can be realized; the industrial application is met. The invention also relates to the hyaluronidase obtained by a variable temperature regulation and control method. The seed culture is transferred into a fermentation culture process for low-temperature induction fermentation; fermentation liquid is subjected to separation purification and freeze drying; hyaluronidase products in different specifications can be obtained. The method has the advantages that the enzyme yield is high; the production period is short; the operation is simple. The molecular weight of the hyaluronidase from the arthrobacter globiformis A152 is determined to be 73.7kDa on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).
Description
Technical field
The invention belongs to biological technical field, be specifically related to the Unidasa of a kind of Arthrobacter globiformis A152 bacterial strain and generation thereof.
Background technology
Hyaluronic acid (Hyaluronicacid, be called for short HA), have another name called Hyaluronic Acid, it is a kind of chain high molecular polymer be alternately formed by connecting by repeating unit [(1 – 3) β-D-N acetylglucosamine-(GlcNAc)-(1 – 4) β-D-Glucose aldehydic acid (GlcUA)], it is the main component forming extracellular matrix, extensively be present in the biological tissues such as skin, cartilage, joint, vitreum, there is important physiologic function.Can be divided into high molecular weight hyaluronic acid (HMWHA) and low-molecular-weight hyaluronic acid (LMWHA) according to its molecular weight difference, HMWHA can suppress the migration of endotheliocyte, anti-angiogenesis activity, promotes human body wound healing; LMWHA promotes the differentiation of endotheliocyte, anti-apoptotic, and promote cartilage, the isocellular migration of endothelium, also have the effect of anti-inflammatory, HA, with the physico-chemical property of its uniqueness, has played important effect in medical science and study of pharmacy.
Unidasa is hyaluronidase again, is the Glycosylase that can make HA that degraded effect occurs, the acidic mucopolysaccharide also can degraded in other body tissues, and it is extensively present in eukaryote and prokaryotic organism, has specific physiologically active.Unidasa is divided into three major types by the sorting technique of generally acknowledging at present: (1) first kind is endo-beta-N-acetyl glucosaminidase (EC3.2.1.35), is mainly derived from the venom of vertebrates and some biology.This fermentoid is lytic enzyme and transglycosylase, by acting on the end product that β-Isosorbide-5-Nitrae glycosidic link degraded substrate obtains based on tetrose.(2) Equations of The Second Kind is inscribe-beta-glucuronidase enzyme (EC3.2.1.36), mainly from sialisterium and the hookworm extraction of leech.This fermentoid is lytic enzyme, Main Function territory β-1,3 glycosidic link, can selective degradation HA, produces the end product based on tetrose or hexose (reducing end is for glucuronic acid); (3) the 3rd classes are hyaluronate lyase (EC4.2.2.1), are mainly produced by fusobacterium, suis, streptomycete, act on β-l, 4 glycosidic links, degraded by β-cancellation mechanism, the end product after degraded HA is the unsaturated disaccharide of 4,5-.
According to the present inventor by known to inspection information, literature search, report is had to derive from the Unidasa of streptomycete (Streptomyceskoganeiensis) at present, the molecular weight 21.6kDa(patent No.: CN102439144B); Derive from the Unidasa of genus bacillus, the molecular weight 123kDa(patent No.: CN103255076A); Derive from the Unidasa of genus bacillus (Bacillusniacini), molecular weight 120kDa(AtsushiKurata etc.); Derive from the Unidasa biting Nicotine Arthrobacter (Artbrobacternicotinovorous), molecular weight 22.1kDa(Su Kang etc.); Derive from the Unidasa of swine streptococcus (Streptococcussuis), molecular weight 130kDa(AndrewG etc.); Derive from the Unidasa of streptococcus pyogenes (Streptococcuspyogenes), molecular weight 40kDa(SudhirKumarSingh etc.); Derive from the Unidasa of streptococcus, molecular weight 111kDa(JohnE.Coligan etc.); Derive from the Unidasa of streptococcus agalactiae (Streptococcusagalactiae), molecular weight 58kDa(MartinOettl etc.).
Retrieved by bulk information, comparison in the molecular weight of microbes producing cellulase and enzyme, finds no identical with the Unidasa that the present inventor studies.
Summary of the invention
The object of this invention is to provide the Unidasa of a kind of Arthrobacter globiformis and generation thereof, separation and purification of the present invention goes out Arthrobacter globiformis (ArthrobacterglobiformisA152) bacterial strain that a strain derives from ocean, and it is carried out to the preservation of biomaterial, its depositary institution: China typical culture collection center, be called for short CCTCC, address: Hongshan District, Wuhan City, Hubei Province Bayi Road Wuhan University; Preservation date: on October 29th, 2015; Preserving number: CCTCCM2015649, utilizes this bacterial strain can obtain by liquid fermenting the Unidasa that vigor is high, good stability, cost are low.
For achieving the above object, the present invention is achieved by the following technical solutions:
The invention provides a kind of Arthrobacter globiformis, its Classification And Nomenclature is Arthrobacter globiformis (ArthrobacterglobiformisA152), its deposit number: CCTCCM2015649.
During this bacterial strain spherical state, cell dia is 0.6-0.8 μm, and during rod state, cell dia is 0.5-0.8 μm × 1-4 μm, and bacterium colony is faint yellow, rounded, the smooth of the edge.
Present invention also offers the Unidasa that described Arthrobacter globiformis alternating temperature regulation and control are produced, it is obtained by following preparation method:
(1) slant strains is inoculated in sterilized seed culture medium, under the condition of culture temperature 31 DEG C-37 DEG C, rotating speed 120-180rpm, cultivates 16-48h, obtain seed culture fluid;
(2) seed culture fluid is inoculated in sterilized fermention medium, under the condition of culture temperature 18 DEG C-30 DEG C, rotating speed 120-180rpm, cultivates 16-48h, obtain Unidasa fermented liquid;
(3) Unidasa fermented liquid described in centrifugal or filtering separation, gets supernatant liquor;
(4) with supernatant liquor described in ammonium sulfate precipitation, centrifugal or collecting by filtration crude protein;
(5) damping fluid redissolves step (4) crude protein collected, and ultrafiltration or dialysis removing small molecular weight impurity, obtain the Unidasa of purifying.
The molecular weight of obtained described Unidasa is 73.7kDa.
Described preparation method also comprises step (6): utilized by the Unidasa obtained in step (5) QFF ion-exchange chromatography or SephadexG100 gel filtration chromatography to refine, obtain electrophoretically pure Unidasa.
Described preparation method also comprises step (7): the Unidasa that step (5) or step (6) obtain is carried out vacuum lyophilization.
Advantage of the present invention and technique effect are: the present invention relates to the Arthrobacter globiformis that a strain derives from ocean.Through gene sequencing technology, genetic marker is the 16SrDNA nucleotide sequence comprised shown in sequence table, by the homology comparison of GenBank database and Physiology and biochemistry qualification, determines that it is Arthrobacter globiformis, called after ArthrobacterglobiformisA152.This bacterial strain cultivates 24h in the fermentation medium, in its fermented liquid, the vigor of Unidasa reaches the highest, with the production bacterial strain of bacterial strain of the present invention as Unidasa, have that Product Activity is high, good stability, the feature that with short production cycle, cost is low, can suitability for industrialized production be realized.
The alternating temperature that the present invention relates to a kind of Unidasa and Unidasa that derive from ArthrobacterglobiformisA152 regulates and controls production method, and the feature of this enzyme is that molecular weight is 73.7kDa, and its molecular weight is different from the Unidasa reported.Collected to the alternating temperature regulation and control of fermented liquid cultivation stage, frozen centrifugation, ammonium sulfate precipitation method by seed liquor cultivation stage, ultrafiltration or dialysis, obtain the Unidasa of purifying, by the Unidasa of purifying by Q-Sepharose FastFlow anion-exchange chromatography and the process of SephadexG-100 gel permeation chromatography, obtain a kind of electrophoretically pure Unidasa.This strain enzyme-producing has the advantages that nutrition scope extensively, is easily cultivated and incubation time is short, the highest 7100U/mL of hyaluronic acid enzyme activity in fermented liquid.
Accompanying drawing explanation
Fig. 1: the Unidasa SDS-PAGE electrophoresis result after purifying.The left side is standard protein molecular weight, and the right is that Arthrobacter globiformis A152 is prepared and purified Unidasa single tape, quantity representation unit: kDa.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described further
1. the screening of a strain ArthrobacterglobiformisA152 bacterial strain and purifying
The acquisition of ArthrobacterglobiformisA152 bacterial strain of the present invention: from seashore tideland, northern Shandong, Qingdao of Shandong province mud, joins and (hyaluronic acid 0.2g, extractum carnis 0.5g, peptone 0.5g, K in the enrichment medium of 25mL is housed
2hPO
41g, MgSO
40.5g, NaCl0.5g, CaCl
20.01g, H
2o1000mL, natural pH) triangular flask in, 28 DEG C, 160rpm shake-flask culture 2d, after then enrichment culture liquid stroke-physiological saline solution suitably being diluted, gets 0.1ml and is coated with primary dcreening operation isolation medium (hyaluronic acid 0.2g, K
2hPO
41g, MgSO
40.5g, NaCl0.5g, CaCl
20.01g, extractum carnis 0.5g, peptone 0.5g, bovine serum albumin: 0.1g, agar powder 18.0g, H
2o1000mL, natural pH) dull and stereotyped.Be inverted for 28 DEG C and cultivate 2d, obtain and choose bacterium colony flat board having transparent circle proterties, obtain the bacterium that a strain can produce Unidasa and be ArthrobacterglobiformisA152 bacterial strain.
2. the qualification of ArthrobacterglobiformisA152 bacterial strain described in
Chelex-100 genome is adopted to extract DNA, forward primer 27F:5 '-AGAGTTTGATCMTGCTCAG-3 ' (SEQIDNo:2), reverse primer 1492R:5 '-ACGGCTACCTTGTTACGACTT-3 ' (SEQIDNo:3).Reaction system 50ul; Reaction conditions: 94 DEG C of denaturation 2min, 94 DEG C of sex change 30s, 55 DEG C of annealing 40s, 72 DEG C extend 1min, and last 72 DEG C extend 10min.The purifying of PCR primer, clone, order-checking are completed by Shanghai biotechnology company limited, 16SrDNA gene order sequencing result (SEQIDNo:1) is carried out homology comparison at GenBank database and determines that it is genus arthrobacter.
Again through the physiology and morphology biochemical identification of Bergey's Mannual genus arthrobacter, it is characterized in that: Gram-positive, obligate aerobic, does not produce gemma, can hydrolyzed starch, reduction nitrate, well-grown in Nutrient meat soup, bacterial strain is not strict to nutritional requirement, can be single nitrogenous source with ammonium chloride, glucose is grow in the minimal medium of carbon source and the energy, does not need to add vitamin H, VitB1, pantothenic acid, B
12.During bacterial strain spherical state, cell dia is about 0.6-0.8 μm, and during rod state, cell dia is 0.5-0.8 μm × 1-4 μm.This strains separation is from coastal waters mud, and 10 DEG C of growths, optimum temperuture is at 25 DEG C-30 DEG C, on nutrient agar, 28 DEG C of cultivation 24h become single bacterium colony, and bacterium colony is faint yellow, circular, the smooth of the edge, determines that it is Arthrobacter globiformis, called after ArthrobacterglobiformisA152 bacterial strain.
Bacterial strain preservation is carried out to this bacterial strain, its depositary institution: China typical culture collection center, be called for short CCTCC, address: Hongshan District, Wuhan City, Hubei Province Bayi Road Wuhan University; Preservation date: on October 29th, 2015; Deposit number: CCTCCM2015649.
3. utilize described ArthrobacterglobiformisA152 bacterial strain alternating temperature to regulate and control to produce Unidasa
By described ArthrobacterglobiformisA152 inoculation to sterilized seed culture medium (hyaluronic acid 0.8g, extractum carnis 15g, peptone 10g, MgSO
40.5g, NaCl5g, FCl
30.1g, H
2o1000mL, natural pH) in, culture temperature 32 DEG C, cultivates rotating speed 160rpm, incubation time 36h, obtains seed culture fluid.Seed culture fluid is inoculated into fermention medium (hyaluronic acid 3g, extractum carnis 5g, peptone 10g, MgSO40.5g, NaCl15g, the CaCl of sterilizing
20.01g, H
2o1000mL, nature pH), culture temperature 22 DEG C (non-alternating temperature regulation and control group culture temperature is 32 DEG C), cultivate rotating speed 180rpm, incubation time 24h, obtain the fermented liquid containing Unidasa, non-alternating temperature regulation and control group fermentation broth enzyme 5461U/ml alive, alternating temperature regulation and control group fermentation broth enzyme is lived as 7100U/ml, and with non-alternating temperature regulation and control group for contrast, alternating temperature regulation and control make fermentation broth enzyme live and improve 30%.
The present invention is all to be related to Unidasa definition alive and measures all with reference to description below:
(1) Unidasa measuring method: 2ml20mMpH6.0 alive contains the phosphate buffered saline buffer of 0.2%HA, adds 20 μ l enzyme liquid, and 37 DEG C of reaction 20min, add 2mlDNS reagent, measure the reducing sugar content reacting and terminate in rear system under 520nm wavelength.
(2) hyaluronic acid enzyme activity definition: at 37 DEG C, under the condition of pH6.0, every min degraded hyaluronic acid enzyme amount produced required for 1 μ g reducing sugar (with glucose meter) is defined as an enzyme activity unit (U)
4. derive from the hyaluronic acid enzyme purification of described ArthrobacterglobiformisA152 bacterial strain
By frozen centrifugation removing fermented liquid thalline, centrifugal condition: temperature 4 DEG C, rotating speed 8000rpm, centrifugation time 10min, obtains the fermented supernatant fluid containing Unidasa.Fermented supernatant fluid is collecting precipitation after 70% saturation ratio ammonium sulfate precipitation, redissolves with the phosphoric acid salt of 20mMpH7.5, through molecular weight cut-off be 3kDa poly (ether-sulfone) ultrafiltration membrane removing small molecular weight impurity, (specific activity is 1.1 × 10 to concentrated 10 times of Unidasas obtaining purifying
4u/mg).
By the Unidasa application of sample of purifying in Q-Sepharose FastFlow post, elutriant, for containing NaCl (0 ~ 1M) 20mM phosphoric acid salt (pH7.5) damping fluid, carries out gradient elution, detects protein peak under 280nm, DNS method is surveyed enzyme and is lived, and collects activated albumen.By the activated protein application of sample after Q-Sepharose FastFlow partial purification in SephadexG100 gel column, elutriant is 20mM phosphoric acid salt (pH7.5) damping fluid, protein peak is detected under 280nm, DNS method is surveyed after enzyme is lived and is collected activated albumen, and (specific activity is 6.9 × 10 to obtain Unidasa refining further
4u/mg).
5. derive from the Unidasa molecular weight determination of described ArthrobacterglobiformisA152 bacterial strain
Concrete grammar: by concentrated 5 times of the described hyaluronidase protein component obtained through gel chromatography chromatography, get 20 μ L, add 8 μ LSampleBuffer in boiling water bath, to heat 5min make its sex change, after taking out cooling, loading electrophoresis together with pre-dyed maker, to dye 45min with coomassie brilliant blue R250 after electrophoresis, the migration distance of bioassay standard protein and sample after decolouring, calculate relative mobility, typical curve is done with method of least squares with the logarithm of relative mobility and each standard protein molecular weight, calculate the molecular weight of this enzyme, the molecular weight that this enzyme shows on SDS-PAGE is: 73.7kDa (see accompanying drawing 1).
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
SEQUENCELISTING
Limited-liability company of <110> Qingdao Haiyang biological medicine research institute
The Unidasa of <120> Arthrobacter globiformis and generation thereof
<130>2015
<160>1
<170>PatentInversion3.3
<210>1
<211>1095
<212>DNA
<213>Arthrobacterglobiformis
<400>1
gagccggatcatattcgacggctcccccacaagggttaggccaccggcttcgggtgttac60
caactttcgtgacttgacgggcggtgtgtacaaggcccgggaacgtattcaccgcagcgt120
tgctgatctgcgattactagcgactccgacttcatggggtcgagttgcagaccccaatcc180
gaactgagaccggctttttgggattagctccacctcacagtatcgcaaccctttgtaccg240
gccattgtagcatgcgtgaagcccaagacataaggggcatgatgatttgacgtcgtcccc300
accttcctccgagttgaccccggcagtctcctatgagtccccgccataacgcgctggcaa360
catagaacgagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctga420
cgacaaccatgcaccacctgtaaaccgaccgcaagcggggcacctgtttccaggtctttc480
cggttcatgtcaagccttggtaaggttcttcgcgttgcatcgaattaatccgcatgctcc540
gccgcttgtgcgggcccccgtcaattcctttgagttttagccttgcggccgtactcccca600
ggcggggcacttaatgcgttagctacggcgcggaaaacgtggaatgtcccccacacctag660
tgcccaacgtttacggcatggactaccagggtatctaatcctgttcgctccccatgcttt720
cgctcctcagcgtcagttacagcccagagacctgccttcgccatcggtgttcctcctgat780
atctgcgcatttcaccgctacaccaggaattccagtctcccctactgcactctagtctgc840
ccgtacccactgcagaaccggagttgagcgccggtctttcacagcagacgcgacaaccgc900
ctacgagctctttacgcccaataattccggataacgcttgcgccctacgtattaccgcgg960
ctgctgcacgtagttaggcggcgcttcttctgcaggtaccgtcactttcgcttcttccta1020
ctgaagaggtttacaacacgaaggcggtcatccctcacgcggcgtcgctgcatcaggctt1080
gcgcccatgtgtgca1095
<210>2
<211>19
<212>DNA
<213> artificial sequence
<400>2
agagtttgatcmtgctcag19
<210>3
<211>21
<212>DNA
<213> artificial sequence
<400>3
acggctaccttgttacgactt21
Claims (6)
1. an Arthrobacter globiformis, it is characterized in that its Classification And Nomenclature is Arthrobacter globiformis ArthrobacterglobiformisA152, its depositary institution: China typical culture collection center, preservation date: on October 29th, 2015, deposit number: CCTCCM2015649.
2. Arthrobacter globiformis according to claim 1, is characterized in that: during bacterial strain spherical state, cell dia is 0.6-0.8 μm, and during rod state, cell dia is 0.5-0.8 μm × 1-4 μm, and bacterium colony is faint yellow, rounded, the smooth of the edge.
3. the Unidasa of Arthrobacter globiformis alternating temperature regulation and control production according to claim 1, is characterized in that it is obtained by following preparation method:
(1) slant strains is inoculated in sterilized seed culture medium, under the condition of culture temperature 31 DEG C-37 DEG C, rotating speed 120-180rpm, cultivates 16-48h, obtain seed culture fluid;
(2) seed culture fluid is inoculated in sterilized fermention medium, under the condition of culture temperature 18 DEG C-30 DEG C, rotating speed 120-180rpm, cultivates 16-48h, obtain Unidasa fermented liquid;
(3) Unidasa fermented liquid described in centrifugal or filtering separation, gets supernatant liquor;
(4) with supernatant liquor described in ammonium sulfate precipitation, centrifugal or collecting by filtration crude protein;
(5) damping fluid redissolves step (4) crude protein collected, and ultrafiltration or dialysis removing small molecular weight impurity, obtain the Unidasa of purifying.
4. Unidasa according to claim 3, is characterized in that: the molecular weight of obtained described Unidasa is 73.7kDa.
5. Unidasa according to claim 3, it is characterized in that: described preparation method also comprises step (6): utilized by the Unidasa obtained in step (5) QFF ion-exchange chromatography or SephadexG100 gel filtration chromatography to refine, obtain electrophoretically pure Unidasa.
6. the Unidasa according to claim 3 or 5, is characterized in that: described preparation method also comprises step (7): the Unidasa that step (5) or step (6) obtain is carried out vacuum lyophilization.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110331178A (en) * | 2019-06-27 | 2019-10-15 | 青岛海洋生物医药研究院股份有限公司 | A kind of enzyme cutting method prepares the method for micromolecule hyaluronic acid and gained micromolecule hyaluronic acid is applied with it |
| CN116731902A (en) * | 2023-03-15 | 2023-09-12 | 甘肃省科学院生物研究所 | Arthrobacter sphaeroides GCG3, application thereof and antibacterial application thereof |
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