CN105567606B - A kind of Arthrobacter globiformis and its hyaluronidase of generation - Google Patents
A kind of Arthrobacter globiformis and its hyaluronidase of generation Download PDFInfo
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- CN105567606B CN105567606B CN201610115793.0A CN201610115793A CN105567606B CN 105567606 B CN105567606 B CN 105567606B CN 201610115793 A CN201610115793 A CN 201610115793A CN 105567606 B CN105567606 B CN 105567606B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
Abstract
The present invention provides a kind of Arthrobacter globiformis and its hyaluronidases of generation.The present invention relates to a kind of Arthrobacter globiformis from intertidal zone sludgeArthrobacter globiformisA152, which has the characteristics that nutritional condition is simple, easily cultivates, produces hyaluronidase using bacterial strain of the invention, has the characteristics that producing enzyme vigor is high, stability is good, at low cost, can be realized large-scale culture, meet industrialized application.Regulate and control the hyaluronidase that production method obtains by alternating temperature the invention further relates to a kind of, seed culture is transferred to during fermented and cultured and carries out low temperature induction fermentation, fermentation liquid is by isolating and purifying, being freeze-dried, the hyaluronic acid enzyme product of different size can be obtained, this method has yield of enzyme high, advantage with short production cycle, easy to operate.It is derived from the present inventionArthrobacter globiformis The hyaluronidase of A152, SDS-PAGE determine that the enzyme molecular weight is 73.7 kDa.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of Arthrobacter globiformis A152 bacterial strain and its generation it is transparent
Matter acid enzyme.
Background technique
Hyaluronic acid (Hyaluronic acid, abbreviation HA) also known as Hyaluronic Acid are by repetitive unit [(1-3) β-D-
N acetylglucosamine-(GlcNAc)-(1-4) β-D-Glucose aldehydic acid (GlcUA)] replace a kind of chain height being formed by connecting
Molecularly Imprinted Polymer is the main component for constituting extracellular matrix, is widely present in the biological group such as skin, cartilage, joint, vitreum
In knitting, there is important physiologic function.According to its molecular weight difference can be divided into high molecular weight hyaluronic acid (HMWHA) and
Low-molecular-weight hyaluronic acid (LMWHA), HMWHA are able to suppress the migration of endothelial cell, and anti-angiogenesis activity promotes human body
Wound healing；LMWHA promotes the differentiation of endothelial cell, and anti-apoptotic promotes cartilage, the isocellular migration of endothelium, also has
Anti-inflammatory effect, HA played an important role in terms of medicine and study of pharmacy with its unique physicochemical property.
Hyaluronidase is called hyaluronidase, is the glycosidase that can make HA that degraded effect occur, can also degrade other
Acid mucopolysaccharide in body tissue, is widely present in eucaryote and prokaryotes, has the physiology of specificity living
Property.Hyaluronidase is divided into three categories by generally acknowledged classification method at present: (1) first kind is endo-beta-N-acetyl aminoglucose
Glycosidase (EC3.2.1.35) is mainly derived from the venom of vertebrate and certain biologies.This fermentoid is hydrolase and turns sugared
Glycosides enzyme obtains the final product based on tetrose by acting on β-Isosorbide-5-Nitrae glycosidic bond degradation substrate.(2) second classes are inscribe-β-Portugals
Grape glycuronide enzyme (EC 3.2.1.36) is mainly extracted from the salivary gland of leech and hookworm.The fermentoid is hydrolase,
Main function domain β -1,3 glycosidic bonds, can selective degradation HA, generate and using tetrose or hexose (reducing end is glucuronic acid) be
Main final product；(3) third class is hyaluronate lyase (EC4.2.2.1), mainly by fusobacterium, streptococcus, streptomycete
It generates, acts on β-l, 4 glycosidic bonds are degraded by β-cancellation mechanism, and the final product after the HA that degrades is 4,5- unsaturated double
Sugar.
According to the present inventor by being had been reported that at present from streptomycete known to inspection information, literature search
(Streptomyces koganeiensis) hyaluronidase, the molecular weight 21.6kDa(patent No.: 102439144 B of CN)；
From the hyaluronidase of bacillus, the molecular weight 123kDa(patent No.: 103255076 A of CN)；From bacillus
(Bacillus niacini) hyaluronidase, molecular weight 120kDa(Atsushi Kurata etc.)；From biting nicotine
Arthrobacterium (Artbrobacter nicotinovorous) hyaluronidase, molecular weight 22.1kDa(Su Kang etc.)；It derives from
Streptococcus suis (Streptococcus suis) hyaluronidase, 130 kDa(Andrew G of molecular weight etc.)；From suppuration
Streptococcus (Streptococcus pyogenes) hyaluronidase, 40 kDa(of molecular weightSudhir Kumar Singh
Deng)；From the hyaluronidase of streptococcus, 111 kDa(John E. Coligan of molecular weight etc.)；From agalasisa chain
Coccus (Streptococcus agalactiae) hyaluronidase, 58 kDa(Martin Oettl of molecular weight etc.).
It is retrieved by bulk information, is compared in terms of the molecular weight of microbes producing cellulase and enzyme, found no and the present inventor
The hyaluronidase of research is identical.
Summary of the invention
The object of the present invention is to provide a kind of Arthrobacter globiformis and its hyaluronidase of generation, the present invention to isolate and purify
Out one plant of source Yu Haiyang Arthrobacter globiformis (Arthrobacter globiformisA152) bacterial strain, and it is given birth to
The preservation of object material, depositary institution: China typical culture collection center, abbreviation CCTCC, address: Wuhan City, Hubei Province flood
Mountain area Bayi Road Wuhan University；Preservation date: on October 29th, 2015；Deposit number: CCTCC M 2015649 utilizes the bacterial strain
The hyaluronidase that vigor is high, stability is good, at low cost can be obtained by liquid fermentation.
For achieving the above object, the present invention is achieved by the following scheme:
The present invention provides a kind of Arthrobacter globiformis, classification naming be Arthrobacter globiformis (Arthrobacter globiformisA152), deposit number: CCTCC M 2015649.
Cell dia is 0.6-0.8 μm when the bacterial strain spherical shape state, and cell dia is 1-4 μm of 0.5-0.8 μ m when rod state,
Bacterium colony is faint yellow, rounded, the smooth of the edge.
The present invention also provides the hyaluronidases of Arthrobacter globiformis alternating temperature regulation production, it passes through following preparation
Method is made:
(1) slant strains are inoculated into sterilized seed culture medium, in 31 DEG C -37 DEG C of cultivation temperature, revolving speed 120-
16-48h is cultivated under conditions of 180rpm, obtains seed culture fluid；
(2) seed culture fluid is inoculated into sterilized fermentation medium, in 18 DEG C -30 DEG C of cultivation temperature, revolving speed
16-48h is cultivated under conditions of 120-180rpm, obtains hyaluronidase fermentation liquid；
(3) it is centrifuged or is separated by filtration the hyaluronidase fermentation liquid, take supernatant；
(4) supernatant described in ammonium sulfate precipitation, is centrifuged or is collected by filtration crude protein；
(5) buffer redissolves the crude protein that step (4) are collected, and ultrafiltration or dialysis remove small molecular weight impurity, purified
Hyaluronidase.
The molecular weight of the hyaluronidase obtained is 73.7 kDa.
The preparation method further includes step (6): hyaluronidase obtained in step (5) is utilized QFF ion exchange
Chromatography or the purification of Sephadex G100 gel filtration chromatography, obtain electrophoretically pure hyaluronidase.
The preparation method further includes step (7): the hyaluronidase that step (5) or step (6) are obtained carries out vacuum
Freeze-drying.
Advantages of the present invention and have the technical effect that the present invention relates to the Arthrobacter globiformis of one plant of source Yu Haiyang.By base
Because of sequencing technologies, genetic marker is comprising 16SrDNA nucleic acid sequence shown in sequence table, by homologous in GenBank database
It compares and Physiology and biochemistry is identified, determine that it is Arthrobacter globiformis, be named asArthrobacter globiformis A152.It should
Bacterial strain is cultivated for 24 hours in the fermentation medium, and the vigor of hyaluronidase reaches highest in fermentation liquid, is made with bacterial strain of the invention
For the production bacterial strain of hyaluronidase, have the characteristics that Product Activity is high, stability is good, with short production cycle, at low cost, Neng Goushi
Existing industrialized production.
The present invention relates to one kind to derive fromArthrobacter globiformis The hyaluronidase and hyalomitome of A152
The alternating temperature of sour enzyme regulates and controls production method, which is characterized in that molecular weight is 73.7kDa, and molecular weight is different from reported
Bright matter acid enzyme.It is received by the alternating temperature regulation of seed liquor cultivation stage to fermentation liquid cultivation stage, refrigerated centrifuge, ammonium sulfate precipitation method
Collection, ultrafiltration or dialysis, obtain the hyaluronidase of purifying, and the hyaluronidase of purifying is passed through Q-Sepharose Fast
Flow anion-exchange chromatography and the processing of Sephadex G-100 gel permeation chromatography, obtain a kind of electrophoretically pure hyaluronic acid
Enzyme.The strain enzyme-producing has the characteristics that nutrition range is wide, be easy culture and incubation time is short, hyaluronic acid enzyme activity in fermentation liquid
Highest 7100U/mL.
Detailed description of the invention
Fig. 1: hyaluronidase SDS-PAGE electrophoresis result after purification.The left side is standard protein molecular weight, and the right is ball
Shape arthrobacterium A152 preparation and purified hyaluronidase single tape, quantity indicate unit: kDa.
Specific embodiment
Technical scheme is described further combined with specific embodiments below
1. one plantArthrobacter globiformis The screening and purifying of A152 bacterial strain
The present inventionArthrobacter globiformis The acquisition of A152 bacterial strain: from Qingdao of Shandong province northern Shandong seashore
Intertidal zone mud, be added in the enriched medium equipped with 25mL (hyaluronic acid 0.2g, beef extract 0.5g, peptone 0.5g,
K2HPO4 1g, MgSO4 0.5g, NaCl 0.5g, CaCl2 0.01g, H2O 1000mL, natural pH) triangular flask in, 28 DEG C,
160rpm shaking flask culture 2d after then suitably diluting enrichment culture liquid with sterile saline, takes 0.1ml coating just screening
From culture medium (hyaluronic acid 0.2g, K2HPO4 1g, MgSO4 0.5g, NaCl 0.5g, CaCl2 0.01g, beef extract 0.5g,
Peptone 0.5g, bovine serum albumin(BSA): 0.1g, agar powder 18.0g, H2O 1000mL, natural pH) plate.28 DEG C of inversion trainings
2d is supported, obtains the bacterium colony chosen and have transparent circle character on plate, obtaining the bacterium that one plant can produce hyaluronidase isArthrobacter globiformis A152 bacterial strain.
Described in 2.Arthrobacter globiformis The identification of A152 bacterial strain
DNA, forward primer 27F:5 '-AGAGTTTGATCMTGCTCAG-3 ' (SEQ are extracted using chelex-100 genome
ID No:2), reverse primer 1492R:5 '-ACGGCTACCTTGTTACGACTT-3 ' (SEQ ID No:3).Reaction system 50ul；
Reaction condition: 94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 40s, 72 DEG C of extension 1min, last 72 DEG C extend
10min.Purifying, clone, the sequencing of PCR product are completed by Shanghai bioengineering Co., Ltd, and 16SrDNA gene order is sequenced
As a result (SEQ ID No:1) carries out homologous comparison in GenBank database and determines that it is Arthrobacter.
It is identified using the form and Physiology and biochemistry of Bergey's Mannual Arthrobacter, it is characterized in that: Gram-positive, obligate good
Oxygen does not generate gemma, can hydrolyze starch, restores nitrate, the well-grown in Nutrient meat soup, bacterial strain is to nutritional requirement
It is not stringent, can with ammonium chloride for single nitrogen source, glucose be carbon source and the energy minimal medium in grow, do not need to add
Add biotin, thiamine, pantothenic acid, B12.Cell dia is about 0.6-0.8 μm when bacterial strain spherical shape state, and cell dia is when rod state
0.5-0.8μm×1-4μm.The strain isolation is grown at 10 DEG C from coastal waters sludge, and optimum temperature is at 25 DEG C -30 DEG C, nutrient agar
For 24 hours at single colonie, bacterium colony is faint yellow for 28 DEG C of cultures on culture medium, and round, the smooth of the edge determines that it is Arthrobacter globiformis, orders
It is entitledArthrobacter globiformis A152 bacterial strain.
Bacterial strain preservation, depositary institution: China typical culture collection center, abbreviation CCTCC, ground are carried out to the bacterial strain
Location: Wuhan City, Hubei Province Hongshan District Bayi Road Wuhan University；Preservation date: on October 29th, 2015；Deposit number: CCTCC M
2015649。
3. described in utilizingArthrobacter globiformis A152 bacterial strain alternating temperature regulation production hyaluronidase
It will be describedArthrobacter globiformis A152 strain inoculated is (saturating to sterilized seed culture medium
Bright matter acid 0.8g, beef extract 15g, peptone 10g, MgSO4 0.5g, NaCl 5g, FCl3 0. 1g, H2O 1000mL is natural
PH in), 32 DEG C of cultivation temperature, revolving speed 160rpm, incubation time 36h is cultivated, obtains seed culture fluid.Seed culture fluid is inoculated with
To fermentation medium (hyaluronic acid 3g, beef extract 5g, peptone 10g, MgSO4 0.5g, the NaCl 15g, CaCl of sterilizing2
0.01g, H2O 1000mL, natural pH), 22 DEG C of cultivation temperature (non-alternating temperature regulation group cultivation temperature is 32 DEG C) cultivates revolving speed
180rpm, incubation time for 24 hours, obtain the fermentation liquid containing hyaluronidase, and non-alternating temperature regulates and controls group fermentation liquid enzyme activity 5461U/ml,
It is 7100U/ml that alternating temperature, which regulates and controls group fermentation liquid enzyme activity, is control with non-alternating temperature regulation group, and alternating temperature regulation makes fermentation liquid enzyme activity
Improve 30%.
All hyaluronic acid enzyme activity that are related to of the present invention are defined with measurement all with reference to following description:
(1) hyaluronic acid enzyme activity measuring method: 6.0 phosphate buffer containing 0.2% HA of 2ml 20mM pH adds
Add 20 μ l enzyme solutions, 37 DEG C of reaction 20min add 2mlDNS reagent, the reduction in system after reaction is measured under 520nm wavelength
Sugared content.
(2) hyaluronic acid enzyme activity defines: under conditions of 37 DEG C, pH 6.0, every min degradation hyaluronic acid generates 1 μ g
Enzyme amount required for reduced sugar (with glucose meter) is defined as an enzyme activity unit (U)
4. from describedArthrobacter globiformis The hyaluronic acid enzyme purification of A152 bacterial strain
Fermentation liquid thallus, centrifugal condition: 4 DEG C of temperature, revolving speed 8000rpm, centrifugation time are removed by refrigerated centrifuge
10min obtains the fermented supernatant fluid containing hyaluronidase.Fermented supernatant fluid collects precipitating after 70% saturation degree ammonium sulfate precipitation,
It is redissolved with the phosphate of 20mM pH7.5, removes small molecular weight impurity, concentration through the poly (ether-sulfone) ultrafiltration membrane that molecular cut off is 3 kDa
10 times of hyaluronidases purified (are 1.1 × 10 than living4U/mg).
The hyaluronidase of purifying is loaded onto Q-Sepharose Fast Flow column, eluent is to contain NaCl
(0~1M) 20mM phosphate (pH 7.5) buffer carries out gradient elution, and protein peak is detected at 280nm, and DNS method surveys enzyme
It is living, collect active albumen.Activated protein after Q-Sepharose Fast Flow partial purification is loaded onto
Sephadex G100 gel column, eluent are 20mM phosphate (pH 7.5) buffer, detect albumen at 280nm
Peak, DNS method collect active albumen after surveying enzyme activity, and obtaining the hyaluronidase further refined (is 6.9 × 10 than living4U/
Mg).
5. from describedArthrobacter globiformis The hyaluronidase molecular weight determination of A152 bacterial strain
Specific method: being concentrated 5 times for the hyaluronidase protein component chromatographed through gel chromatography, take 20 μ L,
8 μ L Sample Buffer are added heating 5min in boiling water bath makes its denaturation, take out it is cooling after, together with pre-dyed maker
Loading electrophoresis dyes 45min with coomassie brilliant blue R250 after electrophoresis, measured after decoloration the migration of standard protein and sample away from
From calculating relative mobility does standard song with the logarithm of relative mobility and each standard protein molecular weight with least square method
Line calculates the molecular weight of this enzyme, the molecular weight that this enzyme is shown on SDS-PAGE are as follows: 73.7 kDa (see attached drawing 1).
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although referring to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
SEQUENCE LISTING
<110>limited liability company, Qingdao Haiyang biological medicine research institute
<120>a kind of Arthrobacter globiformis and its hyaluronidase of generation
<130> 2015
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1095
<212> DNA
<213> Arthrobacter globiformis
<400> 1
gagccggatc atattcgacg gctcccccac aagggttagg ccaccggctt cgggtgttac 60
caactttcgt gacttgacgg gcggtgtgta caaggcccgg gaacgtattc accgcagcgt 120
tgctgatctg cgattactag cgactccgac ttcatggggt cgagttgcag accccaatcc 180
gaactgagac cggctttttg ggattagctc cacctcacag tatcgcaacc ctttgtaccg 240
gccattgtag catgcgtgaa gcccaagaca taaggggcat gatgatttga cgtcgtcccc 300
accttcctcc gagttgaccc cggcagtctc ctatgagtcc ccgccataac gcgctggcaa 360
catagaacga gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga 420
cgacaaccat gcaccacctg taaaccgacc gcaagcgggg cacctgtttc caggtctttc 480
cggttcatgt caagccttgg taaggttctt cgcgttgcat cgaattaatc cgcatgctcc 540
gccgcttgtg cgggcccccg tcaattcctt tgagttttag ccttgcggcc gtactcccca 600
ggcggggcac ttaatgcgtt agctacggcg cggaaaacgt ggaatgtccc ccacacctag 660
tgcccaacgt ttacggcatg gactaccagg gtatctaatc ctgttcgctc cccatgcttt 720
cgctcctcag cgtcagttac agcccagaga cctgccttcg ccatcggtgt tcctcctgat 780
atctgcgcat ttcaccgcta caccaggaat tccagtctcc cctactgcac tctagtctgc 840
ccgtacccac tgcagaaccg gagttgagcg ccggtctttc acagcagacg cgacaaccgc 900
ctacgagctc tttacgccca ataattccgg ataacgcttg cgccctacgt attaccgcgg 960
ctgctgcacg tagttaggcg gcgcttcttc tgcaggtacc gtcactttcg cttcttccta 1020
ctgaagaggt ttacaacacg aaggcggtca tccctcacgc ggcgtcgctg catcaggctt 1080
gcgcccatgt gtgca 1095
<210> 2
<211> 19
<212> DNA
<213>artificial sequence
<400> 2
agagtttgat cmtgctcag 19
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<400> 3
acggctacct tgttacgact t 21
Claims (6)
1. a kind of Arthrobacter globiformis, it is characterised in that its classification naming is Arthrobacter globiformisArthrobacter globiformis
A152, depositary institution: China typical culture collection center, preservation date: on October 29th, 2015, deposit number: CCTCC
M 2015649；Cell dia is 0.6-0.8 μm when bacterial strain spherical shape state, and cell dia is 1-4 μm of 0.5-0.8 μ m when rod state,
Bacterium colony is faint yellow, rounded, the smooth of the edge.
2. the hyaluronidase of Arthrobacter globiformis alternating temperature regulation production described in claim 1, it is characterised in that it passes through following
Preparation method is made:
(1) slant strains are inoculated into sterilized seed culture medium, in 31 DEG C -37 DEG C of cultivation temperature, revolving speed 120-
16-48h is cultivated under conditions of 180rpm, obtains seed culture fluid；
(2) seed culture fluid is inoculated into sterilized fermentation medium, in 18 DEG C -30 DEG C of cultivation temperature, revolving speed 120-
16-48h is cultivated under conditions of 180rpm, obtains hyaluronidase fermentation liquid；
(3) it is centrifuged or is separated by filtration the hyaluronidase fermentation liquid, take supernatant；
(4) supernatant described in ammonium sulfate precipitation, is centrifuged or is collected by filtration crude protein；
(5) buffer redissolves the crude protein that step (4) are collected, and ultrafiltration or dialysis remove small molecular weight impurity, and what is purified is transparent
Matter acid enzyme.
3. hyaluronidase according to claim 2, it is characterised in that: the molecular weight of the hyaluronidase obtained is
73.7 kDa。
4. hyaluronidase according to claim 2, it is characterised in that: the preparation method further includes step (6): will be walked
Suddenly hyaluronidase obtained in (5) is refined using QFF ion-exchange chromatography or Sephadex G100 gel filtration chromatography, is obtained
To electrophoretically pure hyaluronidase.
5. hyaluronidase according to claim 2, it is characterised in that: the preparation method further includes step (6): will be walked
Suddenly the hyaluronidase that (5) obtain carries out vacuum freeze drying.
6. hyaluronidase according to claim 4, it is characterised in that: the preparation method further includes step (7): will be walked
Suddenly the hyaluronidase that (6) obtain carries out vacuum freeze drying.
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