CN101886098A - Application of Arthrobacter nicotinovorans in hyaluronic acid degradation - Google Patents
Application of Arthrobacter nicotinovorans in hyaluronic acid degradation Download PDFInfo
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- CN101886098A CN101886098A CN2010101916036A CN201010191603A CN101886098A CN 101886098 A CN101886098 A CN 101886098A CN 2010101916036 A CN2010101916036 A CN 2010101916036A CN 201010191603 A CN201010191603 A CN 201010191603A CN 101886098 A CN101886098 A CN 101886098A
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Abstract
The invention discloses a new use of Arthrobacter nicotinovorans (the preservation number is CGMCC No.1.1924 and the preservation date is January 16, 2000), and in particular relating to an application of the Arthrobacter nicotinovorans in hyaluronic acid degradation, wherein, the Arthrobacter nicotinovorans has efficient degradation effect on hyaluronic acid under normal temperature. The degradation method of the hyaluronic acid comprises two modes as follows: 1. in the later stage of fermentation production of the hyaluronic acid, adding the Arthrobacter nicotinovorans into a culture, and then carrying out mixed fermentation to obtain low molecular weight hyaluronic acid; and 2. adding the Arthrobacter nicotinovorans to a high molecular weight hyaluronic acid solution to act for 2-24 hours to obtain the low molecular weight hyaluronic acid.
Description
Technical field
The present invention relates to the application of Arthrobacter nicotinovorans in hyaluronic acid degradation, particularly the application of Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) CGMCC NO1.1924 in the degraded hyaluronic acid of Unidasa produced in a strain.
Background technology
Hyaluronic acid has another name called Hyaluronic Acid, is important physical active substance in the body.Hyaluronic acid is in the different metabolism stages, and because of the difference of its relative molecular mass produces different biological activitys, low molecular weight hyaluronic acid has some biological activity that polymer hyaluronic acid does not have, even both have antipodal effect.Low molecular weight hyaluronic acid easily absorbs, and synthetic macromolecule hyaluronic acid again increases the hyaluronic content of endogenous in vivo, can bring into play the effect of health care and beautifying face and moistering lotion.At present fermentation method and the hyaluronic acid relative molecular weight that extracts from animal tissues are generally all more than 1,000,000, and the hyaluronic method of degrading at present mainly comprises: physics method, chemical method and enzyme process.
The enzymic degradation of biomacromolecule does not have an advantage such as by product because its specificity is strong, efficient is high, reaction conditions is gentle, often is used for macromolecular degraded by people's first-selection.But because the source of Unidasa is very limited, cost an arm and a leg, limited their application to a great extent.A lot of Grain-negatives and gram-positive bacteria all produce the hyaluronic enzyme of degradable.The enzyme that gram-negative bacteria produces is present in periplasmic space rather than is discharged into outside the born of the same parents, and is unlikely relevant with pathogenesis, and these enzymes more similarly are chondroitinase.The gram positive organism that produces Unidasa has Streptococcus, Staphylococcus, Clostridium, Propionibacterium, Peptostreptococcus and Streptomyces.Clostridiumperfringens, Propionibacterium acens and Peptostreptococcus are anerobe, and be few to its research.Report about the suis Unidasa is then a lot, and deeply.Other hyaluronic acid degradation enzyme as Treponema pallidum and T.pertenue generation surface association, but seem lytic enzyme, also need further research.Some bacterium of the discovery Candida such as Shimizu of Brazil also produce Unidasa, but not further research reported again afterwards that Paracoccidioides brasiliensis also produced Unidasa.
On the whole, the microorganism that produces Unidasa at present is many, but furthers investigate seldom, mainly based on the suis Unidasa.And the hyaluronic acid enzyme source is limited, costs an arm and a leg.Be badly in need of at present a kind of being easy to and cultivate, enzyme is lived high, and microbial strains safely and efficiently, so that the source of new Unidasa to be provided.
Arthrobacter nicotinovorans (Arthrobacter nicotinovorans), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center by other people, its preservation date is on January 16th, 2000, preserving number is CGMCC NO1.1924, its purposes is mainly degraded histamine alcohol, at present, do not find that it can produce Unidasa and be applied to report in the hyaluronic acid degradation.
Summary of the invention
At above-mentioned prior art, the invention provides a kind of new purposes of Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) CGMCC NO1.1924 (preservation date is on January 16th, 2000), i.e. application in hyaluronic acid degradation, it has Degradation efficiently to hyaluronic acid under normal temperature condition.
During concrete the application, can adopt following two kinds of schemes:
1, in later stage that hyaluronic acid fermentation is produced, be preceding 3-12 hour of fermentation ends, in culture, add Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) the CGMCC NO1.1924 of culture volume 0.1～5%, and mixed fermentation, fermented 3-12 hour, and promptly got the low molecular weight hyaluronic acid of molecular weight between 500-200000Da.With the degraded hyaluronic acid, Arthrobacter nicotinovorans can reduce fermentation broth viscosity, and the degraded hyaluronic acid, and then obtains low molecular weight hyaluronic acid, and the hyaluronan molecule amount that degraded obtains is between 500-200000Da.
2, in polymer hyaluronic acid solution (molecular weight is greater than 1,000,000 Da), add Arthrobacter nicotinovorans (Arthrobacternicotinovorans) CGMCC NO1.1924, act on after 2-24 hour, promptly can obtain the low molecular weight hyaluronic acid of molecular weight between 500-200000Da.
The mass percent concentration of polymer hyaluronic acid is 0.1～5% in the described polymer hyaluronic acid solution; 0.1～5% of the amount preferred solution volume of interpolation Arthrobacter nicotinovorans.
The Arthrobacter nicotinovorans of described interpolation (Arthrobacter nicotinovorans) CGMCC NO1.1924 is the fermented liquid of Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) CGMCC NO1.1924, or the Unidasa of Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) CGMCC NO1.1924 generation.
The present inventor discovers that by experiment Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) has efficient degradation effect (seeing embodiment 2-5 for details) to hyaluronic acid, therefore, can be used to the hyaluronic acid of degrading.
Embodiment
The present invention is further illustrated below in conjunction with embodiment:
Embodiment 1: the evaluation of bacterial classification
From the corrupt liquid of hyaluronic acid, separate obtaining a bacterial strain, it identified with following method:
Extract bacterial chromosomal dna, and pcr amplification bacterial strain 16S rDNA sequence.The forward primer of amplification 16S rDNA is 27F:5 '-AGAGTTTGATC (C/A) TGGCTCAG-3 ' (corresponding to 8-27 base position of E.coil 16S rDNA sequence), reverse primer 1492R:5 '-TACGG (C/T) TACCTTGTTACGACTT-3 ' (corresponding to 1492-1510 the base position of E.coli 16S rDNA).
The PCR reaction system of 100 μ L: 1 * PCR damping fluid, 1.5mmol/L MgCl
2, each 200 μ mol/L of 4 * dNTP mixture, each 0.5 μ mol/L of primer, Taq archaeal dna polymerase 1 μ L (5U/ μ L), 2 μ L DNA stostes.PCR reaction conditions: 96 ℃ of pre-sex change 6min; 94 ℃ of sex change 1min, 50 ℃ of renaturation 1min, 72 ℃ are extended 2min, 30 circulations; Last 72 ℃ of incubation 6min.Pcr amplification product is directly transferred to Shanghai living worker's biotechnology company and is carried out purifying and sequencing after electrophoresis detection.Already present bacterial 16 S rDNA nucleotide sequence carries out the similarity comparative analysis in utilization Blast program and the database.The result shows that (the GenBank accession number: GQ284335.1) sequence similarity is 99% to this bacterial strain, can judge that this bacterial strain is Arthrobacter nicotinovorans (Arthrobacternicotinovorans) CGMCC NO1.1924 with Arthrobacter nicotinovorans (Arthrobacter nicotinovorans).
Embodiment 2: Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) degraded hyaluronic acid
Mass percent is 1% hyaluronic acid solution (the hyaluronan molecule amount is 1,400,000 Da), inoculate 1% Arthrobacter nicotinovorans (Arthrobacter nicotinovorans), 30 ℃ of stir culture 24 hours are cultivated and are finished the back to detect the hyaluronan molecule amount be 1000Da.
Embodiment 3: Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) degraded hyaluronic acid
Mass percent is 3% hyaluronic acid solution (the hyaluronan molecule amount is 1,500,000 Da), inoculate 2% Arthrobacter nicotinovorans (Arthrobacter nicotinovorans), 30 ℃ of stir culture 12 hours are cultivated and are finished the back to detect the hyaluronan molecule amount be 60000Da.
Embodiment 4: Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) degraded hyaluronic acid
Use suis fermentative production hyaluronic acid, in fermentation ends preceding 6 hours, inoculate 1% Arthrobacter nicotinovorans (Arthrobacter nicotinovorans), cultivate and finish the back to detect the hyaluronan molecule amount be 100000Da.
Embodiment 5: Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) degraded hyaluronic acid
Use suis fermentative production hyaluronic acid, in fermentation ends preceding 10 hours, inoculate 0.5% Arthrobacter nicotinovorans (Arthrobacter nicotinovorans), cultivate and finish the back to detect the hyaluronan molecule amount be 60000Da.
Claims (5)
1. the application of Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) CGMCC NO1.1924 in hyaluronic acid degradation.
2. application according to claim 1 is characterized in that: concrete application method is gone into down:
In the later stage that hyaluronic acid fermentation is produced, be preceding 3-12 hour of fermentation ends, in culture, add Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) the CGMCC NO1.1924 of culture volume 0.1～5%, and mixed fermentation, fermented 3-12 hour, and promptly got the low molecular weight hyaluronic acid of molecular weight between 500-200000Da.
3. application according to claim 1 is characterized in that: concrete application method is as follows:
In polymer hyaluronic acid solution, add Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) CGMCCNO1.1924, act on after 2-24 hour, promptly can obtain the low molecular weight hyaluronic acid of molecular weight between 500-200000Da.
4. application according to claim 3 is characterized in that: the mass percent concentration of polymer hyaluronic acid is 0.1～5% in the described polymer hyaluronic acid solution; The amount of adding Arthrobacter nicotinovorans is 0.1～5% of a liquor capacity.
5. according to the described application of claim 1～4, it is characterized in that: the Arthrobacter nicotinovorans of described interpolation (Arthrobacternicotinovorans) CGMCC NO1.1924 is the fermented liquid of Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) CGMCC NO1.1924, or the Unidasa of Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) CGMCCNO1.1924.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567606A (en) * | 2016-03-02 | 2016-05-11 | 青岛海洋生物医药研究院股份有限公司 | Arthrobacter globiformis and hyaluronidase generated by arthrobacter globiformis |
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- 2010-06-04 CN CN2010101916036A patent/CN101886098A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567606A (en) * | 2016-03-02 | 2016-05-11 | 青岛海洋生物医药研究院股份有限公司 | Arthrobacter globiformis and hyaluronidase generated by arthrobacter globiformis |
| CN105567606B (en) * | 2016-03-02 | 2019-03-19 | 青岛海洋生物医药研究院股份有限公司 | A kind of Arthrobacter globiformis and its hyaluronidase of generation |
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Application publication date: 20101117 |