CN102559559A - Bacillus and method of producing hyaluronidase by employing the same - Google Patents
Bacillus and method of producing hyaluronidase by employing the same Download PDFInfo
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- CN102559559A CN102559559A CN2012100391114A CN201210039111A CN102559559A CN 102559559 A CN102559559 A CN 102559559A CN 2012100391114 A CN2012100391114 A CN 2012100391114A CN 201210039111 A CN201210039111 A CN 201210039111A CN 102559559 A CN102559559 A CN 102559559A
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- bacillus
- unidasa
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- hyaluronidase
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 38
- 108010003272 Hyaluronate lyase Proteins 0.000 title abstract description 26
- 229960002773 hyaluronidase Drugs 0.000 title abstract description 10
- 102000001974 Hyaluronidases Human genes 0.000 title abstract 7
- 238000000855 fermentation Methods 0.000 claims abstract description 7
- 230000004151 fermentation Effects 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims description 39
- 239000002609 medium Substances 0.000 claims description 33
- 239000001888 Peptone Substances 0.000 claims description 29
- 108010080698 Peptones Proteins 0.000 claims description 29
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 29
- 235000019319 peptone Nutrition 0.000 claims description 29
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 27
- 239000008103 glucose Substances 0.000 claims description 27
- 238000011218 seed culture Methods 0.000 claims description 24
- 239000001963 growth medium Substances 0.000 claims description 23
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 22
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 16
- 230000001580 bacterial effect Effects 0.000 claims description 16
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 10
- 239000008272 agar Substances 0.000 claims description 10
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 10
- 229920000053 polysorbate 80 Polymers 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 6
- 230000000694 effects Effects 0.000 abstract description 13
- 238000004519 manufacturing process Methods 0.000 abstract description 10
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 abstract description 9
- 229920002674 hyaluronan Polymers 0.000 abstract description 9
- 229960003160 hyaluronic acid Drugs 0.000 abstract description 9
- 241001465754 Metazoa Species 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 239000002054 inoculum Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 4
- 238000006384 oligomerization reaction Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 102000009066 Hyaluronoglucosaminidase Human genes 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 2
- 150000003538 tetroses Chemical class 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241001465677 Ancylostomatoidea Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 206010016275 Fear Diseases 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 241000545744 Hirudinea Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 241000191992 Peptostreptococcus Species 0.000 description 1
- 241000186429 Propionibacterium Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003659 bee venom Substances 0.000 description 1
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- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
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- 210000000416 exudates and transudate Anatomy 0.000 description 1
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- 230000010534 mechanism of action Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002357 osmotic agent Substances 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a (Bacillus) A50 CGMCC No. 5744 and a method of producing hyaluronidase by employing the same. The method is that slant cultivation, seed cultivation and fermentation cultivation are carried out for the (Bacillus) A50 CGMCC No. 5744 so that hyaluronidase is prepared. The hyaluronidase generated by the Bacillus is highly active, up to 105IU/m1. As compared with the prior art, the hyaluronidase prepared from the Bacillus of the invention is high in activity, high in thermal stability and high in pH stability, and can be used for large scale production. Meanwhile, the obtained hyaluronidase can allow high-molecular hyaluronic acid to be degraded, can be used for preparing low-molecular hyaluronic acid and oligomeric hyaluronic acid, enables the cost to be substantially reduced, helps to solve the problem of high cost for hyaluronidase of animal sources, and exhibits a wide application prospect in the field of biochemistry research and for the production of low-molecular hyaluronic acid and oligomeric hyaluronic acid.
Description
Technical field
The method that the present invention relates to a kind of genus bacillus and produce Unidasa with this genus bacillus belongs to technical field of biological fermentation.
Background technology
Unidasa is the hyaluronic class of enzymes of can degrading, and Karl Meyer is divided three classes Unidasa according to the mechanism of action of Unidasa: (1) inscribe β-N-acetylaminoglucosidase is a lytic enzyme; Act on β-1; 4 glycosidic links, end product is mainly tetrose, also can act on chrondroitin or CHS; And having transglycosylase active, testis, bee venom and lysosome Unidasa belong to this type of.(2) Unidasa in leech, hookworm source is the inscribe beta-glucuronidase, acts on β-1,3 glycosidic link, also is lytic enzyme, and main degradation products is a tetrose, specificity degraded mucinase.(3) bacterium Unidasa is also claimed hyaluronate lyase, acts on β-1,4 glycosidic link.
Unidasa in the market mainly is the Unidasa of animal-origin; Be used for the viscosity that human body temporarily reduces intercellular substance; Can impel the transudate or the blood of hypodermoclysis, local repertory to accelerate diffusion and be beneficial to absorption, be a kind of important drug diffusion agent.Clinical in the drug osmotic agent, promote the absorption of medicine, local edema or hemotoncus dissipate after promotion operation and the wound.
Because the Unidasa cost of animal-origin is high, and fears are entertained that risk that animal virus infects, the producer that how tame extraction method prepares hyaluronidase preparation has stopped the production of said preparation, and people hope to use safer inexpensive Unidasa.Microbe-derived Unidasa does not receive source restriction, extracts easily, and purity is high, is difficult for producing immunoreation, can satisfy people's this demand, and therefore how tame research institution is devoted to study microbe fermentation method and prepares Unidasa.Since self-induced transparency matter acid enzyme is found, it is found that multiple mikrobe can produce Unidasa, comprises
Streptococcus, Staphylococcus, Clostridium, Propionibacterium, Peptostreptococcus and StreptomycesDeng, but up to now, it is all lower that the Unidasa enzyme of the production by biological of reporting in the document is lived, and higher enzyme report alive appears among the patent WO2010130810A1, and the mucinase enzyme-producing bacteria is a streptomycete in this patent, and the highest enzyme work is 1.3 * 10
2IU/ml, the enzyme of all the other documents live and all are lower than 100IU/ml, therefore prepare Unidasa and enzyme process prepares low molecular weight hyaluronic acid and the oligomerization mucinase all is in laboratory scale, can not be used for scale operation.
Summary of the invention
Few in order to solve existing Unidasa production technique, and the Unidasa enzyme of gained low problem alive, the invention provides a kind of bacterial classification of producing the high reactivity Unidasa---genus bacillus (
Bacillus) A50.
The present invention also provide adopt this genus bacillus (
Bacillus) A50 produces the method for Unidasa, produces Unidasa with this bacterial classification, enzyme is lived high, can be used for scale operation, and has solved the high problem of Unidasa cost of animal-origin, is convenient to large-scale industrial production.The Unidasa of gained can be used for the degraded macromolecular mucinase, produces low molecular weight hyaluronic acid and oligomerization mucinase, has a extensive future.
Genus bacillus of the present invention (
Bacillus) A50 separates to obtain from air; Obtain because can't repeat; So this bacterial classification has carried out preservation in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), preserving number is CGMCC NO. 5744, and the preservation time is on February 8th, 2012.
The bacterial classification genus bacillus (
Bacillus) acquisition process of A50 CGMCC NO. 5744 is: the plate that enrichment medium will be housed is opened lid, is positioned in the air, collects sedimentation bacterium in the air; After about 1 hour, close the lid, place 25～40 ℃ of incubator aerobics to cultivate; Cultivate after 24 hours, single colony inoculation that separation is obtained in screening culture medium, 25～40 ℃; 150rpm, aerobic was cultivated 12～16 hours, adopted the Chinese Pharmacopoeia method to measure the mucinase enzyme activity; Select the highest bacterial classification of enzyme activity as bacterial classification of the present invention, the bacterial classification enzyme activity can reach 10
5IU/ml.
Above-mentioned each substratum that adopts is formed as follows:
Enrichment medium (100ml): peptone 0.2～2.0g, yeast powder 0.2～2.0g, K
2HPO
43H
2O, MgSO
47H
2O 0.05～0.15g, hyaluronate sodium 0.01～1g, agar powder 2g.
Screening culture medium (100ml): peptone 0.2～2.0g, yeast powder 0.2～2.0g, K
2HPO
43H
2O 0.05～0.15g, MgSO
47H
2O 0.05～0.15g, hyaluronate sodium 0.01～1g.
The genus bacillus of screening gained (
Bacillus) A50 CGMCC NO. 5744 has following characteristic:
1, morphological specificity
Thalline is shaft-like, single or chain.The bacterium colony oyster white has gauffer.
2, molecular biological characteristics
The 16S rDNA sequence of bacterial classification A50 is shown in SEQ NO:1.
Bacterial classification of the present invention is suitable under 25 ~ 40 ℃, carrying out aerobic to be cultivated, and this bacterial classification can be used for producing Unidasa, and method is: with genus bacillus (
Bacillus) A50 CGMCC No. 5744 makes Unidasa through slant culture, seed culture, fermentation culture.Specifically may further comprise the steps:
(1) with genus bacillus (
Bacillus) A50 CGMCC No. 5744 bacterial classifications carry out slant culture, slant strains;
(2) get slant strains and be inoculated in the sterilized seed culture medium, under 25～40 ℃, the condition of 100～200rpm, cultivate 10～24h, seed liquor;
(3) seed liquor is inoculated in the sterilized fermention medium, under 25～40 ℃, the condition of 100～300rpm, cultivates 12～24h, Unidasa.
In the method for above-mentioned production Unidasa, contain following composition in every 100ml slant medium: peptone 0.2～2.0g, yeast powder 0.2～2.0 g, K
2HPO
43H
2O 0.05～0.15 g, MgSO
47H
2O 0.05～0.15 g, glucose 0.5～1.5 g, agar powder 2g, pH transfers to 6.0～8.0, and the temperature of slant culture is 25～40 ℃.
In the method for above-mentioned production Unidasa, contain following composition in every 100ml seed culture medium: peptone 0.2～2.0g, yeast powder 0.2～2.0 g, K
2HPO
43H
2O 0.05～0.15 g, MgSO
47H
2O 0.05～0.15 g, glucose 0.5～1.5 g, pH transfers to 6.0～8.0.
In the method for above-mentioned production Unidasa, contain following composition in every 100ml fermention medium: peptone 0.2～2.0g, yeast powder 0.2～2.0 g, K
2HPO
43H
2O 0.05～0.15 g, MgSO
47H
2O 0.05～0.15 g, glucose 0.5～1.5 g, tween 80 0.05ml, pH transfers to 6.0～8.0.
In the method for above-mentioned production Unidasa, the inoculum size of inclined-plane seed culture and fermentation culture can obtain through prior art, need not pay performing creative labour.The seed liquor that the inoculum size of seed culture medium can reach the required inoculum size of fermentation culture gets final product, and the inoculum size of fermention medium generally gets final product at 3-15%.
In the method for above-mentioned production Unidasa, can adopt one or more adjustings slant medium, seed culture medium and fermention medium pH in hydrochloric acid, sulfuric acid or the phosphoric acid.
The invention provides a kind of highly active genus bacillus bacterial classification, the enrichment from air of this bacterial classification, screening can be used for producing Unidasa, do not appear in the newspapers in the prior art.The hyaluronidase activity that this genus bacillus produced is high, can reach 10
5IU/ml compared with prior art, adopts the hyaluronidase activity of the genus bacillus preparation among the present invention high; Thermally-stabilised height; PH stability is high, can be used for scale operation, the Unidasa degradable macromolecule mucinase that obtains simultaneously; Be used for preparing low molecular weight hyaluronic acid and oligomerization mucinase; Cost significantly reduces, and has solved the high problem of Unidasa cost of animal-origin, is having broad application prospects aspect Biochemical Research field and low molecular weight hyaluronic acid and the hyaluronic production of oligomerization.
Preservation information
Genus bacillus of the present invention (
Bacillus) A50 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on February 8th, 2012, preserving number is CGMCC NO. 5744.
Embodiment
Through specific embodiment the present invention is further set forth below, should be understood that, following explanation only is in order to explain the present invention, its content not to be limited.
Embodiment 1
Slant medium is formed (100ml): peptone 0.2g, yeast powder 2.0g, K
2HPO
43H
2O 0.05g, MgSO
47H
2O 0.05g, glucose 0.5g, agar powder 2g transfers to 6.0 with hydrochloric acid with pH.
Seed culture medium is formed (100ml): peptone 0.2g, yeast powder 2.0g, K
2HPO
43H
2O 0.05g, MgSO
47H
2O 0.05g, glucose 0.5g transfers to 6.0 with hydrochloric acid with pH.
Fermention medium is formed (100ml): peptone 0.2g, yeast powder 2.0g, K
2HPO
43H
2O 0.05g, MgSO
47H
2O 0.05g, glucose 0.5g, Tween80 (tween 80) 0.05ml.All substratum all are solvent with water.
Get slant strains (genus bacillus (
Bacillus) A50 CGMCC NO. 5744) be inoculated in the sterilized seed culture medium, 25 ℃, 150rpm cultivated 24 hours; Then seed liquor is inoculated in the sterilized fermention medium, inoculum size is 10%, 25 ℃; 200rpm cultivated 24 hours, with sulfuric acid pH was maintained 6.0 in the fermenting process, and fermentative prodn obtains fermented liquid; Contain Unidasa in the fermented liquid, can directly use, also can be in order to use again behind the purifying that meets the demands.Adopt the Chinese Pharmacopoeia method to measure mucinase enzyme activity in the fermented liquid, the vigor of Unidasa is 1.2 * 10
5IU/ml.
Embodiment 2
Slant medium is formed (100ml): peptone 1.0g, yeast powder 1.0g, K
2HPO
43H
2O 0.1g, MgSO
47H
2O 0.1g, glucose 1.0g, agar powder 2g transfers to 7.0 with phosphoric acid with pH.
Seed culture medium is formed (100ml): peptone 1.0g, yeast powder 1.0g, K
2HPO
43H
2O 0.1g, MgSO
47H
2O 0.1g, glucose 1.0g transfers to 7.0 with phosphoric acid with pH.
Fermention medium is formed (100ml): peptone 1.0g, yeast powder 1.0g, K
2HPO
43H
2O 0.1g, MgSO
47H
2O 0.1g, glucose 1.0g, Tween80 0.05ml.
Get slant strains (genus bacillus (
Bacillus) A50 CGMCC NO. 5744) be inoculated in the sterilized seed culture medium, 30 ℃, 100rpm cultivated 15 hours; Then seed liquor is inoculated in the sterilized fermention medium, inoculum size is 10%, 35 ℃; 300rpm cultivated 16 hours, with sulfuric acid pH was maintained 7.0 in the fermenting process, and fermentative prodn obtains Unidasa; Adopt the Chinese Pharmacopoeia method to measure mucinase enzyme activity in the fermented liquid, the vigor of Unidasa is 2.8 * 10
5IU/ml.
Embodiment 3
Slant medium is formed (100ml): peptone 1.5g, yeast powder 1.5g, K
2HPO
43H
2O 0.15g, MgSO
47H
2O 0.15g, glucose 1.5g, agar powder 2g transfers to 8.0 with sulfuric acid with pH.
Seed culture medium is formed (100ml): peptone 1.5g, yeast powder 1.5g, K
2HPO
43H
2O 0.15g, MgSO
47H
2O 0.15g, glucose 1.5g transfers to 8.0 with sulfuric acid with pH.
Fermention medium is formed (100ml): peptone 0.5g, yeast powder 1.5g, K
2HPO
43H
2O 0.1g, MgSO
47H
2O 0.05g, glucose 1.5g, Tween80 0.05ml.
Get slant strains (genus bacillus (
Bacillus) A50 CGMCC NO. 5744) be inoculated in the sterilized seed culture medium, 35 ℃, 200rpm cultivated 13 hours; Then seed liquor is inoculated in the sterilized fermention medium, inoculum size is 10%, 40 ℃; 100rpm cultivated 12 hours, with hydrochloric acid pH was maintained 7.0 in the fermenting process, and fermentative prodn obtains Unidasa; Adopt the Chinese Pharmacopoeia method to measure mucinase enzyme activity in the fermented liquid, the vigor of Unidasa is 1.8 * 10
5IU/ml.
Embodiment 4
Slant medium is formed (100ml): peptone 2.0g, yeast powder 0.5g, K
2HPO
43H
2O 0.05g, MgSO
47H
2O 0.05g, glucose 1.0g, agar powder 2g transfers to 6.5 with sulfuric acid with pH.
Seed culture medium is formed (100ml): peptone 2.0g, yeast powder 0.5g, K
2HPO
43H
2O 0.05g, MgSO
47H
2O 0.05g, glucose 1.0g transfers to 6.5 with sulfuric acid with pH.
Fermention medium is formed (100ml): peptone 1.5g, yeast powder 0.2g, K
2HPO
43H
2O0.15g, MgSO
47H
2O 0.15g, glucose 1.5g, Tween80 0.05ml.
Get slant strains (genus bacillus (
Bacillus) A50 CGMCC NO. 5744) be inoculated in the sterilized seed culture medium, 40 ℃, 180rpm cultivated 10 hours; Then seed liquor is inoculated in the sterilized fermention medium, inoculum size is 10%, 36 ℃; 280rpm cultivated 15 hours, with phosphoric acid pH was maintained 8.0 in the fermenting process, and fermentative prodn obtains Unidasa; Adopt the Chinese Pharmacopoeia method to measure mucinase enzyme activity in the fermented liquid, the vigor of Unidasa is 1.5 * 10
5IU/ml.
Embodiment 5
Slant medium is formed (100ml): peptone 0.5g, yeast powder 1.5g, K
2HPO
43H
2O 0.15g, MgSO
47H
2O 0.1g, glucose 0.5g, agar powder 2g transfers to 7.5 with phosphoric acid with pH.
Seed culture medium is formed (100ml): peptone 0.5g, yeast powder 1.5g, K
2HPO
43H
2O 0.15g, MgSO
47H
2O 0.1g, glucose 0.5g transfers to 7.5 with phosphoric acid with pH.
Fermention medium is formed (100ml): peptone 2.0g, yeast powder 0.2g, K
2HPO
43H
2O 0.05g, MgSO
47H
2O 0.05g, glucose 0.5g, Tween80 0.05ml.
Get slant strains (genus bacillus (
Bacillus) A50 CGMCC NO. 5744) be inoculated in the sterilized seed culture medium, 36 ℃, 120rpm cultivated 14 hours; Then seed liquor is inoculated in the sterilized fermention medium, inoculum size is 10%, 30 ℃; 180rpm cultivated 20 hours, with phosphoric acid pH was maintained 7.5 in the fermenting process, and fermentative prodn obtains Unidasa; Adopt the Chinese Pharmacopoeia method to measure mucinase enzyme activity in the fermented liquid, the vigor of Unidasa is 2.5 * 10
5IU/ml.
Embodiment 6
Slant medium is formed (100ml): peptone 1.0g, yeast powder 1.0g, K
2HPO
43H
2O 0.1g, MgSO
47H
2O 0.15g, glucose 1.5g, agar powder 2g transfers to 7.0 with hydrochloric acid with pH.
Seed culture medium is formed (100ml): peptone 1.0g, yeast powder 1.0g, K
2HPO
43H
2O 0.1g, MgSO
47H
2O 0.15g, glucose 1.5g transfers to 7.0 with hydrochloric acid with pH.
Fermention medium is formed (100ml): peptone 1.5g, yeast powder 0.5g, K
2HPO
43H
2O 0.05g, MgSO
47H
2O 0.15g, glucose 1.5g, Tween80 0.05ml.
Get slant strains (genus bacillus (
Bacillus) A50 CGMCC NO. 5744) be inoculated in the sterilized seed culture medium, 32 ℃, 150rpm cultivated 18 hours; Then seed liquor is inoculated in the sterilized fermention medium, inoculum size is 10%, 28 ℃; 200rpm cultivated 22 hours, with hydrochloric acid pH was maintained 8.0 in the fermenting process, and fermentative prodn obtains Unidasa; Adopt the Chinese Pharmacopoeia method to measure mucinase enzyme activity in the fermented liquid, the vigor of Unidasa is 1.6 * 10
5IU/ml.
Embodiment 7
Slant medium is formed (100ml): peptone 2.0g, yeast powder 1.5g, K
2HPO
43H
2O 0.05g, MgSO
47H
2O 0.15g, glucose 0.5g, agar powder 2g transfers to 7.0 with phosphoric acid with pH.
Seed culture medium is formed (100ml): peptone 2.0g, yeast powder 1.5g, K
2HPO
43H
2O 0.05g, MgSO
47H
2O 0.15g, glucose 0.5g transfers to 7.0 with phosphoric acid with pH.
Fermention medium is formed (100ml): peptone 0.5g, yeast powder 1.5g, K
2HPO
43H
2O0.15g, MgSO
47H
2O 0.1g, glucose 1.0g, Tween80 0.05ml.
Get slant strains (genus bacillus (
Bacillus) A50 CGMCC NO. 5744) be inoculated in the sterilized seed culture medium, 30 ℃, 200rpm cultivated 20 hours; Then seed liquor is inoculated in the sterilized fermention medium, inoculum size is 10%, 34 ℃; 220rpm cultivated 14 hours, with phosphoric acid pH was maintained 7.5 in the fermenting process, and fermentative prodn obtains Unidasa; Adopt the Chinese Pharmacopoeia method to measure mucinase enzyme activity in the fermented liquid, the vigor of Unidasa is 2.1 * 10
5IU/ml.
The nucleotides sequence tabulation
< 110>Shandong Freda Biopharm Co., Ltd.
< 120>a kind of genus bacillus and adopt this bacterial classification to produce the method for Unidasa
<160>1
<210>1
<211>1418
<212>DNA
< 213>genus bacillus (Bacillus) A50 CGMCC No. 5744
<400>1
gcggctggct?ccttacggtt?accccaccga?cttcgggtgt?tacaaactct?cgtggtgtga?60
cgggcggtgt?gtacaaggcc?cgggaacgta?ttcaccgcgg?catgctgatc?cgcgattact?120
agcgattccg?gcttcatgca?ggcgagttgc?agcctgcaat?ccgaactgag?aatggtttta?180
tgggattggc?taaacctcgc?ggtcttgcag?ccctttgtac?catccattgt?agcacgtgtg?240
tagcccaggt?cataaggggc?atgatgattt?gacgtcatcc?ccaccttcct?ccggtttgtc?300
accggcagtc?accttagagt?gcccaactga?atgctggcaa?ctaagatcaa?gggttgcgct?360
cgttgcggga?cttaacccaa?catctcacga?cacgagctga?cgacaaccat?gcaccacctg?420
tcactctgtc?ccccgaaggg?gaacgtccta?tctctaggag?tgtcagagga?tgtcaagacc?480
tggtaaggtt?cttcgcgttg?cttcgaatta?aaccacatgc?tccaccgctt?gtgcgggccc?540
ccgtcaattc?ctttgagttt?cagccttgcg?gccgtactcc?ccaggcggag?tgcttaatgc?600
gttagctgca?gcactaaagg?gcggaaaccc?tctaacactt?agcactcatc?gtttacggcg?660
tggactacca?gggtatctaa?tcctgtttgc?tccccacgct?ttcgcgcctc?agcgtcagtt?720
acagaccaga?aagccgcctt?cgccactggt?gttcctccac?atctctacgc?atttcaccgc?780
tacacgtgga?attccgcttt?cctcttctgt?actcaagtcc?cccagtttcc?aatgaccctc?840
cacggttgag?ccgtgggctt?tcacatcaga?cttaaaggac?cgcctgcgcg?cgctttacgc?900
ccaataattc?cggacaacgc?ttgccaccta?cgtattaccg?cggctgctgg?cacgtagtta?960
gccgtggctt?tctggttagg?taccgtcaag?gtaccggcag?ttactccggt?acttgttctt?1020
ccctaacaac?agagctttac?gacccgaagg?ccttcatcgc?tcacgcggcg?ttgctccgtc?1080
agactttcgt?ccattgcgga?agattcccta?ctgctgcctc?ccgtaggagt?ctgggccgtg?1140
tctcagtccc?agtgtggccg?atcaccctct?caggtcggct?acgcatcgtc?gccttggtga?1200
gccgttacct?caccaactag?ctaatgcgcc?gcgggcccat?ctgtaagtgt?cagcgtaaac?1260
cgactttcag?cttttcctca?tgagaggaaa?aggattatcc?ggtattagct?ccggtttccc?1320
gaagttatcc?cagtcttaca?ggcaggttgc?ccacgtgtta?ctcacccgtc?cgccgctaac?1380
caagaggtgc?aagcacctca?agattcgctc?gacttgca 1418
Claims (7)
- A genus bacillus ( Bacillus) A50 CGMCC NO.5744.
- 2. the working method of a Unidasa is characterized in that: adopt the genus bacillus described in the claim 1 ( Bacillus) A50 CGMCC NO. 5744, make Unidasa through slant culture, seed culture, fermentation culture.
- 3. working method according to claim 2 is characterized in that, specifically may further comprise the steps:(1) with genus bacillus ( Bacillus) A50 CGMCC NO. 5744 bacterial classifications carry out slant culture, slant strains;(2) get slant strains and be inoculated in the sterilized seed culture medium, under 25～40 ℃, the condition of 100～200rpm, cultivate 10～24h, seed liquor;(3) seed liquor is inoculated in the sterilized fermention medium, under 25～40 ℃, the condition of 100～300rpm, cultivates 12～24h, Unidasa.
- 4. working method according to claim 3 is characterized in that: contain following composition in every 100ml slant medium: peptone 0.2～2.0g, yeast powder 0.2～2.0 g, K 2HPO 43H 2O 0.05～0.15 g, MgSO 47H 2O 0.05～0.15 g, glucose 0.5～1.5 g, agar powder 2g, pH transfers to 6.0～8.0.
- 5. working method according to claim 3 is characterized in that: contain following composition in every 100ml seed culture medium: peptone 0.2～2.0g, yeast powder 0.2～2.0 g, K 2HPO 43H 2O 0.05～0.15 g, MgSO 47H 2O 0.05～0.15 g, glucose 0.5～1.5 g, pH transfers to 6.0～8.0.
- 6. working method according to claim 3 is characterized in that: contain following composition in every 100ml fermention medium: peptone 0.2～2.0g, yeast powder 0.2～2.0 g, K 2HPO 43H 2O 0.05～0.15 g, MgSO 47H 2O 0.05～0.15 g, glucose 0.5～1.5 g, tween 80 0.05ml, pH transfers to 6.0～8.0.
- 7. working method according to claim 3 is characterized in that: the pH that adopts one or more adjusting slant medium, seed culture medium and fermention mediums in hydrochloric acid, sulfuric acid and the phosphoric acid.
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| CN2012100391114A CN102559559A (en) | 2012-02-21 | 2012-02-21 | Bacillus and method of producing hyaluronidase by employing the same |
| PCT/CN2012/085503 WO2013123791A1 (en) | 2012-02-21 | 2012-11-29 | Bacillus, hyaluronic acid enzyme, and uses thereof |
| KR1020147026162A KR101736790B1 (en) | 2012-02-21 | 2012-11-29 | Bacillus, hyaluronic acid enzyme, and uses thereof |
| JP2014557976A JP5957096B2 (en) | 2012-02-21 | 2012-11-29 | Bacillus, hyaluronic acid enzyme and use thereof |
| CN201510427147.3A CN105055440B (en) | 2012-02-21 | 2012-11-29 | Purposes of oligomerization hyaluronic acid or oligomerization hyaluronate and combinations thereof |
| US14/379,862 US9493755B2 (en) | 2012-02-21 | 2012-11-29 | Bacillus, hyaluronidase, and uses thereof |
| ES12869142.5T ES2665556T3 (en) | 2012-02-21 | 2012-11-29 | Bacillus, hyaluronic acid enzyme and uses thereof |
| EP12869142.5A EP2818543B8 (en) | 2012-02-21 | 2012-11-29 | Bacillus, hyaluronic acid enzyme, and uses thereof |
| CN201210497017.3A CN103255076B (en) | 2012-02-21 | 2012-11-29 | A kind of bacillus, a kind of hyaluronidase and its preparation method and application |
| CN201210499375.8A CN103255187B (en) | 2012-02-21 | 2012-11-29 | Low molecular hyaluronate, preparation method and purpose thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013123791A1 (en) * | 2012-02-21 | 2013-08-29 | 华熙福瑞达生物医药有限公司 | Bacillus, hyaluronic acid enzyme, and uses thereof |
| CN103834593A (en) * | 2014-03-05 | 2014-06-04 | 青岛农业大学 | Paracoccus and application thereof |
| WO2020177456A1 (en) * | 2019-03-05 | 2020-09-10 | 山东安华生物医药股份有限公司 | Mutagenic strain of lactobacillus plantarum, mutagenesis method therefor and application thereof |
| CN111849814A (en) * | 2020-07-20 | 2020-10-30 | 江南大学 | A kind of Acinetobacter degrading hyaluronic acid and its culture method and application |
| CN114317496A (en) * | 2021-12-14 | 2022-04-12 | 华熙生物科技股份有限公司 | Hyaluronidase liquid preparation and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030092118A1 (en) * | 1997-10-31 | 2003-05-15 | Deangelis Paul L. | Hyaluronan synthase genes and expression thereof in bacillus hosts |
| CN1636052A (en) * | 2001-12-21 | 2005-07-06 | 诺维信生物聚合物公司 | Method for producing hyaluronic acid in recombinant host cells |
| US20050221446A1 (en) * | 2004-03-31 | 2005-10-06 | Novozymes Biopolymer A/S. | Methods for producing hyaluronic acid in a Bacillus cell |
| CN101384724A (en) * | 2006-02-15 | 2009-03-11 | 诺维信生物聚合物公司 | Production of low molecular weight hyaluronic acid |
| CN101709285A (en) * | 2001-06-13 | 2010-05-19 | 俄克拉何马大学董事会 | Hyaluronan synthase genes and expression thereof |
-
2012
- 2012-02-21 CN CN2012100391114A patent/CN102559559A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030092118A1 (en) * | 1997-10-31 | 2003-05-15 | Deangelis Paul L. | Hyaluronan synthase genes and expression thereof in bacillus hosts |
| CN101709285A (en) * | 2001-06-13 | 2010-05-19 | 俄克拉何马大学董事会 | Hyaluronan synthase genes and expression thereof |
| CN1636052A (en) * | 2001-12-21 | 2005-07-06 | 诺维信生物聚合物公司 | Method for producing hyaluronic acid in recombinant host cells |
| US20050221446A1 (en) * | 2004-03-31 | 2005-10-06 | Novozymes Biopolymer A/S. | Methods for producing hyaluronic acid in a Bacillus cell |
| CN101384724A (en) * | 2006-02-15 | 2009-03-11 | 诺维信生物聚合物公司 | Production of low molecular weight hyaluronic acid |
Non-Patent Citations (4)
| Title |
|---|
| LIANG-JUNG CHIEN AND CHENG-KANG LEE: "Enhanced Hyaluronic Acid Production in Bacillus subtilis by Coexpressing Bacterial Hemoglobin", 《BIOTECHNOLOGY PROGRESS》 * |
| MEI-FANG HSU: "Stimulating effects of Bacillus subtilis natto-fermented Radix astragali on hyaluronic acid production in human skin cells", 《JOURNAL OF ETHNOPHARMACOLOGY》 * |
| 石艳丽等: "发酵法生产玻璃酸研究概况", 《医药生物技术》 * |
| 郭学平等: "发酵法制备透明质酸", 《日用化学工业》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013123791A1 (en) * | 2012-02-21 | 2013-08-29 | 华熙福瑞达生物医药有限公司 | Bacillus, hyaluronic acid enzyme, and uses thereof |
| US9493755B2 (en) | 2012-02-21 | 2016-11-15 | Bloomage Freda Biopharm Co., Ltd. | Bacillus, hyaluronidase, and uses thereof |
| CN103834593A (en) * | 2014-03-05 | 2014-06-04 | 青岛农业大学 | Paracoccus and application thereof |
| WO2020177456A1 (en) * | 2019-03-05 | 2020-09-10 | 山东安华生物医药股份有限公司 | Mutagenic strain of lactobacillus plantarum, mutagenesis method therefor and application thereof |
| CN111849814A (en) * | 2020-07-20 | 2020-10-30 | 江南大学 | A kind of Acinetobacter degrading hyaluronic acid and its culture method and application |
| CN114317496A (en) * | 2021-12-14 | 2022-04-12 | 华熙生物科技股份有限公司 | Hyaluronidase liquid preparation and application thereof |
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