CN106190854A - A kind of desert pseudocyst bacterium and the preparation method of oritavancin intermediate - Google Patents
A kind of desert pseudocyst bacterium and the preparation method of oritavancin intermediate Download PDFInfo
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Abstract
The invention discloses a kind of desert pseudocyst bacterium and the preparation method of oritavancin intermediate.Bacterial strain desert pseudocyst bacterium HS807-AN-2396 (Kibdelosporangium aridum) of described high yield oritavancin intermediate, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.10576.The preparation method of described oritavancin intermediate is to be fermented in the fermentation medium by described desert pseudocyst bacterium CGMCC No.10576, obtains oritavancin intermediate A 82846B from fermentation liquid.This preparation method can be at 60m3The industrialization realizing the high yield of oritavancin intermediate fermentation titer 2315mg/L, beneficially oritavancin on tank produces.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of desert pseudocyst bacterium and oritavancin intermediate
Preparation method.
Background technology
Aerobic gram positive coccus is the important pathogen of bacterial infection, from late 1980s with
Coming, this bacterioid is caused to be infected in the trend persistently risen.But gram positive coccus drug resistance is increasingly
Seriously, severe challenge is brought to the treatment of infectious disease.Teicoplanin and vancomycin are to be currently used for
The conventional sugar peptide medicament for the treatment of drug resistance gram-positive bacterial infections.But, the widest along with clinical practice
General, the most in succession occur in that drug resistance of vancomycin S. aureus L-forms (VISA, VRSA) and through the ages
Mycin drug resistance enterococcus (VRE).Therefore, teicoplanin and vancomycin can not fully meet clinic
Need, it is necessary to the glycopeptide antibiotics medicine that exploitation application is new.
On August 6th, 2014, U.S. FDA ratifies the oritavancin listing of Medicines company exploitation
(Oritavancin, LY-333328), trade name Orbactiv, it is the teicoplanin and the most mould of continuing
The second generation of glycopeptide antibiotics of exploitation after element.Orbactiv is that U.S. FDA is ratified for acute bacterial
Resisting of the first and unique single-dose regimen that skin and skin structure infection (ABSSSIs) are treated
Raw element.Controlling of the ABSSSIs adult patient that Orbactiv injection causes for gram positive bacteria sensitive strain
Treat, including: staphylococcus aureus (Staphylococcus aureus, methicillin-sensitivity and methoxy west
Woods Resistant strain), micrococcus scarlatinae (Mlicrococcus scarlatinae), streptococcus agalactiae
(Streptococcus agalactiae), streptococcus dysgalactiae (Streptococcus dysgalactiae), angina
Hammer flora (Streptococcus anginosus, Streptococcus intermedius and Streptococcus
And enterococcus faecalis (Enterococcus faecalis, vancomycin sensitive strain) constellatus).
The chemosynthesis of oritavancin needs key intermediate A82846B, and it is by desert pseudocyst bacterium
(Kibdelosporangium aridum) ferments generation.At present, both at home and abroad to oritavancin pharmaceutically active
Carry out more research, and the research and development and production report to intermediate A82846B are few.Last century 90
Age Li Lai United States Patent (USP)s US5843437 (grant date 1998 on December 1) that obtain of company etc. are right
Produce the strain of A82846B, fermentation and extraction process to be described, but its fermentation titer is low.And
Improve the fermentation level of A82846B, oritavancin industrialization is had important using value.
ARTP (Atmospheric and Room Temperature Plasma) is atmospheric pressure at room plasma
Body, is a kind of new plasma source grown up in recent years, it is possible to produce under normal pressure (1atm)
Raw temperature, between 25～40 DEG C, has high activity particle and (is such as in the helium atom of excited state, oxygen former
Son, nitrogen-atoms, OH-Free radical etc.) plasma jet of concentration.Research shows, in plasma
The suitably active particle of dosage acts on microorganism, it is possible to make microorganism wall and the structure of cell membrane and
Permeability changes, and cause gene damage, and then make microbial gene sequences and metabolism network thereof significantly become
Change, ultimately result in microorganism and produce sudden change.Compared with classic mutagenesis method, use atmospheric pressure at room plasma
Body effectively causes the sudden change high, that easily acquisition hereditary stability is good of multifarious DNA damage, mutation rate
The advantages such as strain；Compared with molecule manipulation means or other chemomorphosis means, atmospheric pressure at room plasma enters
Row microorganism mutation breeding has easy and simple to handle, low cost, participates in mutagenic processes etc. without poisonous and harmful substance
Advantage.
NTG (N-methyl-N '-nitro-N nitrosoguanidine) is alkylating agent conventional in Microbial Breeding, has
The title of super mutagenic agent.Alkylating agent is with active al, and it is high that this group can transfer to other electron density
Molecule up, make to add on base many positions alkyl, thus change hydrogen bond in many-side.Rely on
The sudden change of NTG induction is mainly GC-AT conversion, the excision of the least scope, frameshift mutation and GC
To disappearance.
Summary of the invention
The technical problem to be solved is, for current oritavancin intermediate (A82846B)
The defect that fermentation titer is low, it is provided that a kind of desert pseudocyst bacterium and fermentation thereof obtain the preparation of A82846B
Method, described desert pseudocyst bacterium can produce oritavancin intermediate with high yield, and fermentation efficiency is high；
Described preparation method can obtain substantial amounts of oritavancin intermediate, beneficially industrialization at lower cost
Produce.
Shown in the structural formula of A82846B of the present invention such as formula (1):
The present inventor combines the mutation means of ARTP and NTG both physics and chemistry to carry out
Mutation screening, it is thus achieved that fermentation titer is high, the sudden change of the product A82846B of stabilization characteristics of genetics
Desert pseudocyst bacterium.And the fermentation of desert pseudocyst bacterium is obtained the fermentation medium of the method for A82846B
Formula, technological parameter etc. are optimized.
One of technical scheme is: a kind of desert pseudocyst bacterium (Kibdelosporangium
Aridum), it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number
For: CGMCC No.10576.
Separate from the pedotheque of Haiti and obtain strain A.orientalis A82846, obtain through natural separation
Bacterial strain HS807-O-13, bacterial strain HS807-O-13 after mutagenesis screening, obtain bacterial strain HS807-A-639,
Described bacterial strain HS807-A-639 is through ARTP and NTG Mutation screening, it is thus achieved that have high yield
The bacterial strain CGMCC No.10576 of A82846B.Identifying this bacterial strain, result is desert pseudocyst
Bacterium (Kibdelosporangium aridum), named HS807-AN-2396.This bacterial strain is in 2015
On March 11, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and receives
Register on the books numbering CGMCC No.10576 to preservation center, and it has a following Microbiological Characteristics:
1, morphologic characteristic
When examining under a microscope the bacterial strain cultivated in solid medium, this bacterium is different in size thread,
Hyphal diameter is 0.1-1.0 μm, produces white spore, and Gram’s staining is positive.
2, the characteristic learned is cultivated
When in temperature 14～37 DEG C, pH5.0～7.5 times, colony growth is good, exceedes this scope and then can go out
Now growth difference or situation about not growing.
3, physiological property
Can utilize well D-Glucose, maltose, D-lactose as carbon source, but do not utilize D-wood
Sugar, L-arabinose and glycine.Ammonium salt class nitrogen source can be effectively utilized；It can be difficult to utilize nitric acid
Salt nitrogen source.Can on the degradation product in addition to adenine well-grown；In terms of degraded, it is only capable of
Degraded casein and tyrosine.And gelatin liquefaction can be carried out and hydrogen sulfide can be produced.
The two of technical scheme are: a kind of method preparing oritavancin intermediate A 82846B,
Described desert pseudocyst bacterium CGMCC No.10576 is fermented at fermentation medium, from fermentation liquid
Obtain oritavancin intermediate A 82846B.
The time of fermentation of the present invention is the time that this area is conventional, preferably 168～240 hours,
It is more preferably 192～212 hours, is most preferably 202～210 hours.The temperature of fermentation of the present invention is
The temperature that this area is conventional, is more preferably 28～32 DEG C by preferably 25～34 DEG C.
Described fermentation is carried out in shaking flask, and the volume of described shaking flask is the volume that this area is conventional, preferably
For 250mL.Rotating speed is the rotating speed that this area is conventional, preferably 250rpm.Described amplitude is ability
The amplitude that territory is conventional, preferably 5cm.The humidity of described fermentation is the humidity that this area is conventional, preferably
Ground is 40～60%.
Described fermentation is carried out in lab scale fermentation tank, and the volume of described lab scale fermentation tank is that this area is conventional
Volume, preferably 50L.Speed of agitator is the rotating speed that this area is conventional, preferably 200～600rpm.
Dissolved oxygen amount is the dissolved oxygen amount that this area is conventional, and preferably 30～50%, described percentage ratio is this area
Routine, the preferably oxygen content of the fermentation cylinder for fermentation liquid percentage ratio of saturated oxygen content at a temperature of accounting for this.
Air mass flow is the air mass flow that this area is conventional, preferably 0.5～1.0vvm.
Described fermentation is carried out in pilot scale fermentation tank, and the volume of described pilot scale fermentation tank is that this area is conventional
Volume, preferably 5000L.Speed of agitator is the rotating speed that this area is conventional, preferably 30～150rpm.
Dissolved oxygen amount is the dissolved oxygen amount that this area is conventional, and preferably 30～40%, described percentage ratio is this area
Routine, the preferably oxygen content of the fermentation cylinder for fermentation liquid percentage ratio of saturated oxygen content at a temperature of accounting for this.
Air mass flow is the air mass flow that this area is conventional, preferably 0.2～1.0vvm.
Described fermentation is carried out in industrialization fermentation tank, and the volume of described industrialization fermentation tank is that this area is normal
The volume of rule, preferably 60m3.Speed of agitator is the rotating speed that this area is conventional, preferably
30～120rpm.Dissolved oxygen amount is the dissolved oxygen amount that this area is conventional, preferably 30～35%, described percentage
More conventional than for this area, the preferably oxygen content of fermentation cylinder for fermentation liquid saturated oxygen content at a temperature of accounting for this
Percentage ratio.Air mass flow is the air mass flow that this area is conventional, preferably 0.1～0.8vvm.
The inoculum concentration of the seed liquor of described fermentation is the inoculum concentration that this area is conventional, preferably 10～12%,
Described percentage ratio is percent by volume.Described seed liquor is obtained by the method including the steps: will
Described desert pseudocyst bacterium CGMCC No.10576 cultivates in seed culture medium.
Wherein, the time of described cultivation is the time that this area is conventional, preferably 37～44 hours, more
It it is 41～43 hours goodly.The temperature of described cultivation is the temperature that this area is conventional, preferably 28～32 DEG C.
Described cultivation is carried out in shaking flask, and the volume of described shaking flask is the volume that this area is conventional, preferably
For 250mL.Rotating speed is the rotating speed that this area is conventional, preferably 250rpm.Described amplitude is ability
The amplitude that territory is conventional, preferably 5cm.The humidity of described fermentation is the humidity that this area is conventional, preferably
Ground is 40～60%.
Described cultivation is carried out in lab scale seed tank, and the volume of described lab scale seed tank is that this area is conventional
Volume, preferably 15L.Speed of agitator is the rotating speed that this area is conventional, preferably 200～600rpm.
Dissolved oxygen amount is the dissolved oxygen amount that this area is conventional, and preferably 30～50%, described percentage ratio is this area
The oxygen content of seed culture medium percentage of saturated oxygen content at a temperature of accounting for this in routine, preferably seed tank
Ratio.Air mass flow is the air mass flow that this area is conventional, preferably 0.5～1.5vvm.
Described cultivation is carried out in pilot scale seed tank, and the volume of described pilot scale seed tank is that this area is conventional
Volume, preferably 500L.Speed of agitator is the rotating speed that this area is conventional, preferably 30～150rpm.
Dissolved oxygen amount is the dissolved oxygen amount that this area is conventional, and preferably 30～40%, described percentage ratio is this area
The oxygen content of seed culture medium percentage of saturated oxygen content at a temperature of accounting for this in routine, preferably seed tank
Ratio.Air mass flow is the air mass flow that this area is conventional, preferably 0.2～1.0vvm.
Described cultivation is carried out in industrialization seed tank, described industrialization seed tank conventional for this area
Volume, preferably 15m3.Speed of agitator is the rotating speed that this area is conventional, preferably 50～200rpm.
Dissolved oxygen amount is the dissolved oxygen amount that this area is conventional, and preferably 30～50%, described percentage ratio is this area
The oxygen content of seed culture medium percentage of saturated oxygen content at a temperature of accounting for this in routine, preferably seed tank
Ratio.Air mass flow is the air mass flow that this area is conventional, preferably 0.2～1.0vvm.
Described seed culture medium is the seed culture medium that this area is conventional, it is preferred that it includes following
Constituent (g/L): glucose 5.0～15.0, soluble starch 10.0～30.0, soybean cake powder 10.0～30.0,
Extraction from yeast powder 2.0～8.0, caseinhydrolysate 2.0～8.0, sodium chloride 0.1～0.5 and calcium carbonate 0.9～1.5,
PH6.8～7.5；More preferably include following constituent (g/L): glucose 10.0, soluble starch
20.0, soybean cake powder 20.0, extraction from yeast powder 5.0, caseinhydrolysate 5.0, sodium chloride 0.3 and carbonic acid
Calcium 1.2, pH 7.2.
It is preferred that described fermentation is carried out in lab scale fermentation tank, pilot scale fermentation tank or industrialization fermentation tank,
It also comprises the following steps that adds TYR；And/or, add Valine.
Wherein, described TYR is 0.0～30.0g/L at the content of described fermentation medium, preferably
It is 4.0～6.0g/L.Described Valine is 0.0～20.0g/L at the content of described fermentation medium, preferably
Ground is 2.0～4.0g/L.
The three of technical scheme are: a kind of desert pseudocyst bacterium CGMCC for fermenting described
No.10576 obtains the fermentation medium of oritavancin intermediate A 82846B, and it includes following composition
Composition (g/L): organic carbon source 75.0～155.0, organic nitrogen source 24.0～58.0 and inorganic salt 1.9～6.3；
Described organic carbon source is the one in glucose, maltodextrin, sucrose, molasses and corn starch or many
Kind；Described organic nitrogen source is soybean cake powder, cottonseed meal, caseinhydrolysate, extraction from yeast powder and egg
One or more in white peptone；Described inorganic salt is one or both in sodium chloride, calcium carbonate.
It is preferred that the fermentation medium described in described fermentation medium also includes following constituent
(g/L) one or more in: trace element 0.04～0.22, aminoacid 0.0～50.0 and/or defoamer
0.2～0.8.Described trace element is one or both in four water manganese sulfates and cobalt chloride hexahydrate.Described
Aminoacid be one or more in TYR, Valine, Pidolidone and L-Leu, relatively
Goodly, described aminoacid is one or both in TYR and Valine.Described froth breaking
Agent is fermentation defoamer, it is preferred that described defoamer is fermentation defoamer THIX-298, purchases
From Yantai Thinking Finechem Technology Co., Ltd..
The pH of described fermentation medium is the pH that this area is conventional, preferably 6.0～7.5, more preferably
Ground is 6.5～7.8.
More preferably, described fermentation medium includes following constituent (g/L): glucose 10.0～30.0,
Maltodextrin 60.0～100.0, molasses 5.0～25.0, caseinhydrolysate 2.0～10.0, TYR 0～30.0,
Valine 0～20.0, extraction from yeast powder 2.0～8.0, cottonseed meal 20.0～40.0, sodium chloride 0.4～0.8,
Calcium carbonate 1.5～5.5, four water manganese sulfates 0.02～0.14, cobalt chloride hexahydrate 0.02～0.08 and defoamer 0.2～0.8,
PH6.5～7.8；Most preferably, described fermentation medium includes following constituent (g/L): glucose
20.0, maltodextrin 80.0, molasses 15.0, caseinhydrolysate 6.0, TYR 12.0, Valine
3.0, extraction from yeast powder 5.0, cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate 3.5, four water manganese sulfate
0.08, cobalt chloride hexahydrate 0.05 and THIX-2980.2, pH7.5.
The four of technical scheme are: a kind of obtain described desert pseudocyst bacterium CGMCC
The method of No.10576, it comprises the following steps, cultivates described desert pseudocyst bacterium in the medium
CGMCC No.10576.
Wherein, described culture medium is the culture medium that this area is conventional, it is possible to the desert described in growth intends spore
Capsule bacterium CGMCC No.10576, preferably ISP2, Gause I, YMS or ISP9 train
Support base, more preferably for the fermentation medium described in description of the invention, most preferably for description of the invention institute
The seed culture medium stated.The temperature of described cultivation is the temperature that this area is conventional, it is possible to the famine described in growth
Unconcerned pseudocyst bacterium CGMCC No.10576, it is preferred that be 14～37 DEG C, is more preferably 25～34 DEG C,
It is most preferably 28～32 DEG C.The pH of described cultivation is the pH that this area is conventional, it is possible to the famine described in growth
Unconcerned pseudocyst bacterium CGMCC No.10576, preferably 5.0～7.5, be more preferably 6.0～7.5,
It is most preferably 6.5～7.8.
On the basis of meeting common sense in the field, above-mentioned each optimum condition, can combination in any, i.e. get Ben Fa
Bright each preferred embodiments.
Agents useful for same of the present invention and raw material are the most commercially.
The most progressive effect of the present invention is: ARTP Yu the NTG complex mutation that the present invention provides
A82846B superior strain CGMCC No.10576, stabilization characteristics of genetics, favorable reproducibility, amplification is prone to
Realize, there is excellent industrial applications and be worth.Fermentation of the present invention manufactures the side of A82846B
Method, structure adjusting fermentating formula, fermentating controling process, and for TYR and Valine
Carry out adding and residual quantity control, it is achieved 60m3The high yield of tank top fermentation titer 2315mg/L, on rule
Reporting higher than existing document on mould and in technical merit, the beneficially industrialization of oritavancin produces.
Biomaterial preservation information
The HS807-AN-2396 of the present invention, is deposited in China Microbiological bacterium on March 11st, 2015
Plant preservation administration committee common micro-organisms center (CGMCC), preservation address: north, Chaoyang District, Beijing City
Occasion West Road 1 institute 3, postcode: 100101, deposit number is: CGMCC No.10576, biological
Material (strain) is HS807-AN-2396, and Classification And Nomenclature is Kibdelosporangium aridum.
Accompanying drawing explanation
Fig. 1 is desert pseudocyst bacterium HS807-AN-2396 selection-breeding pedigree, and the numeral of Fig. 1 represents this bacterium
Strain corresponding A82846B fermentation titer.
Fig. 2 is the fermentation liquid HPLC collection of illustrative plates (Rt of A82846B is 5.588) of embodiment 1.
Fig. 3 is the fermentation liquid ESI/MS collection of illustrative plates of embodiment 1.
Fig. 4 is the 60m of embodiment 153The fermentating metabolism curve of tank.
Detailed description of the invention
Further illustrate the present invention below by the mode of embodiment, but the most therefore limit the present invention to
Among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, according to often
Rule method and condition, or select according to catalogue.
Embodiment 1 superior strain desert pseudocyst bacterium HS807-AN-2396 (Kibdelosporangium
Aridum) preparation
Separate from the pedotheque of Haiti and obtain strain A.orientalis A82846 (strain A.orientalis
A82846 sees: Oliver Puk, Petra Huber, Daniel Bischoff, etc., Glycopeptide
Biosynthesis in Amycolatopsis mediterranei DSM5908:Function of a
Halogenase and a Haloperoxidase/Perhydrolase, Chemistry&Biology, Vol.9,
225 235, February, 2002), then natural separation obtains HS807-O-13 bacterial strain, HS807-O-13
Bacterial strain obtains the strain HS807-A-639 that sets out after mutagenesis screening.Prepare the bacteria suspension of HS807-A-639,
And with filter paper filtering, microscopy cell dispersion reaches more than 95%, it is ensured that major part is unicellular, then
For mutagenic treatment.
ARTP mutagenic treatment: adjusting plasma generation gas-helium gas flow is 12.5L/min；Plasma
Body emission port and the distance contained between sample iron plate are 2mm；The irradiation power of plasma is 100w,
Irradiation time is 30s, obtains the illuminated bacteria suspension of ARTP.
The NTG accurately weighing 10.0mg is dissolved in 1mL acetone, adds the illuminated bacterium of above-mentioned ARTP
Suspension makes the final concentration of NTG reach 1.0mg/L.4 DEG C of slow concussions process 20min, are centrifuged and abandon supernatant,
Break up with physiological saline solution, repeated washing 2 times so that it is terminate reaction.
Draw fluid medium containing mutation to containing 750mg/L A82846B (from the positive medicine in sea, Zhejiang
At industry limited company obtain) culture dish and containing 2.0g/L TYR culture dish in carry out
Coating is cultivated.Cultivation temperature 28～32 DEG C, cultivation cycle is 8d.The cause of result of calculation display complex mutation
Dead rate reaches 89%.
Picking list bacterium colony resistant panel after complex mutation, enters seed bottle and cultivates, wherein seed culture
Base is (g/L): glucose 10.0, soluble starch 20.0, soybean cake powder 20.0, extraction from yeast powder 5.0,
Caseinhydrolysate 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2,30 DEG C cultivates 3d, then at 250mL
Fermentation shake flask cultivate, wherein, fermentation medium is (g/L): glucose 20.0, maltodextrin 80.0,
Molasses 15.0, caseinhydrolysate 6.0, TYR 12.0, Valine 3.0, extraction from yeast powder 5.0,
Cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate 3.5, four water manganese sulfate 0.08, cobalt chloride hexahydrate 0.05
And THIX-2980.2, pH7.5.30 DEG C of fermentations 8d, HPLC detect A82846B content.Obtain one
Strain high productive mutant HS807-AN-2396, shake flask fermentation titer is 1325mg/L, multiple sieve (i.e. according to
Identical condition shaking flask again, carries out fermentation culture) shake flask fermentation titer 1263mg/L, its HPLC afterwards
Collection of illustrative plates is as shown in Figure 2.Fermentation liquid fermentation obtained carries out separating-purifying, and (step of separating-purifying sees
Tsuji N.；T.Kamigauchi,M.Kobayashi&Y.Terui:New glycopeptide antibiotics:
II.The isolation and structures of chloroorienticins.J.Antibiotics 41:1506-1510,
1988), the ESI/MS collection of illustrative plates of the A82846B after purification is as shown in Figure 3.
To be set out strain HS807-A-639, picking list bacterium colony, enter seed bottle and cultivate, wherein seed culture
Base is (g/L): glucose 10.0, soluble starch 20.0, soybean cake powder 20.0, extraction from yeast powder 5.0,
Caseinhydrolysate 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2,30 DEG C cultivates 3d, then at 250mL
Fermentation shake flask cultivate, wherein, fermentation medium is (g/L): glucose 20.0, maltodextrin 80.0,
Molasses 15.0, caseinhydrolysate 6.0, TYR 12.0, Valine 3.0, extraction from yeast powder 5.0,
Cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate 3.5, four water manganese sulfate 0.08, cobalt chloride hexahydrate 0.05
And THIX-2980.2, pH7.5.30 DEG C of fermentations 8d, HPLC detect A82846B content.Shaking flask is sent out
Ferment titer is 683mg/L, multiple sieve (i.e. according to identical condition shaking flask again, carry out fermentation culture)
Rear shake flask fermentation titer is 656mg/L.From the point of view of fermentation results, high productive mutant HS807-AN-2396
Fermentation titer improve 93% than starting strain HS807-A-639.
By above-mentioned high productive mutant HS807-AN-2396, on March 11st, 2015 in Chinese micro-life
Thing culture presevation administration committee's common micro-organisms center (CGMCC) preservation, it is thus achieved that deposit number is:
CGMCC No.10576, biomaterial (strain) is HS807-AN-2396, and Classification And Nomenclature is
Kibdelosporangium aridum。
The morphology of embodiment 2 bacterial strain HS807-AN-2396 (CGMCC No.10576) and cultivation are learned
Feature
With reference to " streptomycete identification handbook ", " actinomycetic classification and qualification ", " common bacteria system identification
Handbook " and the book such as " Molecular Cloning: A Laboratory guide " in relevant content test.
Related symbol represents explanation: 0: without growth；1: grow the most weak；2: can grow, have a small amount of spore
Son；3: well-grown, there is a large amount of spore；4: grow best, there is abundant spore；+: positive；-:
Negative.
Cultural characteristic: use ISP1, ISP2, ISP3, ISP4, ISP5, Gause I, calcium malate,
Nine kinds of culture medium (obtaining at Haizheng Medicine Stock Co., Ltd., Zhejiang Prov) of YMS and Cha Shi, 28 DEG C of cultivations
After 6～8 days, observe mycelial color and pigment situation.
Table 1 bacterial strain HS807-AN-2396 cultural characteristic in 9 kinds of culture medium
The result explanation of table 1, this improvement strain well-grown on ISP2, Gause I, YMS,
And in different culture medium, show different outward appearance, pigments and produce spore situation.
The physiological and biochemical property of embodiment 3 bacterial strain HS807-AN-2396 (CGMCC No.10576)
Condition of culture is 28 DEG C and cultivates 6～8 days.
A) carbon source: using culture medium based on ISP9, the final concentration of various carbon sources is 1.0%, institute
The percentage ratio stated is mass percent.
B) inorganic nitrogen-sourced: using culture medium based on ISP9, the concentration of potassium nitrate and ammonium sulfate is
0.1%.
Carbon source and inorganic nitrogen-sourced utilization power are shown in Table 2, the result explanation of table 2, bacterial strain
HS807-AN-2396 is different to different utilization of carbon source degree, can utilize well D-Glucose,
Maltose, D-lactose, but do not utilize D-xylose, L-arabinose and glycine.Can effective land productivity
With ammonium salt class nitrogen source；It can be difficult to utilize Nitrates nitrogen source.
The carbon source of table 2 bacterial strain HS807-AN-2396 and the utilization power in nitrogen source
| Carbon source | Growing state | Carbon source | Growing state | Inorganic nitrogen-sourced | Growing state |
| D-Glucose | 3 | Salicin | 2 | Ammonium sulfate | + |
| D-Raffinose | 2 | D-lactose | 3 | Potassium nitrate | - |
| D-xylose | 0 | Galactose | 2 | ||
| D-glucitol | 2 | Inositol | 2 | ||
| L-arabinose | 0 | Mannitol | 2 | ||
| Glycerol | 2 | Glycine | 0 | ||
| Maltose | 3 | Xylan | 2 | ||
| D-Fructose | 1 | Inulin | 2 | ||
| D-sucrose | 2 | Rhamnose | 2 |
C) Degrading experiment: employing basal medium is GYEA (pH6.8), the concentration of various degradation products
It is shown in Table 3.Table 3 illustrates in addition to adenine, and bacterial strain HS807-AN-2396 can be at other several degradation products
Upper well-grown；In terms of degraded, bacterial strain HS807-AN-2396 is only capable of degraded casein and cheese ammonia
Acid.Percentage ratio described in table 3 is mass percent.
The Degrading experiment result of table 3 bacterial strain HS807-AN-2396
| Degradation product | Degradate concentrations | Result | Degradation product | Degradate concentrations | Result |
| Adenine | 0.5% | 2 ,- | Casein | 1.0% | 4 ,+ |
| Guanine | 0.5% | 4 ,- | Tyrosine | 1.0% | 4 ,+ |
| Xylan | 0.4% | 4 ,- | Tween-40 | 1.0% | 4 ,- |
| Hypoxanthine | 0.4% | 4 ,- | Tween-60 | 1.0% | 4 ,- |
| Tween-80 | 1.0% | 4 ,- |
D) catalase test uses YMS culture medium.
E) M.R and V-P experiment uses " common bacteria system identification handbook " method.Table 4 illustrates,
Bacterial strain HS807-AN-2396 can carry out gelatin liquefaction and can produce hydrogen sulfide.
The physiological and biochemical property that table 4 bacterial strain HS807-AN-2396 is main
| Pilot project | Result | Pilot project | Result | Pilot project | Result |
| Gelatin liquefaction | + | Milk peptonizes | - | Cellulose utilization | - |
| Starch Hydrolysis | - | Nitrate reduction | - | Catalase | - |
| Milk solidifies | - | Hydrogen sulfide produces | + | ||
| V.P tests | - | M.R tests | - |
Being illustrated by the data of embodiment 2～3, described desert pseudocyst bacterium CGMCC No.10576 has
Following Microbiological Characteristics:
1, morphologic characteristic
When examining under a microscope the bacterial strain cultivated in solid medium, cell is different in size thread,
Size is 0.4-1.0 μm, produces white spore, and Gram’s staining is positive.
2, the characteristic learned is cultivated
When in temperature 14～37 DEG C, pH 5.0～7.5 times, colony growth is good, exceedes this scope and then can go out
Now growth difference or situation about not growing.
3, physiological property
Can utilize well D-Glucose, maltose, D-lactose as carbon source, but do not utilize D-wood
Sugar, L-arabinose and glycine.Ammonium salt class nitrogen source can be effectively utilized；It can be difficult to utilize nitric acid
Salt nitrogen source.Can on the degradation product in addition to adenine well-grown；In terms of degraded, it is only capable of
Degraded casein and tyrosine.And gelatin liquefaction can be carried out and hydrogen sulfide can be produced.
The 16S rDNA sequence of embodiment 4 bacterial strain HS807-AN-2396 (CGMCC No.10576)
Analyze
16S rDNA sequence analysis: the 16S rDNA sequence of bacterial strain HS807-AN-2396 and with
In GenBank, correlated series carries out BLAST and compares, and the results are shown in Table 5.
Table 5 bacterial strain HS807-AN-2396 and the homology of related strain
Tested by the appearance features of bacterial strain HS807-AN-2396 (CGMCC No.10576), find
The classification relevant parameter of this bacterial strain and desert pseudocyst bacterium (Kibdelosporangium aridum) connects very much
Closely, bacterial strain HS807-AN-2396 is carried out 16S rDNA order-checking, sequencing result such as sequence table simultaneously
Shown in SEQ ID No.1.By the bacterial strain HS807-AN-2396's as shown in sequence table SEQ ID No.1
16S rDNA sequence and the 16S rDNA of desert pseudocyst bacterium (Kibdelosporangium aridum)
Sequence is compared, and comparison result finds, bacterial strain HS807-AN-2396 and desert pseudocyst bacterium
The homology of (Kibdelosporangium aridum) reaches as high as 98.9%: bacterial strain
The evolution of Kibdelosporangium aridum strain DSM 43828 and bacterial strain HS807-AN-2396
Parent's source relation closest to, both homologys are 98.9%.Therefore bacterial strain HS807-AN-2396 is accredited as
Desert pseudocyst bacterium (Kibdelosporangium aridum).
Embodiment 5 bacterial strain HS807-AN-2396 (CGMCC No.10576) is in 250mL shaking flask
Without the fermentation in amino acid media
Seed culture medium (g/L): glucose 10.0, soluble starch 20.0, hot moulding soybean cake powder 20.0,
Extraction from yeast powder 5.0, caseinhydrolysate 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2.
In seed bottle, the loading amount of seed culture medium is 20mL/250mL, cultivation temperature 32 DEG C, cultivates wet
Degree 50～60%, shaking table amplitude 5cm, shaking speed 250rpm, cultivation cycle 39h.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolysis cheese egg
White 6.0, extraction from yeast powder 5.0, cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate 3.5, four water sulphuric acid
Manganese 0.08 and cobalt chloride hexahydrate 0.05, pH7.3.
Seed bottle is 10% to the subcultivation amount of fermentation flask, and described percentage ratio is percent by volume.
In fermentation flask, the loading amount of fermentation medium is 20mL/250mL, cultivation temperature 28 DEG C, cultivates wet
Degree 40～50%, shaking table amplitude 5cm, shaking speed 250rpm, cultivation cycle 212h.
After fermentation ends, HPLC detects fermentation titer, and A82846B reaches 923mg/L.
Embodiment 6 bacterial strain HS807-AN-2396 (CGMCC No.10576) is in 250mL shaking flask
Add the fermentation in TYR, Valine culture medium.
Seed culture medium (g/L): glucose 10.0, soluble starch 20.0, hot moulding soybean cake powder 20.0,
Extraction from yeast powder 5.0, caseinhydrolysate 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2.
The loading amount of seed bottle is 20ml/250ml, cultivation temperature 28 DEG C, cultivates humidity 40～50%, shaking table
Amplitude 5cm, shaking speed 250rpm, cultivation cycle 37h.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolysis cheese egg
White 6.0, TYR 12.0, Valine 3.0, extraction from yeast powder 5.0, cottonseed meal 30.0, chlorine
Change sodium 0.6, calcium carbonate 3.5, four water manganese sulfate 0.08 and cobalt chloride hexahydrate 0.05, pH7.3.
Seed bottle is 10% to the subcultivation amount of fermentation flask, and described percentage ratio is percent by volume.
In fermentation flask, the loading amount of fermentation medium is 20mL/250mL, cultivation temperature 32 DEG C, cultivates wet
Degree 50～60%, shaking table amplitude 5cm, shaking speed 250rpm, cultivation cycle 210h.
After fermentation ends, HPLC detects fermentation titer, and A82846B reaches 1210mg/L.
Embodiment 7 bacterial strain HS807-AN-2396 (CGMCC No.10576) sending out on 50L tank
Ferment lab scale craft is studied
Based on the technological parameter that 250ml shaking flask obtains, according to bacterial strain described in embodiment 1
The characteristic of HS807-AN-2396, design 50L tank fermentation technology is also optimized, and investigates mutant growth
Metabolic condition and the optimal of A82846B produce element ability.
Seed culture medium (g/L): glucose 10.0, soluble starch 20.0, hot moulding soybean cake powder 20.0,
Extraction from yeast powder 5.0, caseinhydrolysate 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-298 (are purchased from
Yantai Thinking Finechem Technology Co., Ltd.) 0.2, pH 7.2.Wherein, the dress of seed culture medium in seed tank
Amount is 10L/15L.
Seed tank control technique: cultivation temperature 28～32 DEG C, speed of agitator 200～600rpm, dissolved oxygen
30%～50%, air mass flow 0.5～1.5vvm, cultivation cycle 43h.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolysis cheese egg
White 6.0, TYR 12.0, Valine 3.0, extraction from yeast powder 5.0, cottonseed meal 30.0, chlorine
Change sodium 0.6, calcium carbonate 3.5, four water manganese sulfate 0.08, cobalt chloride hexahydrate 0.05, THIX-2980.6,
pH7.3。
Seed tank is 12% to the subcultivation amount of fermentation tank, and described percentage ratio is percent by volume.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, cultivation temperature 28～32 DEG C, speed of agitator
200～600rpm, dissolved oxygen 30～50%, air mass flow 0.5～1.0vvm, cultivation cycle 206h.
Feeding medium during fermentation controls: stream adds TYR, controls TYR residual quantity 4.0～6.0g/L.
Sweat carries out total sugar, reducing sugar, amino nitrogen, mycelial concentration and fermentation titer etc. simultaneously
Detection.After fermentation ends, HPLC detects fermentation titer, and A82846B reaches 2156mg/L.
Embodiment 8 bacterial strain HS807-AN-2396 (CGMCC No.10576) sending out on 50L tank
Ferment lab scale craft is studied
Seed culture medium (g/L): glucose 5.0, soluble starch 10.0, soybean cake powder 10.0, ferment
Female extracting powder 2.0, caseinhydrolysate 2.0, sodium chloride 0.1, calcium carbonate 0.9, THIX-2980.2, pH
7.5。
Seed tank control technique: cultivation temperature 28～32 DEG C, speed of agitator 200～600rpm, dissolved oxygen
30%～50%, air mass flow 0.5～1.5vvm, cultivation cycle 41h.Wherein, seed culture in seed tank
The loading amount of base is 10L/15L.
Fermentation medium (g/L): glucose 10.0, corn starch 100.0, sucrose 5.0, hydrolysis cheese egg
White 2.0, Pidolidone 30.0, L-Leu 20.0, extraction from yeast powder 2.0, soybean cake powder 20.0, carbon
Acid calcium 5.5, four water manganese sulfate 0.02, THIX-2980.8, pH6.5.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, cultivation temperature 28～32 DEG C, speed of agitator
200～600rpm, dissolved oxygen 30～50%, air mass flow 0.5～1.0vvm, cultivation cycle 168h.
Feeding medium during fermentation controls: stream adds Valine, controls Valine residual quantity 0.0～20.0g/L.
Sweat carries out total sugar, reducing sugar, amino nitrogen, mycelial concentration and fermentation titer etc. simultaneously
Detection, above-mentioned fermentation condition can ferment and obtain A82846B.HPLC detection fermentation after fermentation ends
Titer, A82846B reaches 2110mg/L.
Embodiment 9 bacterial strain HS807-AN-2396 (CGMCC No.10576) sending out on 50L tank
Ferment lab scale craft is studied
Seed culture medium (g/L): glucose 15.0, soluble starch 30.0, soybean cake powder 30.0, ferment
Female extracting powder 8.0, caseinhydrolysate 8.0, sodium chloride 0.5, calcium carbonate 1.5, THIX-2980.2, pH
6.8。
Seed tank control technique: cultivation temperature 28～32 DEG C, speed of agitator 200～600rpm, dissolved oxygen
30%～50%, air mass flow 0.5～1.5vvm, cultivation cycle 41h.Wherein, seed culture in seed tank
The loading amount of base is 10L/15L.
Fermentation medium (g/L): glucose 30.0, maltodextrin 60.0, molasses 25.0, hydrolysis cheese egg
White 10.0, extraction from yeast powder 8.0, cottonseed meal 40.0, sodium chloride 0.8, cobalt chloride hexahydrate 0.02,
THIX-2980.8, pH7.8.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, cultivation temperature 28～32 DEG C, speed of agitator
200～600rpm, dissolved oxygen 30～50%, air mass flow 0.5～1.0vvm, cultivation cycle 240h.
Feeding medium during fermentation controls: stream adds TYR, controls TYR residual quantity 0.0～30.0g/L.
Sweat carries out total sugar, reducing sugar, amino nitrogen, mycelial concentration and fermentation titer etc. simultaneously
Detection, above-mentioned fermentation condition can ferment and obtain A82846B.HPLC detection fermentation after fermentation ends
Titer, A82846B reaches 1967mg/L.
Embodiment 10 bacterial strain HS807-AN-2396 (CGMCC No.10576) sending out on 50L tank
Ferment lab scale craft is studied
Seed culture medium (g/L): glucose 15.0, soluble starch 30.0, soybean cake powder 30.0, ferment
Female extracting powder 8.0, caseinhydrolysate 8.0, sodium chloride 0.5, calcium carbonate 1.5, THIX-2980.2, pH
6.8。
Seed tank control technique: cultivation temperature 28～32 DEG C, speed of agitator 200～600rpm, dissolved oxygen
30%～50%, air mass flow 0.5～1.5vvm, cultivation cycle 41h.Wherein, seed culture in seed tank
The loading amount of base is 10L/15L.
Fermentation medium (g/L): glucose 30.0, maltodextrin 100.0, molasses 5.0, hydrolysis cheese egg
White 10.0, extraction from yeast powder 8.0, cottonseed meal 20.0, sodium chloride 0.4, calcium carbonate 1.5, four water sulfur
Acid manganese 0.14, cobalt chloride hexahydrate 0.08, pH7.5.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, cultivation temperature 28～32 DEG C, speed of agitator
200～600rpm, dissolved oxygen 30～50%, air mass flow 0.5～1.0vvm, cultivation cycle 240h.
Feeding medium during fermentation controls: stream adds TYR, controls TYR residual quantity 0.0～30.0g/L.
Sweat carries out total sugar, reducing sugar, amino nitrogen, mycelial concentration and fermentation titer etc. simultaneously
Detection, above-mentioned fermentation condition can ferment and obtain A82846B.HPLC detection fermentation after fermentation ends
Titer, A82846B reaches 2016mg/L.
Embodiment 11 bacterial strain HS807-AN-2396 (CGMCC No.10576) sending out on 50L tank
Ferment lab scale craft is studied
Seed culture medium (g/L): glucose 15.0, soluble starch 30.0, soybean cake powder 30.0, ferment
Female extracting powder 8.0, caseinhydrolysate 8.0, sodium chloride 0.5, calcium carbonate 1.5, THIX-2980.2, pH
6.8。
Seed tank control technique: cultivation temperature 28～32 DEG C, speed of agitator 200～600rpm, dissolved oxygen
30%～50%, air mass flow 0.5～1.5vvm, cultivation cycle 41h.Wherein, seed culture in seed tank
The loading amount of base is 10L/15L.
Fermentation medium (g/L): glucose 30.0, maltodextrin 60.0, molasses 25.0, hydrolysis cheese egg
White 10.0, extraction from yeast powder 8.0, cottonseed meal 40.0, sodium chloride 0.4, calcium carbonate 1.5, pH5.0.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, cultivation temperature 28～32 DEG C, speed of agitator
200～600rpm, dissolved oxygen 30～50%, air mass flow 0.5～1.0vvm, cultivation cycle 240h.
Feeding medium during fermentation controls: stream adds TYR, controls TYR residual quantity 2.0～6.0g/L.
Sweat carries out total sugar, reducing sugar, amino nitrogen, mycelial concentration and fermentation titer etc. simultaneously
Detection, above-mentioned fermentation condition can ferment and obtain A82846B.HPLC detection fermentation after fermentation ends
Titer, A82846B reaches 1954mg/L.
Embodiment 12 bacterial strain HS807-AN-2396 (CGMCC No.10576) sending out on 50L tank
Ferment lab scale craft is studied
Seed culture medium (g/L): glucose 10.0, soluble starch 20.0, hot moulding soybean cake powder 20.0,
Extraction from yeast powder 5.0, caseinhydrolysate 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-298 (are purchased from
Yantai Thinking Finechem Technology Co., Ltd.) 0.2, pH 7.2.Wherein, the dress of seed culture medium in seed tank
Amount is 10L/15L.
Seed tank control technique: cultivation temperature 28～32 DEG C, speed of agitator 200～600rpm, dissolved oxygen
30%～50%, air mass flow 0.5～1.5vvm, cultivation cycle 43h.Wherein, seed culture in seed tank
The loading amount of base is 10L/15L.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolysis cheese egg
White 6.0, TYR 3.0, Valine 3.0, Pidolidone 3.0, L-Leu 3.0, extraction from yeast
Powder 5.0, cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate 3.5, four water manganese sulfate 0.08, six water chlorine
Change cobalt 0.05, THIX-2980.2, pH7.5.
Seed tank is 12% to the subcultivation amount of fermentation tank, and described percentage ratio is percent by volume.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, cultivation temperature 25 DEG C, speed of agitator
200～600rpm, dissolved oxygen 30～50%, air mass flow 0.5～1.0vvm, cultivation cycle 206h.
Feeding medium during fermentation controls: stream adds TYR, controls TYR residual quantity 4.0～6.0g/L.
Sweat carries out total sugar, reducing sugar, amino nitrogen, mycelial concentration and fermentation titer etc. simultaneously
Detection.After fermentation ends, HPLC detects fermentation titer, and A82846B reaches 1791mg/L.
Embodiment 13 bacterial strain HS807-AN-2396 (CGMCC No.10576) sending out on 50L tank
Ferment lab scale craft is studied
Seed culture medium (g/L): glucose 10.0, soluble starch 20.0, hot moulding soybean cake powder 20.0,
Extraction from yeast powder 5.0, caseinhydrolysate 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-298 (are purchased from
Yantai Thinking Finechem Technology Co., Ltd.) 0.2, pH 7.2.Wherein, the dress of seed culture medium in seed tank
Amount is 10L/15L.
Seed tank control technique: cultivation temperature 28～32 DEG C, speed of agitator 200～600rpm, dissolved oxygen
30%～50%, air mass flow 0.5～1.5vvm, cultivation cycle 43h.Wherein, seed culture in seed tank
The loading amount of base is 10L/15L.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, hydrolysis cheese egg
White 6.0, TYR 30.0, Valine 20.0, peptone 5.0, cottonseed meal 30.0, sodium chloride
0.6, calcium carbonate 3.5, four water manganese sulfate 0.08, cobalt chloride hexahydrate 0.05, THIX-2980.2, pH6.0.
Seed tank is 12% to the subcultivation amount of fermentation tank, and described percentage ratio is percent by volume.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, cultivation temperature 34 DEG C, speed of agitator
200～600rpm, dissolved oxygen 30～50%, air mass flow 0.5～1.0vvm, cultivation cycle 206h.Fermentation is mended
Material controls: stream adds TYR, controls TYR residual quantity 4.0～6.0g/L.
Sweat carries out total sugar, reducing sugar, amino nitrogen, mycelial concentration and fermentation titer etc. simultaneously
Detection.After fermentation ends, HPLC detects fermentation titer, and A82846B reaches 1563mg/L.
Embodiment 14 bacterial strain HS807-AN-2396 (CGMCC No.10576) is on 5000L tank
Fermentation pilot process research
Based on the technological parameter that 50L fermentation tank obtains, according to bacterial strain described in embodiment 1
The characteristic of HS807-AN-2396, design 5000L tank fermentation technology is also optimized, investigates mutant raw
Long metabolic condition and the optimal of A82846B produce element ability.
Seed culture medium (g/L): glucose 10.0, soluble starch 20.0, hot moulding soybean cake powder 20.0,
Extraction from yeast powder 5.0, caseinhydrolysate 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-2980.2,
pH 7.2。
Seed tank control technique: cultivation temperature 28～32 DEG C, speed of agitator 30～150rpm, dissolved oxygen 30～40%,
Air mass flow 0.2～0.1vvm, cultivation cycle 41h.Wherein, in seed tank, the loading amount of seed culture medium is
300L/500L。
Fermentation medium (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0, hydrolysis cheese egg
White 6.0, TYR 3.0, extraction from yeast powder 5.0, cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate
3.5, four water manganese sulfates 0.08, cobalt chloride hexahydrate 0.05 and defoamer THIX-2980.4, pH7.5.
Seed tank is 10% to the subcultivation amount of fermentation tank, and described percentage ratio is percent by volume.Fermentation
In tank, the loading amount of fermentation medium is 3500L: cultivation temperature 28～32 DEG C, speed of agitator 30～150rpm,
Dissolved oxygen 30～40%, air mass flow 0.2～1.0vvm, cultivation cycle 202h.
Feeding medium during fermentation controls: stream adds TYR, controls TYR residual quantity 4.0～6.0g/L.
Sweat carries out total sugar, reducing sugar, amino nitrogen, mycelial concentration and fermentation titer etc. simultaneously
Detection.After fermentation ends, HPLC detects fermentation titer, and A82846B reaches 2199mg/L.
Embodiment 15 bacterial strain HS807-AN-2396 (CGMCC No.10576) is at 60m3Sending out on tank
Ferment industrialization is amplified
Based on the technological parameter that 5000L fermentation tank obtains, according to bacterial strain described in embodiment 1
The characteristic of HS807-AN-2396, designs 60m3Tank fermentation technology is also optimized, and investigates mutant raw
Long metabolic condition and the optimal of A82846B produce element ability.
Seed culture medium (g/L): glucose 10.0, soluble starch 20.0, hot moulding soybean cake powder 20.0,
Extraction from yeast powder 5.0, caseinhydrolysate 5.0, sodium chloride 0.3, calcium carbonate 1.2, THIX-2980.2,
pH 7.2。
Seed tank control technique: cultivation temperature 28～32 DEG C, speed of agitator 50～200rpm, dissolved oxygen 30～
50%, air mass flow 0.2～1.0vvm, cultivation cycle 41h.Wherein, seed culture medium in seed tank
Loading amount is 5m3/15m3。
Fermentation medium (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0, hydrolysis cheese egg
White 6.0, TYR 12.0, Valine 3.0, extraction from yeast powder 5.0, cottonseed meal 30.0, chlorine
Change sodium 0.6, calcium carbonate 3.5, four water manganese sulfate 0.08, cobalt chloride hexahydrate 0.05, THIX-2980.2,
pH7.5。
Seed tank is 12% to the subcultivation amount of fermentation tank, and described percentage ratio is percent by volume.Fermentation
In tank, the loading amount of fermentation medium is 42m3: cultivation temperature 28～32 DEG C, speed of agitator 30～120rpm,
Dissolved oxygen 30～35%, air mass flow 0.1～0.8vvm, cultivation cycle 192h.
Feeding medium during fermentation controls: stream adds TYR, controls TYR residual quantity 4.0～6.0g/L.
Sweat carries out total sugar, reducing sugar, amino nitrogen, mycelial concentration and fermentation titer etc. simultaneously
Detection.60m3By the fermentating metabolism of above-mentioned cultivation and fermentation condition fermentation strain HS807-AN-2396 in tank
Curve sees Fig. 4.After fermentation ends, HPLC detects fermentation titer, and A82846B reaches 2315mg/L.
Should be understood that, after the foregoing having read the present invention, those skilled in the art can be to this
Bright making various changes or modifications, these equivalent form of values fall within the application appended claims equally and are limited
Scope.
Claims (20)
1. a desert pseudocyst bacterium (Kibdelosporangium aridum), it is characterised in that it is protected
Ensconcing China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is: CGMCC
No.10576。
2. the method preparing oritavancin intermediate A 82846B, it is characterised in that will be such as right
Require that the desert pseudocyst bacterium CGMCC No.10576 described in 1 ferments in the fermentation medium, from sending out
Ferment liquid obtains oritavancin intermediate A 82846B.
3. method as claimed in claim 2, it is characterised in that the time of described fermentation is 168～240
Hour, preferably 192～212 hours, be more preferably 202～210 hours；And/or, described fermentation
Temperature be 25～34 DEG C, preferably 28～32 DEG C.
4. method as claimed in claim 3, it is characterised in that described fermentation is carried out in shaking flask,
Rotating speed is 250rpm；Amplitude is 5cm；And/or, the humidity of described fermentation is 40～60%.
5. method as claimed in claim 3, it is characterised in that described fermentation is in lab scale fermentation tank
Carry out；Speed of agitator is 200～600rpm；Dissolved oxygen amount is 30～50%；And/or, air mass flow is
0.5～1.0vvm.
6. method as claimed in claim 3, it is characterised in that described fermentation is in pilot scale fermentation tank
Carrying out, speed of agitator is 30～150rpm；Dissolved oxygen amount is 30～40%；And/or, air mass flow is
0.2～1.0vvm.
7. method as claimed in claim 3, it is characterised in that described fermentation is at industrialization fermentation tank
In carry out, speed of agitator is 30～120rpm；Dissolved oxygen amount is 30～35%；And/or, air mass flow is
0.1～0.8vvm.
8. method as claimed in claim 2, it is characterised in that the inoculation of the seed liquor of described fermentation
Amount is 10～12%, and described percentage ratio is percent by volume；The seed liquor of described fermentation is as follows by including
Step method obtain: by desert pseudocyst bacterium CGMCC No.10576 as claimed in claim 1
Seed culture medium is cultivated.
9. method as claimed in claim 8, it is characterised in that the time of described cultivation is 37～44
Hour, preferably 41～43 hours；And/or, the temperature of described cultivation is 28～32 DEG C.
10. method as claimed in claim 9, it is characterised in that described cultivation is carried out in shaking flask,
Rotating speed is 250rpm；Amplitude is 5cm；And/or, the humidity of described fermentation is 40～60%.
11. methods as claimed in claim 9, it is characterised in that described cultivation is in lab scale seed tank
Carrying out, speed of agitator is 200～600rpm；The dissolved oxygen amount of described lab scale fermentation tank is 30～50%；With/
Or, air mass flow is 0.5～1.5vvm.
12. methods as claimed in claim 9, it is characterised in that described cultivation is in pilot scale seed tank
Carrying out, speed of agitator is 30～150rpm；Dissolved oxygen amount is 30～40%；And/or, air mass flow is
0.2～1.0vvm.
13. methods as claimed in claim 9, it is characterised in that described cultivation is at industrialization seed tank
In carry out, speed of agitator is 50～200rpm；Dissolved oxygen amount is 30～50%；And/or, air mass flow is
0.2～1.0vvm.
14. methods as claimed in claim 8, it is characterised in that described seed culture medium include as
Under constituent (g/L): glucose 5.0～15.0, soluble starch 10.0～30.0, soybean cake powder
10.0～30.0, extraction from yeast powder 2.0～8.0, caseinhydrolysate 2.0～8.0, sodium chloride 0.1～0.5 and carbonic acid
Calcium 0.9～1.5, pH6.8～7.5；More preferably include following constituent (g/L): glucose 10.0,
Soluble starch 20.0, soybean cake powder 20.0, extraction from yeast powder 5.0, caseinhydrolysate 5.0, sodium chloride
0.3, defoamer THIX-2980.2, pH 7.2 are used in calcium carbonate 1.2 and fermentation.
15. methods as claimed in claim 2, it is characterised in that described fermentation lab scale fermentation tank,
Carrying out in pilot scale fermentation tank or industrialization fermentation tank, it also comprises the following steps that
Add TYR；And/or, add Valine.
16. methods as claimed in claim 15, it is characterised in that described TYR accounts for described sending out
The content of ferment culture medium is 0.0～30.0g/L, preferably 4.0～6.0g/L；And/or, described L-figured silk fabrics ammonia
It is 0.0～20.0g/L that acid accounts for the content of described fermentation medium, preferably 2.0～4.0g/L.
17. 1 kinds are used for the desert pseudocyst bacterium CGMCC No.10576 as claimed in claim 1 that ferments
Obtain the fermentation medium of oritavancin intermediate A 82846B, it is characterised in that it includes following
Constituent (g/L): organic carbon source 75.0～155.0, organic nitrogen source 24.0～58.0 and inorganic salt 1.9～6.3；
Described organic carbon source is the one in glucose, maltodextrin, sucrose, molasses and corn starch or many
Kind；Described organic nitrogen source is soybean cake powder, cottonseed meal, caseinhydrolysate, extraction from yeast powder and egg
One or more in white peptone；Described inorganic salt is one or both in sodium chloride, calcium carbonate.
18. fermentation medium as claimed in claim 17, it is characterised in that described fermentation culture
Base also includes one or more in following constituent (g/L): trace element 0.04～0.22, ammonia
Base acid 0.0～50.0 and/or defoamer 0.2～0.8；Described trace element is four water manganese sulfates and six water chlorine
Change in cobalt one or both；Described aminoacid be TYR, Valine, Pidolidone and
One or both in one or more in L-Leu, preferably TYR and Valine；
Described defoamer is fermentation defoamer；The pH of described fermentation medium is 6.0～7.5, preferably
It is 6.5～7.8；And/or, described defoamer is fermentation defoamer THIX-298.
19. fermentation medium as claimed in claim 18, it is characterised in that described fermentation culture
Base includes following constituent (g/L): glucose 10.0～30.0, maltodextrin 60.0～100.0, sugar
Honey 5.0～25.0, caseinhydrolysate 2.0～10.0, TYR 0～30.0, Valine 0～20.0, ferment
Female extracting powder 2.0～8.0, cottonseed meal 20.0～40.0, sodium chloride 0.4～0.8, calcium carbonate 1.5～5.5, four
Water manganese sulfate 0.02～0.14, cobalt chloride hexahydrate 0.02～0.08 and defoamer 0.2～0.8, pH6.5～7.8；
It is preferred that include following constituent (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0,
Caseinhydrolysate 6.0, TYR 12.0, Valine 3.0, extraction from yeast powder 5.0, cottonseed meal
30.0, sodium chloride 0.6, calcium carbonate 3.5, four water manganese sulfate 0.08, cobalt chloride hexahydrate 0.05 and THIX-298
0.2, pH7.5.
20. one kind obtains desert pseudocyst bacterium CGMCC No.10576's as claimed in claim 1
Method, it is characterised in that cultivate desert pseudocyst bacterium as claimed in claim 1 in the medium
CGMCC No.10576；It is preferred that described culture medium is training as claimed in claim 14
Support base.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109504624A (en) * | 2018-11-20 | 2019-03-22 | 广东肇庆星湖生物科技股份有限公司 | A kind of screening technique of leucine producing strain |
| CN113174419A (en) * | 2021-05-13 | 2021-07-27 | 丽珠集团福州福兴医药有限公司 | Feeding method for improving fermentation yield of oritavancin intermediate A82846B |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN87106483A (en) * | 1986-09-19 | 1988-06-08 | 伊莱利利公司 | Process for preparing glycopeptide antibiotics |
| US5821099A (en) * | 1996-09-13 | 1998-10-13 | Eli Lilly And Company | Glycosyltransferase gene GtfA from Amycolatopsis orientalis |
| US6087143A (en) * | 1997-09-05 | 2000-07-11 | Eli Lilly And Company | Glycosyltransferase gene gtfA from Amycolatopsis orientalis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN87106483A (en) * | 1986-09-19 | 1988-06-08 | 伊莱利利公司 | Process for preparing glycopeptide antibiotics |
| US5821099A (en) * | 1996-09-13 | 1998-10-13 | Eli Lilly And Company | Glycosyltransferase gene GtfA from Amycolatopsis orientalis |
| US6087143A (en) * | 1997-09-05 | 2000-07-11 | Eli Lilly And Company | Glycosyltransferase gene gtfA from Amycolatopsis orientalis |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109504624A (en) * | 2018-11-20 | 2019-03-22 | 广东肇庆星湖生物科技股份有限公司 | A kind of screening technique of leucine producing strain |
| CN113174419A (en) * | 2021-05-13 | 2021-07-27 | 丽珠集团福州福兴医药有限公司 | Feeding method for improving fermentation yield of oritavancin intermediate A82846B |
| CN113174419B (en) * | 2021-05-13 | 2024-01-30 | 丽珠集团福州福兴医药有限公司 | Feeding method for improving fermentation yield of Oriwaxy intermediate A82846B |
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