CN106190854B - A kind of preparation method of desert pseudocyst bacterium and oritavancin intermediate - Google Patents
A kind of preparation method of desert pseudocyst bacterium and oritavancin intermediate Download PDFInfo
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Abstract
The invention discloses the preparation methods of a kind of desert pseudocyst bacterium and oritavancin intermediate.The bacterial strain desert pseudocyst bacterium HS807-AN-2396 (Kibdelosporangium aridum) of the high yield oritavancin intermediate, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.10576.The preparation method of the oritavancin intermediate is that the desert pseudocyst bacterium CGMCC No.10576 ferments in the fermentation medium, and oritavancin intermediate A 82846B is obtained from fermentation liquid.The preparation method can be in 60m3The high yield that oritavancin intermediate fermentation titer 2315mg/L is realized on tank, is conducive to the industrialization production of oritavancin.
Description
Technical field
The invention belongs to field of medicaments, and in particular to a kind of preparation side of desert pseudocyst bacterium and oritavancin intermediate
Method.
Background technique
Aerobic gram-positive cocci is the important pathogen of bacterial infection, and since late 1980s, such is thin
Infection caused by bacterium is in continuous upward trend.However gram-positive cocci drug resistance is on the rise, to controlling for infectious diseases
Treatment brings severe challenge.Teicoplanin and vancomycin are the common glycopeptide classes currently used for treating drug resistance gram-positive bacterial infections
Drug.But it is increasingly extensive with clinical application, also occur in succession all over the world drug resistance of vancomycin S. aureus L-forms (VISA,
) and Vancomycin-resistant Enterococcus (VRE) VRSA.Therefore, teicoplanin and vancomycin cannot fully meet clinical need
It wants, it is necessary to the new glycopeptide antibiotics drug of development and application.
On August 6th, 2014, U.S. FDA ratify Medicines company exploitation oritavancin listing (Oritavancin,
LY-333328), trade name Orbactiv, it is the second generation glycopeptide class antibiosis developed after teicoplanin and vancomycin
Element.Orbactiv be U.S. FDA approval for treat first of acute bacterial skin and skin structure infection (ABSSSIs) and
The antibiotic of unique single-dose regimen.Orbactiv injection is used for ABSSSIs caused by gram positive bacteria sensitive strain
The treatment of adult patient, comprising: staphylococcus aureus (Staphylococcus aureus, methicillin-sensitivity and methoxy west
Woods antibody-resistant bacterium), micrococcus scarlatinae (Mlicrococcus scarlatinae), Streptococcusagalactiae (Streptococcus
Agalactiae), streptococcus dysgalactiae (Streptococcus dysgalactiae), streptococcus anginosus group
(Streptococcus anginosus, Streptococcus intermedius and Streptococcus
) and enterococcus faecalis (Enterococcus faecalis, vancomycin sensitive strain) constellatus.
The chemical synthesis of oritavancin needs key intermediate A82846B, by desert pseudocyst bacterium
(Kibdelosporangium aridum) fermentation generates.Currently, having carried out more grind to oritavancin pharmaceutical activity both at home and abroad
Study carefully, and the research and development and production report to intermediate A82846B are few.The United States Patent (USP) that last century the nineties Li Lai company obtains
The strain of production A82846B, fermentation and extraction technique is described in US5843437 (grant date on December 1st, 1998) etc.,
But its fermentation titer is low.And the fermentation level of A82846B is improved, there is important application value to oritavancin industrialization.
ARTP (Atmospheric and Room Temperature Plasma) is atmospheric pressure at room plasma, is close
The new plasma source of the one kind in the past few years to grow up, can generate temperature between 25~40 DEG C at normal pressure (1atm)
, with high activity particle (helium atom, oxygen atom, nitrogen-atoms, OH as being in excitation state-Free radical etc.) concentration plasma
Body jet stream.Studies have shown that the active particle of suitable dosage acts on microorganism in plasma, can make microorganism wall and
The structure and permeability changes of cell membrane, and cause gene damage, and then keep microbial gene sequences and its metabolism network significant
Variation eventually leads to microorganism and generates mutation.Compared with classic mutagenesis method, effectively caused using atmospheric pressure at room plasma more
The advantages that DNA damage of sample, mutation rate are high, the good mutant strain that easily obtains genetic stability；With molecule manipulation means or its
He compares chemical mutagenesis means, and atmospheric pressure at room plasma carries out microorganism mutation breeding, and there is easy to operate, at low cost, nothing to have
Malicious harmful substance participates in the advantages that mutagenic processes.
NTG (N- methyl-N '-nitro-N nitrosoguanidine) is common alkylating agent in Microbial Breeding, there is super mutagens
Title.Alkylating agent has active al, this group can be transferred to other electron-dense molecules up, make many positions of base
It sets and increases alkyl, to change hydrogen bond in various aspects.It is mainly GC-AT conversion by the mutation that NTG induces, in addition also
Small range excision, frameshift mutation and GC pairs of missing.
Summary of the invention
The technical problem to be solved by the present invention is to low for current oritavancin intermediate (A82846B) fermentation titer
Defect, a kind of desert pseudocyst bacterium is provided and its fermentation obtains the preparation method of A82846B, the desert pseudocyst bacterium can
Oritavancin intermediate is produced with high yield, fermentation efficiency is high；The preparation method can obtain largely at lower cost
Oritavancin intermediate, is conducive to industrialization production.
Shown in the structural formula of A82846B of the present invention such as formula (1):
The present inventor, which combines the mutagenesis means of both physics of ARTP and NTG and chemistry, carries out complex mutation sieve
Choosing obtains the desert pseudocyst bacterium of the mutation of high, stabilization characteristics of genetics the production A82846B of fermentation titer.And spore is intended to desert
Fermentative medium formula, the technological parameter of method etc. that the fermentation of capsule bacterium obtains A82846B optimize.
Technical solution of the present invention first is that: a kind of desert pseudocyst bacterium (Kibdelosporangium aridum),
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC No.10576.
Separation obtains strain A.orientalis A82846, the bacterial strain obtained through natural separation from the pedotheque of Haiti
HS807-O-13, bacterial strain HS807-O-13 obtain bacterial strain HS807-A-639, the bacterial strain HS807-A-639 after mutagenesis screening
Through ARTP and NTG Mutation screening, the bacterial strain CGMCC No.10576 with high yield A82846B is obtained.The bacterial strain is carried out
Identification, result are desert pseudocyst bacterium (Kibdelosporangium aridum), are named as HS807-AN-2396.The bacterial strain
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 11st, 2015, and receives preservation
Register on the books number CGMCC No.10576 at center, with Microbiological Characteristics below:
1, morphologic characteristic
When observing the bacterial strain cultivated in solid medium under the microscope, which is in filiform different in size, and mycelia is straight
Diameter is 0.1-1.0 μm, produces white spore, and Gram's staining is positive.
2, the characteristic that culture is learned
Under 14~37 DEG C of temperature, pH5.0~7.5, bacterium colony well-grown, more than the range then will appear growth difference or
Non-growing situation.
3, physiological property
Can be well using D-Glucose, maltose, D- lactose as carbon source, but do not utilize D- xylose, L- Arabic
Sugar and glycine.Ammonium salt class nitrogen source can be effectively utilized；It can be difficult to utilizing Nitrates nitrogen source.Can except adenine with
Well-grown on outer degradation product；In terms of degradation, can only degrade casein and tyrosine.And it is able to carry out gelatin liquefaction
And hydrogen sulfide can be generated.
Technical solution of the present invention second is that: a method of oritavancin intermediate A 82846B is prepared, by the famine
Unconcerned pseudocyst bacterium CGMCC No.10576 ferments in fermentation medium, and oritavancin intermediate A 82846B is obtained from fermentation liquid.
The time of fermentation of the present invention be this field routine time, preferably 168~240 hours, more preferably for
192~212 hours, be most preferably 202~210 hours.The temperature of fermentation of the present invention is the temperature of this field routine, preferably
Ground is 25~34 DEG C, is more preferably 28~32 DEG C.
The fermentation carries out in shaking flask, and the volume of the shaking flask is the volume of this field routine, preferably 250mL.
Revolving speed is the revolving speed of this field routine, preferably 250rpm.The amplitude is the amplitude of this field routine, preferably 5cm.
The humidity of the fermentation be this field routine humidity, preferably 40~60%.
The fermentation carries out in lab scale fermentor, and the volume of the lab scale fermentor is the volume of this field routine, compared with
It goodly is 50L.Speed of agitator is the revolving speed of this field routine, preferably 200~600rpm.Dissolved oxygen amount is that this field is conventional
Dissolved oxygen amount, preferably 30~50%, the percentage are that this field is conventional, and preferably fermentation cylinder for fermentation liquid is oxygen-containing
Amount accounts for the percentage for being saturated oxygen content at this temperature.Air mass flow be this field routine air mass flow, preferably 0.5~
1.0vvm。
The fermentation carries out in pilot scale fermentation tank, and the volume of the pilot scale fermentation tank is the volume of this field routine, compared with
It goodly is 5000L.Speed of agitator is the revolving speed of this field routine, preferably 30~150rpm.Dissolved oxygen amount is that this field is conventional
Dissolved oxygen amount, preferably 30~40%, the percentage is that this field is conventional, and preferably fermentation cylinder for fermentation liquid contains
Oxygen amount accounts for the percentage for being saturated oxygen content at this temperature.Air mass flow be this field routine air mass flow, preferably 0.2~
1.0vvm。
The fermentation carries out in industrialization fermentor, and the volume of the industrialization fermentor is the body of this field routine
Product, preferably 60m3.Speed of agitator is the revolving speed of this field routine, preferably 30~120rpm.Dissolved oxygen amount is this field
Conventional dissolved oxygen amount, preferably 30~35%, the percentage is that this field is conventional, preferably fermentation cylinder for fermentation liquid
Oxygen content account at this temperature be saturated oxygen content percentage.Air mass flow is the air mass flow of this field routine, preferably
0.1~0.8vvm.
The inoculum concentration of the seed liquor of the fermentation be this field routine inoculum concentration, preferably 10~12%, it is described
Percentage is percent by volume.The seed liquor is obtained by the method included the following steps, namely: by the desert pseudocyst bacterium
CGMCC No.10576 is cultivated in seed culture medium.
Wherein, the time of the culture is the time of this field routine, preferably 37~44 hours, more preferably for 41~
43 hours.The temperature of the culture be this field routine temperature, preferably 28~32 DEG C.
The culture carries out in shaking flask, and the volume of the shaking flask is the volume of this field routine, preferably 250mL.
Revolving speed is the revolving speed of this field routine, preferably 250rpm.The amplitude is the amplitude of this field routine, preferably 5cm.
The humidity of the fermentation be this field routine humidity, preferably 40~60%.
The culture carries out in lab scale seeding tank, and the volume of the lab scale seeding tank is the volume of this field routine, compared with
It goodly is 15L.Speed of agitator is the revolving speed of this field routine, preferably 200~600rpm.Dissolved oxygen amount is that this field is conventional
Dissolved oxygen amount, preferably 30~50%, the percentage are that this field is conventional, seed culture medium preferably in seeding tank
Oxygen content accounts for the percentage for being saturated oxygen content at this temperature.Air mass flow be this field routine air mass flow, preferably 0.5
~1.5vvm.
The culture carries out in pilot scale seeding tank, and the volume of the pilot scale seeding tank is the volume of this field routine, compared with
It goodly is 500L.Speed of agitator is the revolving speed of this field routine, preferably 30~150rpm.Dissolved oxygen amount is that this field is conventional
Dissolved oxygen amount, preferably 30~40%, the percentage are that this field is conventional, seed culture medium preferably in seeding tank
Oxygen content accounts for the percentage for being saturated oxygen content at this temperature.Air mass flow be this field routine air mass flow, preferably 0.2
~1.0vvm.
The culture carries out in industrialization seeding tank, and the industrialization seeding tank is the volume of this field routine, compared with
It goodly is 15m3.Speed of agitator is the revolving speed of this field routine, preferably 50~200rpm.Dissolved oxygen amount is that this field is conventional
Dissolved oxygen amount, preferably 30~50%, the percentage are that this field is conventional, seed culture medium preferably in seeding tank
Oxygen content accounts for the percentage for being saturated oxygen content at this temperature.Air mass flow be this field routine air mass flow, preferably 0.2
~1.0vvm.
The seed culture medium is the seed culture medium of this field routine, preferably comprising 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；It more preferably include such as
Under constituent (g/L): glucose 10.0, soluble starch 20.0, soybean cake powder 20.0, extraction from yeast powder 5.0, hydrolysis junket
Albumen 5.0, sodium chloride 0.3 and calcium carbonate 1.2, pH 7.2.
Preferably, it is described fermentation carried out in lab scale fermentor, pilot scale fermentation tank or industrialization fermentor, further include as
Under step: add l-tyrosine；And/or add Valine.
Wherein, the l-tyrosine the fermentation medium content be 0.0~30.0g/L, preferably 4.0~
6.0g/L.The Valine is 0.0~20.0g/L, preferably 2.0~4.0g/L in the content of the fermentation medium.
Technical solution of the present invention third is that: it is a kind of to be obtained for the desert pseudocyst bacterium CGMCC No.10576 that ferments
Obtain the fermentation medium of oritavancin intermediate A 82846B comprising following constituent (g/L): organic carbon source 75.0~
155.0, organic nitrogen source 24.0~58.0 and inorganic salts 1.9~6.3；The organic carbon source is glucose, maltodextrin, sugarcane
One of sugar, molasses and cornstarch are a variety of；The organic nitrogen source be soybean cake powder, cottonseed meal, caseinhydrolysate,
One of extraction from yeast powder and peptone are a variety of；The inorganic salts are one or both of sodium chloride, calcium carbonate.
Preferably, fermentation medium described in the fermentation medium further includes in following constituent (g/L)
It is one or more: microelement 0.04~0.22, amino acid 0.0~50.0 and/or defoaming agent 0.2~0.8.The micro member
Element is one or both of four water manganese sulfates and CoCL2 6H2O.The amino acid is l-tyrosine, Valine, L- paddy
One of propylhomoserin and L-Leu are a variety of, preferably, the amino acid is one of l-tyrosine and Valine
Or two kinds.The defoaming agent is fermentation defoaming agent, preferably, the defoaming agent is fermentation defoaming agent THIX-298,
Purchased from Yantai Thinking Finechem Technology Co., Ltd..
The pH of the fermentation medium is the pH of this field routine, preferably 6.0~7.5, more preferably for 6.5~
7.8。
More preferably, the fermentation medium includes following constituent (g/L): glucose 10.0~30.0, malt
Dextrin 60.0~100.0, molasses 5.0~25.0, caseinhydrolysate 2.0~10.0, l-tyrosine 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 sulfate 0.02~0.14, CoCL2 6H2O 0.02~0.08 and defoaming agent 0.2~0.8, pH6.5~7.8；Most preferably, described
Fermentation medium include following constituent (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0, hydrolysis junket egg
White 6.0, l-tyrosine 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 sulfates 0.08, CoCL2 6H2O 0.05 and THIX-2980.2, pH7.5.
Technical solution of the present invention fourth is that: the side of desert pseudocyst bacterium CGMCC No.10576 described in acquisition a kind of
Method comprising following step cultivates the desert pseudocyst bacterium CGMCC No.10576 in the medium.
Wherein, the culture medium is the culture medium of this field routine, can grow the desert pseudocyst bacterium CGMCC
No.10576, preferably ISP2, Gause I, YMS or ISP9 culture medium, more preferably for described in description of the invention
Fermentation medium is most preferably seed culture medium described in description of the invention.The temperature of the culture is that this field is conventional
Temperature can grow the desert pseudocyst bacterium CGMCC No.10576, preferably, be 14~37 DEG C, more preferably for
25~34 DEG C, be most preferably 28~32 DEG C.The pH of the culture is the pH of this field routine, can grow the desert and intend spore
Capsule bacterium CGMCC No.10576, preferably 5.0~7.5, it is more preferably 6.0~7.5, is most preferably 6.5~7.8.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that: ARTP provided by the invention and NTG complex mutation A82846B superior strain
CGMCC No.10576, stabilization characteristics of genetics, favorable reproducibility, amplification are easily achieved, and have excellent industrial application value.This
The method of the invention fermentation manufacture A82846B, structure adjusting fermentating formula, fermentating controling process, and it is directed to L- junket
Propylhomoserin and Valine are added to be controlled with residual quantity, realizes 60m3The high yield of tank top fermentation potency 2315mg/L, is being advised
It is higher than existing literature on mould and in technical level to report, is conducive to the industrialization production of oritavancin.
Biomaterial preservation information
HS807-AN-2396 of the invention has been deposited in Chinese microorganism strain preservation management committee on March 11st, 2015
Member's meeting common micro-organisms center (CGMCC), preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode: 100101,
Deposit number are as follows: CGMCC No.10576, biomaterial (strain) are HS807-AN-2396, and classification naming is
Kibdelosporangium aridum。
Detailed description of the invention
Fig. 1 is desert pseudocyst bacterium HS807-AN-2396 breeding pedigree, and the digital representation of Fig. 1 bacterial strain is corresponding
A82846B fermentation titer.
Fig. 2 is the fermentation liquid HPLC map of embodiment 1 (Rt of A82846B is 5.588).
Fig. 3 is the fermentation liquid ESI/MS map of embodiment 1.
Fig. 4 is the 60m of embodiment 153The fermentating metabolism curve of tank.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
1 superior strain desert pseudocyst bacterium HS807-AN-2396's (Kibdelosporangium aridum) of embodiment
Preparation
Separation obtains strain A.orientalis A82846 (strain A.orientalis from the pedotheque of Haiti
A82846 is referring to 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, and HS807-O-13 bacterial strain obtains the strain that sets out after mutagenesis screening
HS807-A-639.The bacteria suspension of HS807-A-639 is prepared, and is filtered with filter paper, microscopy cell dispersion degree reaches 95% or more,
Ensure largely to be unicellular, is subsequently used for mutagenic treatment.
ARTP mutagenic treatment: it is 12.5L/min that gas-helium gas flow, which occurs, for adjustment plasma；Plasma emission mouth with
Containing the distance between sample iron plate is 2mm；The irradiation power of plasma is 100w, irradiation time 30s, obtains ARTP and is irradiated
Bacteria suspension.
The NTG for accurately weighing 10.0mg is dissolved in 1mL acetone, and the irradiated bacteria suspension of above-mentioned ARTP, which is added, keeps the end of NTG dense
Degree reaches 1.0mg/L.4 DEG C slowly shake processing 20min, and centrifugation is abandoned supernatant, broken up with sterile saline, wash repeatedly 2 times,
It is set to terminate reaction.
Drawing the fluid nutrient medium containing mutagenesis, (from Zhejiang, Haizheng Pharmaceutical Co share has to 750mg/L A82846B is contained
Obtained at limit company) culture dish and l-tyrosine culture dish containing 2.0g/L in be coated culture.Cultivation temperature 28~
32 DEG C, cultivation cycle 8d.Calculated result shows that the lethality of complex mutation reaches 89%.
From picking single colonie in the resistant panel after complex mutation, into seed bottle culture, wherein seed culture medium 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 1.2,7.2,30 DEG C of culture 3d of pH, then in 250mL fermentation shake flask culture, wherein fermentation medium is (g/
L): glucose 20.0, maltodextrin 80.0, molasses 15.0, caseinhydrolysate 6.0, l-tyrosine 12.0, Valine 3.0, ferment
Mother extracting powder 5.0, cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate 3.5, four water manganese sulfates 0.08,0.05 and of CoCL2 6H2O
THIX-2980.2, pH7.5.30 DEG C of fermentations 8d, HPLC detect A82846B content.Obtain one plant of high productive mutant HS807-AN-
2396, shake flask fermentation potency is 1325mg/L, is shaken after secondary screening (i.e. according to identical condition shaking flask again, carrying out fermented and cultured)
Bottle fermentation titer 1263mg/L, HPLC map is as shown in Figure 2.The fermentation liquid that fermentation is obtained carries out separating-purifying, and (separation mentions
Pure step is referring to 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 figure of the A82846B after purification
Spectrum is as shown in Figure 3.
To set out strain HS807-A-639, picking single colonie, and into seed bottle culture, wherein seed culture medium 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
With calcium carbonate 1.2,7.2,30 DEG C of culture 3d of pH, then in 250mL fermentation shake flask culture, wherein fermentation medium is (g/L):
Glucose 20.0, maltodextrin 80.0, molasses 15.0, caseinhydrolysate 6.0, l-tyrosine 12.0, Valine 3.0, yeast
Extract powder 5.0, cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate 3.5, four water manganese sulfates 0.08,0.05 and of CoCL2 6H2O
THIX-2980.2, pH7.5.30 DEG C of fermentations 8d, HPLC detect A82846B content.Shake flask fermentation potency is 683mg/L, secondary screening
(i.e. according to identical condition shaking flask again, carrying out fermented and cultured) shake flask fermentation potency is 656mg/L afterwards.Come from fermentation results
It sees, the fermentation titer of high productive mutant HS807-AN-2396 improves 93% than starting strain HS807-A-639.
By above-mentioned high productive mutant HS807-AN-2396, on March 11st, 2015 in Chinese microorganism strain preservation pipe
The reason committee common micro-organisms center (CGMCC) preservation obtains deposit number are as follows: CGMCC No.10576, biomaterial (strain)
For HS807-AN-2396, classification naming is Kibdelosporangium aridum.
Feature is learned in the morphology of 2 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) and culture
Referring to " streptomycete identification handbook ", " classification and identification of actinomyces ", " common bacteria system identification handbook " and " divide
Sub- cloning experimentation guide " etc. related content in books tested.
Related symbol indicates to illustrate: 0: without growth；1: growth is very weak；2: can grow, there is a small amount of spore；3: well-grown,
There are a large amount of spores；4: growth is best, there is abundant spore；+: it is positive；: it is negative.
Cultural characteristic: using ISP1, ISP2, ISP3, ISP4, ISP5, Gause I, calcium malate, YMS and nine kinds of Cha Shi
Culture medium (obtains) from Haizheng Medicine Stock Co., Ltd., Zhejiang Prov, 28 DEG C culture 6~8 days after, observe mycelial color and
Pigment situation.
Cultural characteristic of the 1 bacterial strain HS807-AN-2396 of table on 9 kinds of culture mediums
The result explanation of table 1, improvement strain well-grown on ISP2, Gause I, YMS, and in different trainings
It supports on base, show different appearances, pigment and produces spore situation.
The physiological and biochemical property of 3 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576)
Condition of culture is 28 DEG C and cultivates 6~8 days.
A) carbon source: using ISP9 as basic culture medium, the final concentration of various carbon sources is 1.0%, the percentage
For mass percent.
B) inorganic nitrogen-sourced: using the basic culture medium of ISP9 conduct, the concentration of potassium nitrate and ammonium sulfate is 0.1%.
Carbon source and inorganic nitrogen-sourced utilization power are shown in Table 2, and the result explanation of table 2, bacterial strain HS807-AN-2396 is to different
Utilization of carbon source degree is different, can utilize D-Glucose, maltose, D- lactose well, but does not utilize D- xylose, L- Arabic
Sugar and glycine.Ammonium salt class nitrogen source can be effectively utilized；It can be difficult to utilizing Nitrates nitrogen source.
The carbon source of 2 bacterial strain HS807-AN-2396 of table and the utilization power of nitrogen source
| Carbon source | Growing state | Carbon source | Growing state | It is inorganic nitrogen-sourced | Growing state |
| D-Glucose | 3 | Salicin | 2 | Ammonium sulfate | + |
| D- gossypose | 2 | D- lactose | 3 | Potassium nitrate | - |
| D- xylose | 0 | Galactolipin | 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: using basal medium for GYEA (pH6.8), and the concentration of various degradation products is shown in Table 3.3 explanation of table
In addition to adenine, bacterial strain HS807-AN-2396 can on other several degradation products well-grown；In terms of degradation, bacterial strain
HS807-AN-2396 can only degrade casein and tyrosine.Percentage described in table 3 is mass percent.
The Degrading experiment result of 3 bacterial strain HS807-AN-2396 of table
| Degradation product | Degradate concentrations | As a result | Degradation product | Degradate concentrations | As a 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.The explanation of table 4, bacterial strain HS807-AN-
2396 can be carried out gelatin liquefaction and can generate hydrogen sulfide.
The 4 main physiological and biochemical property of bacterial strain HS807-AN-2396 of table
| Pilot project | As a result | Pilot project | As a result | Pilot project | As a result |
| Gelatin liquefaction | + | Milk peptonizes | - | Cellulose utilization | - |
| Starch Hydrolysis | - | Nitrate reduction | - | Catalase | - |
| Milk solidification | - | Hydrogen sulfide generates | + | ||
| V.P experiment | - | M.R experiment | - |
Illustrate that the desert pseudocyst bacterium CGMCC No.10576 has microorganism below by the data of embodiment 2~3
Learn characteristic:
1, morphologic characteristic
When observing the bacterial strain cultivated in solid medium under the microscope, cell is in filiform different in size, and size is
0.4-1.0 μm, white spore is produced, Gram's staining is positive.
2, the characteristic that culture is learned
At 14~37 DEG C of temperature, pH 5.0~7.5, it is poor then to will appear growth more than the range for bacterium colony well-grown
Or non-growing situation.
3, physiological property
Can be well using D-Glucose, maltose, D- lactose as carbon source, but do not utilize D- xylose, L- Arabic
Sugar and glycine.Ammonium salt class nitrogen source can be effectively utilized；It can be difficult to utilizing Nitrates nitrogen source.Can except adenine with
Well-grown on outer degradation product；In terms of degradation, can only degrade casein and tyrosine.And it is able to carry out gelatin liquefaction
And hydrogen sulfide can be generated.
The 16S rDNA sequence of 4 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) is analyzed
16S rDNA sequence analysis: the 16S rDNA sequence of bacterial strain HS807-AN-2396 and with correlated series in GenBank
BLAST comparison is carried out, the results are shown in Table 5.
The homology of table 5 bacterial strain HS807-AN-2396 and related strain
It is tested by the appearance features of bacterial strain HS807-AN-2396 (CGMCC No.10576), finds the bacterial strain and desert
The classification relevant parameter of pseudocyst bacterium (Kibdelosporangium aridum) is very close, while to bacterial strain HS807-AN-
2396 carry out 16S rDNA sequencing, and sequencing result is as shown in sequence table SEQ ID No.1.It will be such as sequence table SEQ ID No.1 institute
The 16S rDNA sequence of the bacterial strain HS807-AN-2396 shown and desert pseudocyst bacterium (Kibdelosporangium aridum)
16S rDNA sequence is compared, comparison result discovery, bacterial strain HS807-AN-2396 and desert pseudocyst bacterium
The homology of (Kibdelosporangium aridum) reaches as high as 98.9%: bacterial strain Kibdelosporangium aridum
The evolution parent source relationship of strain DSM 43828 and bacterial strain HS807-AN-2396 is closest, and the homology of the two is
98.9%.Therefore bacterial strain HS807-AN-2396 is accredited as desert pseudocyst bacterium (Kibdelosporangium aridum).
5 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) is in 250mL shaking flask without in amino acid media
Fermentation
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 culture medium is 20mL/250mL in seed bottle, 32 DEG C of cultivation temperature, cultivates humidity 50~60%, shakes
Bed amplitude 5cm, shaking speed 250rpm, cultivation cycle 39h.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, caseinhydrolysate 6.0, yeast are taken out
Powder 5.0, cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate 3.5, four water manganese sulfates 0.08 and CoCL2 6H2O 0.05 are mentioned,
pH7.3。
The culture transferring amount of seed bottle to fermentation flask is 10%, and the percentage is percent by volume.
The loading amount of fermentation medium is 20mL/250mL in fermentation flask, 28 DEG C of cultivation temperature, cultivates humidity 40~50%, shakes
Bed amplitude 5cm, shaking speed 250rpm, cultivation cycle 212h.
HPLC detects fermentation titer after fermentation, and A82846B reaches 923mg/L.
6 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) adds l-tyrosine, L- in 250mL shaking flask
Fermentation in 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, 28 DEG C of cultivation temperature, cultivates humidity 40~50%, shaking table amplitude 5cm shakes
Bed revolving speed 250rpm, cultivation cycle 37h.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, caseinhydrolysate 6.0, L- junket ammonia
Sour 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 sulfates
0.08 and CoCL2 6H2O 0.05, pH7.3.
The culture transferring amount of seed bottle to fermentation flask is 10%, and the percentage is percent by volume.
The loading amount of fermentation medium is 20mL/250mL in fermentation flask, 32 DEG C of cultivation temperature, cultivates humidity 50~60%, shakes
Bed amplitude 5cm, shaking speed 250rpm, cultivation cycle 210h.
HPLC detects fermentation titer after fermentation, and A82846B reaches 1210mg/L.
Fermentation lab scale craft research of the 7 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) on 50L tank
Based on the technological parameter that 250ml shaking flask obtains, according to the spy of bacterial strain HS807-AN-2396 described in embodiment 1
Property, it designs 50L tank fermentation technology and optimizes, investigate the plain ability of best production of mutant strain growth metabolism situation and A82846B.
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 (being purchased from Yantai Thinking Finechem Technology Co., Ltd.)
0.2, pH 7.2.Wherein, the loading amount of seed culture medium is 10L/15L in seeding tank.
Seeding tank control technique: 28~32 DEG C of cultivation temperature, 200~600rpm of speed of agitator, dissolved oxygen 30%~50% is empty
0.5~1.5vvm of throughput, cultivation cycle 43h.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, caseinhydrolysate 6.0, L- junket ammonia
Sour 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 sulfates
0.08, CoCL2 6H2O 0.05, THIX-2980.6, pH7.3.
The culture transferring amount of seeding tank to fermentor is 12%, and the percentage is percent by volume.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, 28~32 DEG C of cultivation temperature, speed of agitator 200~600rpm, molten
Oxygen 30~50%, 0.5~1.0vvm of air mass flow, cultivation cycle 206h.
Feeding medium during fermentation control: stream plus l-tyrosine control l-tyrosine residual quantity in 4.0~6.0g/L.
The detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc. is carried out in fermentation process simultaneously.Fermentation
After HPLC detect fermentation titer, A82846B reaches 2156mg/L.
Fermentation lab scale craft research of the 8 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) on 50L tank
Seed culture medium (g/L): glucose 5.0, soluble starch 10.0, soybean cake powder 10.0, extraction from yeast powder 2.0,
Caseinhydrolysate 2.0, sodium chloride 0.1, calcium carbonate 0.9, THIX-2980.2, pH7.5.
Seeding tank control technique: 28~32 DEG C of cultivation temperature, 200~600rpm of speed of agitator, dissolved oxygen 30%~50% is empty
0.5~1.5vvm of throughput, cultivation cycle 41h.Wherein, the loading amount of seed culture medium is 10L/15L in seeding tank.
Fermentation medium (g/L): glucose 10.0, cornstarch 100.0, sucrose 5.0, caseinhydrolysate 2.0, L- paddy ammonia
Sour 30.0, L-Leu 20.0, extraction from yeast powder 2.0, soybean cake powder 20.0, calcium carbonate 5.5, four water manganese sulfates 0.02, THIX-
2980.8 pH6.5.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, 28~32 DEG C of cultivation temperature, speed of agitator 200~600rpm, molten
Oxygen 30~50%, 0.5~1.0vvm of air mass flow, cultivation cycle 168h.
Feeding medium during fermentation control: stream plus Valine control Valine residual quantity in 0.0~20.0g/L.
The detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc. is carried out in fermentation process simultaneously, it is above-mentioned
Fermentation condition can ferment and obtain A82846B.HPLC detects fermentation titer after fermentation, and A82846B reaches 2110mg/L.
Fermentation lab scale craft research of the 9 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) on 50L tank
Seed culture medium (g/L): glucose 15.0, soluble starch 30.0, soybean cake powder 30.0, extraction from yeast powder 8.0,
Caseinhydrolysate 8.0, sodium chloride 0.5, calcium carbonate 1.5, THIX-2980.2, pH 6.8.
Seeding tank control technique: 28~32 DEG C of cultivation temperature, 200~600rpm of speed of agitator, dissolved oxygen 30%~50% is empty
0.5~1.5vvm of throughput, cultivation cycle 41h.Wherein, the loading amount of seed culture medium is 10L/15L in seeding tank.
Fermentation medium (g/L): glucose 30.0, maltodextrin 60.0, molasses 25.0, caseinhydrolysate 10.0, yeast
Extract powder 8.0, cottonseed meal 40.0, sodium chloride 0.8, CoCL2 6H2O 0.02, THIX-2980.8, pH7.8.
The loading amount of fermentation cylinder for fermentation culture medium is 35L, 28~32 DEG C of cultivation temperature, speed of agitator 200~600rpm, molten
Oxygen 30~50%, 0.5~1.0vvm of air mass flow, cultivation cycle 240h.
Feeding medium during fermentation control: stream plus l-tyrosine control l-tyrosine residual quantity in 0.0~30.0g/L.
The detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc. is carried out in fermentation process simultaneously, it is above-mentioned
Fermentation condition can ferment and obtain A82846B.HPLC detects fermentation titer after fermentation, and A82846B reaches 1967mg/L.
Fermentation lab scale craft research of the 10 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) on 50L tank
Seed culture medium (g/L): glucose 15.0, soluble starch 30.0, soybean cake powder 30.0, extraction from yeast powder 8.0,
Caseinhydrolysate 8.0, sodium chloride 0.5, calcium carbonate 1.5, THIX-2980.2, pH6.8.
Seeding tank control technique: 28~32 DEG C of cultivation temperature, 200~600rpm of speed of agitator, dissolved oxygen 30%~50% is empty
0.5~1.5vvm of throughput, cultivation cycle 41h.Wherein, the loading amount of seed culture medium is 10L/15L in seeding tank.
Fermentation medium (g/L): glucose 30.0, maltodextrin 100.0, molasses 5.0, caseinhydrolysate 10.0, yeast
Powder 8.0, cottonseed meal 20.0, sodium chloride 0.4, calcium carbonate 1.5, four water manganese sulfates 0.14, CoCL2 6H2O 0.08 are extracted,
pH7.5。
The loading amount of fermentation cylinder for fermentation culture medium is 35L, 28~32 DEG C of cultivation temperature, speed of agitator 200~600rpm, molten
Oxygen 30~50%, 0.5~1.0vvm of air mass flow, cultivation cycle 240h.
Feeding medium during fermentation control: stream plus l-tyrosine control 0.0~30.0g/L of l-tyrosine residual quantity.
The detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc. is carried out in fermentation process simultaneously, it is above-mentioned
Fermentation condition can ferment and obtain A82846B.HPLC detects fermentation titer after fermentation, and A82846B reaches 2016mg/L.
Fermentation lab scale craft research of the 11 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) on 50L tank
Seed culture medium (g/L): glucose 15.0, soluble starch 30.0, soybean cake powder 30.0, extraction from yeast powder 8.0,
Caseinhydrolysate 8.0, sodium chloride 0.5, calcium carbonate 1.5, THIX-2980.2, pH6.8.
Seeding tank control technique: 28~32 DEG C of cultivation temperature, 200~600rpm of speed of agitator, dissolved oxygen 30%~50% is empty
0.5~1.5vvm of throughput, cultivation cycle 41h.Wherein, the loading amount of seed culture medium is 10L/15L in seeding tank.
Fermentation medium (g/L): glucose 30.0, maltodextrin 60.0, molasses 25.0, caseinhydrolysate 10.0, yeast
Extract 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, 28~32 DEG C of cultivation temperature, speed of agitator 200~600rpm, molten
Oxygen 30~50%, 0.5~1.0vvm of air mass flow, cultivation cycle 240h.
Feeding medium during fermentation control: stream plus l-tyrosine control l-tyrosine residual quantity in 2.0~6.0g/L.
The detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc. is carried out in fermentation process simultaneously, it is above-mentioned
Fermentation condition can ferment and obtain A82846B.HPLC detects fermentation titer after fermentation, and A82846B reaches 1954mg/L.
Fermentation lab scale craft research of the 12 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) on 50L tank
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 (being purchased from Yantai Thinking Finechem Technology Co., Ltd.)
0.2, pH 7.2.Wherein, the loading amount of seed culture medium is 10L/15L in seeding tank.
Seeding tank control technique: 28~32 DEG C of cultivation temperature, 200~600rpm of speed of agitator, dissolved oxygen 30%~50% is empty
0.5~1.5vvm of throughput, cultivation cycle 43h.Wherein, the loading amount of seed culture medium is 10L/15L in seeding tank.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, caseinhydrolysate 6.0, L- junket ammonia
Sour 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 sulfates 0.08, CoCL2 6H2O 0.05, THIX-2980.2, pH7.5.
The culture transferring amount of seeding tank to fermentor is 12%, and the percentage is percent by volume.
The loading amount of fermentation cylinder for fermentation culture medium be 35L, 25 DEG C of cultivation temperature, 200~600rpm of speed of agitator, dissolved oxygen 30
~50%, 0.5~1.0vvm of air mass flow, cultivation cycle 206h.
Feeding medium during fermentation control: stream plus l-tyrosine control l-tyrosine residual quantity in 4.0~6.0g/L.
The detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc. is carried out in fermentation process simultaneously.Fermentation
After HPLC detect fermentation titer, A82846B reaches 1791mg/L.
Fermentation lab scale craft research of the 13 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) on 50L tank
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 (being purchased from Yantai Thinking Finechem Technology Co., Ltd.)
0.2, pH 7.2.Wherein, the loading amount of seed culture medium is 10L/15L in seeding tank.
Seeding tank control technique: 28~32 DEG C of cultivation temperature, 200~600rpm of speed of agitator, dissolved oxygen 30%~50% is empty
0.5~1.5vvm of throughput, cultivation cycle 43h.Wherein, the loading amount of seed culture medium is 10L/15L in seeding tank.
Fermentation medium (g/L): glucose 20.0, maltodextrin 70.0, molasses 15.0, caseinhydrolysate 6.0, L- junket ammonia
Sour 30.0, Valine 20.0, peptone 5.0, cottonseed meal 30.0, sodium chloride 0.6, calcium carbonate 3.5, four water manganese sulfates
0.08, CoCL2 6H2O 0.05, THIX-2980.2, pH6.0.
The culture transferring amount of seeding tank to fermentor is 12%, and the percentage is percent by volume.
The loading amount of fermentation cylinder for fermentation culture medium be 35L, 34 DEG C of cultivation temperature, 200~600rpm of speed of agitator, dissolved oxygen 30
~50%, 0.5~1.0vvm of air mass flow, cultivation cycle 206h.Feeding medium during fermentation control: stream plus l-tyrosine control L- junket ammonia
Sour residual quantity is in 4.0~6.0g/L.
The detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc. is carried out in fermentation process simultaneously.Fermentation
After HPLC detect fermentation titer, A82846B reaches 1563mg/L.
Fermentation pilot process of the 14 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) on 5000L tank is ground
Study carefully
Based on the technological parameter that 50L fermentor obtains, according to the spy of bacterial strain HS807-AN-2396 described in embodiment 1
Property, it designs 5000L tank fermentation technology and optimizes, investigate the plain energy of best production of mutant strain growth metabolism situation and A82846B
Power.
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.
Seeding tank control technique: 28~32 DEG C of cultivation temperature, 30~150rpm of speed of agitator, dissolved oxygen 30~40%, air
0.2~0.1vvm of flow, cultivation cycle 41h.Wherein, the loading amount of seed culture medium is 300L/500L in seeding tank.
Fermentation medium (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0, caseinhydrolysate 6.0, L- junket ammonia
Sour 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, six water chlorinations
Cobalt 0.05 and defoaming agent THIX-2980.4, pH7.5.
The culture transferring amount of seeding tank to fermentor is 10%, and the percentage is percent by volume.Fermentation cylinder for fermentation training
The loading amount for supporting base is 3500L: 28~32 DEG C of cultivation temperature, 30~150rpm of speed of agitator, dissolved oxygen 30~40%, air mass flow
0.2~1.0vvm, cultivation cycle 202h.
Feeding medium during fermentation control: stream plus l-tyrosine control l-tyrosine residual quantity in 4.0~6.0g/L.
The detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc. is carried out in fermentation process simultaneously.Fermentation
After HPLC detect fermentation titer, A82846B reaches 2199mg/L.
15 bacterial strain HS807-AN-2396 of embodiment (CGMCC No.10576) is in 60m3Fermentation industrialization amplification on tank
Based on the technological parameter that 5000L fermentor obtains, according to bacterial strain HS807-AN-2396 described in embodiment 1
Characteristic designs 60m3Tank fermentation technology simultaneously optimizes, and investigates the plain energy of best production of mutant strain growth metabolism situation and A82846B
Power.
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.
Seeding tank control technique: 28~32 DEG C of cultivation temperature, 50~200rpm of speed of agitator, dissolved oxygen 30~50%, air
0.2~1.0vvm of flow, cultivation cycle 41h.Wherein, the loading amount of seed culture medium is 5m in seeding tank3/15m3。
Fermentation medium (g/L): glucose 20.0, maltodextrin 80.0, molasses 15.0, caseinhydrolysate 6.0, L- junket ammonia
Sour 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 sulfates
0.08, CoCL2 6H2O 0.05, THIX-2980.2, pH7.5.
The culture transferring amount of seeding tank to fermentor is 12%, and the percentage is percent by volume.Fermentation cylinder for fermentation training
The loading amount for supporting base is 42m3: 28~32 DEG C of cultivation temperature, 30~120rpm of speed of agitator, dissolved oxygen 30~35%, air mass flow 0.1
~0.8vvm, cultivation cycle 192h.
Feeding medium during fermentation control: stream plus l-tyrosine control l-tyrosine residual quantity in 4.0~6.0g/L.
The detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc. is carried out in fermentation process simultaneously.60m3
In tank referring to fig. 4 by the fermentating metabolism curve of above-mentioned cultivation and fermentation condition fermentation strain HS807-AN-2396.After fermentation
HPLC detects fermentation titer, and A82846B reaches 2315mg/L.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention
Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (17)
1. a kind of desert pseudocyst bacterium (Kibdelosporangium aridum), which is characterized in that it is deposited in the micro- life of China
Object culture presevation administration committee common micro-organisms center, deposit number are as follows: CGMCC No.10576.
2. a kind of method for preparing oritavancin intermediate A 82846B, which is characterized in that by desert as described in claim 1
Pseudocyst bacterium CGMCC No.10576 ferments in the fermentation medium, and oritavancin intermediate A 82846B is obtained from fermentation liquid.
3. method according to claim 2, which is characterized in that the time of the fermentation is 168~240 hours；The fermentation
Temperature be 25~34 DEG C.
4. method as claimed in claim 3, which is characterized in that the fermentation carries out in shaking flask, revolving speed 250rpm；Amplitude
For 5cm；The humidity of the fermentation is 40~60%.
5. method as claimed in claim 3, which is characterized in that the fermentation carries out in lab scale fermentor；Speed of agitator is
200~600rpm；Dissolved oxygen amount is 30~50%；Air mass flow is 0.5~1.0vvm.
6. method as claimed in claim 3, which is characterized in that the fermentation carries out in pilot scale fermentation tank, and speed of agitator is
30~150rpm；Dissolved oxygen amount is 30~40%；Air mass flow is 0.2~1.0vvm.
7. method as claimed in claim 3, which is characterized in that the fermentation carries out in industrialization fermentor, speed of agitator
For 30~120rpm；Dissolved oxygen amount is 30~35%；Air mass flow is 0.1~0.8vvm.
8. method according to claim 2, which is characterized in that the inoculum concentration of the seed liquor of the fermentation is 10~12%, institute
The percentage stated is percent by volume；The seed liquor of the fermentation is obtained by the method included the following steps, namely: will be wanted such as right
Desert pseudocyst bacterium CGMCC No.10576 described in asking 1 is cultivated in seed culture medium.
9. method according to claim 8, which is characterized in that the time of the culture is 37~44 hours, the culture
Temperature is 28~32 DEG C.
10. method as claimed in claim 9, which is characterized in that the culture carries out in shaking flask, revolving speed 250rpm；Vibration
Width is 5cm；The humidity of the fermentation is 40~60%.
11. method as claimed in claim 9, which is characterized in that the culture carries out in lab scale seeding tank, and speed of agitator is
200~600rpm；The dissolved oxygen amount of the lab scale fermentor is 30~50%；Air mass flow is 0.5~1.5vvm.
12. method as claimed in claim 9, which is characterized in that the culture carries out in pilot scale seeding tank, and speed of agitator is
30~150rpm；Dissolved oxygen amount is 30~40%；Air mass flow is 0.2~1.0vvm.
13. method as claimed in claim 9, which is characterized in that the culture carries out in industrialization seeding tank, speed of agitator
For 50~200rpm；Dissolved oxygen amount is 30~50%；Air mass flow is 0.2~1.0vvm.
14. method according to claim 8, which is characterized in that the seed culture medium 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.
15. method according to claim 2, which is characterized in that the fermentation is in lab scale fermentor, pilot scale fermentation tank or industry
Change and carried out in fermentor, is include thed steps that as follows:
Add l-tyrosine；Or, adding Valine.
16. a kind of method for obtaining desert pseudocyst bacterium CGMCC No.10576 as described in claim 1, which is characterized in that
Desert pseudocyst bacterium CGMCC No.10576 as described in claim 1 is cultivated in the medium.
17. method as claimed in claim 14, which is characterized in that the seed culture medium includes 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, chlorination
Sodium 0.3, calcium carbonate 1.2 and fermentation defoaming agent THIX-2980.2, pH7.2.
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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 |
|---|---|---|---|---|
| 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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