CN106497794B - One plant of naked born of the same parents' shell of structure nest for producing echinocandin B and its application - Google Patents

Abstract

The naked born of the same parents' shell of structure nest for producing echinocandin B the invention discloses one plant and its application.The present invention discloses the naked born of the same parents' shell of one plant of structure nest (Emericella nidulans), and deposit number is CGMCC No.10988.Tank top fermentation is carried out using the naked born of the same parents' shell of the structure nest for producing echinocandin B disclosed by the invention, the yield of echinocandin B can reach 4016mg/L.The structure nest naked born of the same parents' shell fermentation titer disclosed by the invention for producing echinocandin B is high, has good application value.

Description

One plant of naked born of the same parents' shell of structure nest for producing echinocandin B and its application

Technical field

The invention belongs to Microbial Breeding and field of fermentation engineering, are related to the naked born of the same parents' shell of structure nest of one plant of production echinocandin B (Emericella nidulans) and its application.

Background technique

Currently, the antifungal drug clinically used mainly have polyenoid class (Polyene), lipopeptid class (Lipopeptid), The drugs such as ucleosides (Nucleoside), azole (Azole).Due in terms of safety, many antifungal drugs are also needed The functions such as liver, kidney are monitored in the course for the treatment of, therefore clinically need further to develop a series of antifungals efficiently, less toxic Object.The lipopeptid class echinocandin drug listed in recent years is exactly most effective one kind.

It is reported that lipopeptid class antifungal drug specifically includes that Caspofungin (Caspofungin), anidulafungin (Anidulafungin), mikafen (Micafungin), amino Kangding (Aminocandin) etc..These types of antifungal drug It is all that reactive intermediate is generated by microbial fermentation, it is obtained using semi-synthetic modification.Lipopeptid class antifungal drug is main Inhibit β -1 by specificity, 3-D- glucan synthase reaches antimycotic sense so as to cause cell wall breakage and cell death The purpose of dye.Since β-(1,3)-D- glucan is not present in mammal, therefore their influences to body are small, therefore, patient couple The tolerance of such drug is preferable.

Anidulafungin is developed by Vicuron Pharmaceutical company, the U.S., and 2005 VicuronPharmaceutical is purchased by Pfizer (Pfizer), is also included into Pfizer together with anidulafungin product.2006 Year, Food and Drug Adminstration of the US (FDA) has approved the new antifungal anidulafungin of Pfizer, trade name (Eraxis), for treating the certain infection being led to by candida albicans.Anidulafungin is by the naked born of the same parents' shell (Emericella of structure nest Nidulans) the lipopeptide compound echinocandin B (Echinocandin B) that fermentation generates is through semi-synthetic gained.Clinical test Confirm that anidulafungin can fight Mycotoruloides mould, including (such as to azole antifungi drug and polyenoid class antifungi drug Nystatin the Candida glabrata) to develop drug resistance and its separation bacterium pearl.Anidulafungin is approved at present for treating non-white blood Ball low adult's candidemia, candida albicans peritonitis and the intraperitoneal purulence ulcer of candida albicans.Anidulafungin oral absorption is poor, is only capable of Intravenous infusion administration.

Predominantly Lilly (Eli Lilly) company that the research and development of anidulafungin precursor substance echinocandin B appear in the newspapers. After finding Echinocandin B compound from the 1970s, the similar group such as Echinocandin C, D is had found successively Point, the structure of common Echinocandin compound and the like is currently known as shown in following general formula and table 1:

The R of the specific substance formula of of table 1₁-R₄The specific group indicated

Echinocandin B passes through the digestion and the semi-synthetic modification of a few steps of acylase, can just obtain anidulafungin, this structure Change so that anidulafungin has bigger distribution volume and broader spectrum of antibacterial activity compared with other echinocandin antifungal agents.

Fig. 1 is anidulafungin process route.

Mutagens NTG (N- methyl-N '-nitro-N nitrosoguanidine) is common alkylating agent in Microbial Breeding, is had super The title of mutagens.Alkylating agent has active al, this group can be transferred to other electron-dense molecules up, make base Many positions on increase alkyl, to have in many-sided ability for changing hydrogen bond.It is mainly by the mutation that NTG induces GC-AT conversion, in addition there are also small range excision, frameshift mutation and GC pairs of missings.

The existing strain NRRL8112 for producing echinocandin B is quite original, and fermentation titer is in 1000mg/L hereinafter, therefore not Have industrialization value, need to be transformed the strain, reduces the fermenting and producing cost of echinocandin B, can be used for reaching Commercialized purpose.

Summary of the invention

The technical problem to be solved by the present invention is to prepare the bacterial strain for producing echinocandin B.

In order to solve the above technical problems, the present invention provides the naked born of the same parents' shell of one plant of structure nest (Emericella nidulans), incited somebody to action It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as: CGMCC) on June 17th, 2015 (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), strain number 709-N5253, deposit number CGMCC No.10988。

In order to solve the above technical problems, the present invention also provides a kind of methods for preparing echinocandin B, including by above-mentioned structure nest Naked born of the same parents' shell carries out fermented and cultured in the fermentation medium, obtains fermentation liquid.

In the above method, the condition of the fermented and cultured are as follows: 23-27 DEG C of cultivation temperature, speed of agitator 30-600rpm, molten Oxygen is greater than 10%, air mass flow 0.2-1.5vvm, cultivation cycle 192-288h, pH 4.5-6.5；

The cultivation temperature is specially 25-27 DEG C, then specially 26 DEG C；

The speed of agitator is specially 30-500rpm, then specially 30-150rpm；

The dissolved oxygen is specially 20-40%, then specially 20-30%；

The air mass flow is specially 0.2-1.0vvm, then specially 0.5-1.0vvm；

The cultivation cycle is specially 212-283h, then specially 228-252h.

In the above method, in the fermentation medium organic carbon source be glucose, lactose, sucrose, dextrin, cornstarch, One of soya-bean oil, rapeseed oil and methyl oleate are a variety of, and organic nitrogen source is soybean cake powder, peptone, cottonseed meal and yeast One of powder is a variety of.

In any of the above-described method, the fermentation medium contains glucose 10.0-30.0g/L, maltodextrin 5.0-15.0g/L, soya-bean oil 50.0-100.0g/L, cottonseed meal 20.0-40.0g/L, soybean cake powder 10.0-30.0g/L, yeast Extract powder 1.0-6.0g/L, ammonium sulfate 1.0-5.0g/L, calcium carbonate 2.0-8.0g/L, four hydrated manganese sulfate 0.01-0.03g/L, Cobalt chloride hexahydrate 0.03-0.06g/L, defoaming agent 0.2-0.8g/L, pH5.8-6.8；

The fermentation medium specifically contains glucose 15.0g/L, maltodextrin 10.0g/L, soya-bean oil 80.0g/L, cottonseed Cake powder 25.0g/L, soybean cake powder 18.0g/L, extraction from yeast powder 3.0g/L, ammonium sulfate 2.5g/L, calcium carbonate 5.0g/L, four hydrations Manganese sulfate 0.02g/L, cobalt chloride hexahydrate 0.04g/L, defoaming agent 0.6g/L, pH 6.3；Or specifically contain glucose 15.0g/ L, maltodextrin 10.0g/L, soya-bean oil 100.0g/L, cottonseed meal 25.0g/L, soybean cake powder 18.0g/L, extraction from yeast powder 3.0g/L, ammonium sulfate 2.5g/L, calcium carbonate 5.0g/L, four hydrated manganese sulfate 0.02g/L, cobalt chloride hexahydrate 0.04g/L, defoaming Agent 0.8g/L, pH 6.5；

The fermentation medium is specifically made of above-mentioned each ingredient and water, i.e., the surplus of the described fermentation medium is water.

In any of the above-described method, the naked born of the same parents' shell of structure nest be by seed liquor culture transferring into the fermentation medium Carry out the fermented and cultured；

The seed liquor is that the naked born of the same parents' shell of above-mentioned structure nest is carried out seed culture to obtain in seed culture medium；

The condition of the seed culture are as follows: 23-27 DEG C of cultivation temperature, speed of agitator 30-600rpm, dissolved oxygen is greater than 10%, Cultivation cycle 30-70h, air mass flow 0.2-1.5vvm；

The cultivation temperature is specially 25-27 DEG C, then specially 25 DEG C；

The speed of agitator is specially 50-600rpm, then specially 50-200rpm；

The dissolved oxygen is specially 30-50%, then specially 30-40%；

The air mass flow is specially 0.3-1.5vvm, then specially 0.3-1.0vvm；

The cultivation cycle is specially 43-63h, then specially 48-52h.

In the above method, the seed culture medium contain glucose 8.0-12.0g/L, soluble starch 18-22.0g/L, Hot moulding soybean cake powder 18-22.0g/L, cottonseed meal 12.0-17.0g/L, extraction from yeast powder 2.0-4.0g/L, dipotassium hydrogen phosphate 3.0-4.0g/L, calcium carbonate 2.0-4.0g/L, pH 6.5-7.0；

The seed culture medium specifically contains glucose 10.0g/L, soluble starch 20.0g/L, hot moulding soybean cake powder 20.0g/L, cottonseed meal 15.0g/L, extraction from yeast powder 3.0g/L, dipotassium hydrogen phosphate 3.5g/L, calcium carbonate 3.0g/L, pH 6.8；

The seed culture medium is specifically made of above-mentioned each ingredient and water, i.e., the surplus of the described seed culture medium is water.

In any of the above-described method, the seed liquor is according to volumn concentration 8-12% culture transferring to the fermentation In culture medium；

The seed liquor is specifically according to 10% culture transferring of volumn concentration into the fermentation medium.

In order to solve the above technical problems, the application the present invention also provides the naked born of the same parents' shell of above-mentioned structure nest in preparation echinocandin B.

Shown in the structural formula of echinocandin B of the present invention such as formula (1):

The present invention carries out NTG mutagenesis screening using NRRL8112 as starting strain, by NRRL8112, obtains one plant of white bacterium of production spine The naked born of the same parents' shell of the structure nest of plain B (Emericella nidulans) (deposit number CGMCCNo.10988), with following biology Learn characteristic:

1, morphologic characteristic

It is observed under the microscope when solid medium turns out the mycelium come, for cell in the filiform separated, cell is straight Diameter is 5-8 μm.

2, the characteristic that culture is learned

When 14~45 DEG C of temperature, pH 3.5~7.5 culture when, bacterium colony well-grown, be more than the range it is possible that Growth difference or non-growing situation.

3, physiological property

Can xylan not be utilized using D-Glucose, D- gossypose, D- xylose, D- sucrose as carbon source well. Ammonium salt can be effectively utilized and Nitrates are inorganic nitrogen-sourced.Can the purine substances such as adenine, guanine and tyrosine, Well-grown on the substances such as casein, and it is able to carry out gelatin liquefaction and nitrate reduction.

Beneficial effects of the present invention:

1, (deposit number is the naked born of the same parents' shell of structure nest (Emericella nidulans) provided by the invention for producing echinocandin B CGMCC No.10988) stabilization characteristics of genetics.

2, (deposit number is the naked born of the same parents' shell of structure nest (Emericella nidulans) provided by the invention for producing echinocandin B CGMCC No.10988) favorable reproducibility.The fermentation effect of echinocandin B on 250ml shaking flask, 50L fermentor and 5000L fermentor Valence respectively reaches 4325mg/L, 3789mg/L and 4016mg/L, it was demonstrated that the strain produces antibiotic ability in amplification process to be had Good reproducibility.

3, in the prior art, application No. is 201310477670.8 Chinese patent applications to disclose a kind of fermentation method preparation The method and bacterial strain of echinocandin B, the fermentation titer of the echinocandin B of report are 3033.9mg/L；Application No. is 201310478251.6 Chinese patent application disclose a kind of raising anidulafungin precursor compound Echinocandin B and produce The method of amount, the fermentation titer of the echinocandin B of report are 1237mg/L；Application No. is 201210041876.1 China specially Benefit application discloses echinocandin superior strain and its application, and the fermentation titer of the echinocandin B of report is 800mg/L；Shen It number please disclose a kind of for producing the culture medium of echinocandin B for 201210015967.8 Chinese patent application, report Echinocandin B fermentation titer be 908mg/L.And the structure nest naked born of the same parents' shell provided by the invention for producing echinocandin B (Emericella nidulans) (deposit number be CGMCC No.10988) using fermentating formula provided by the present invention, Under fermentating controling process, the fermentation titer of echinocandin B can reach 4016mg/L on 5000L tank, be higher than in technical level Existing literature report.

In conclusion the naked born of the same parents' shell of structure nest provided by the invention has good application value.

Preservation explanation

Strain name: the naked born of the same parents' shell of structure nest

Latin name: Emericella nidulans

Strain number: 709-N5253

Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center

Preservation mechanism abbreviation: CGMCC

Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3

Preservation date: on June 17th, 2015

Collection is registered on the books number: CGMCC No.10988

Detailed description of the invention

Fig. 1 is anidulafungin process route.

Fig. 2 is the HPLC map of echinocandin B standard items.

Fig. 3 is the HPLC map of the fermentation liquid of 709-N5253 in embodiment 2.

Fig. 4 is the ESI/MS map of the echinocandin B of the fermentation liquid of 709-N5253 in embodiment 2.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this Invention is not for limiting the scope of the invention.

Bacterial strain NRRL8112 provides for North America agricultural Organism Depositary.

Soluble starch is sea salt six and starch Chemical Industry Co., Ltd product.

Hot moulding soybean cake powder and soya-bean oil are the Jiangnan Ningbo City Bei Lun grease Co., Ltd product.

Cottonseed meal is Beijing Kang Mingwei culture medium technology Co., Ltd product.

Extraction from yeast powder is Zhejiang east into pharmaceutcal corporation, Ltd's product.

Maltodextrin is Shandong Xiwang Sugar Co., Ltd's product.

Soybean cake powder is Qingdao Creat Medium Co., Ltd.'s product.

Defoaming agent is Yantai Thinking Finechem Technology Co., Ltd.'s product.

Echinocandin B standard items are the bacterial strain NRRL8112's obtained with the step of the embodiment of the present invention 1 one to step 4 Fermentation is raw material, and according to document, " Wang Haiyan, Li Xiaolu, Jiang Qin, Li Ning, Zhang Xuexia: Echinocandin B's isolates and purifies work Skill research China's antibiotic magazine 2012,37 (3): method disclosed in 216-219. " is prepared.

Culture medium in following embodiments is formulated by water.

The fermentation of embodiment 1, bacterial strain NRRL8112 in 250ml shaking flask

One, the preparation of seed culture medium

Seed culture medium: glucose 10.0g/L, soluble starch 20.0g/L, hot moulding soybean cake powder 20.0g/L, cottonseed cake Powder 15.0g/L, extraction from yeast powder 3.0g/L, dipotassium hydrogen phosphate 3.5g/L and calcium carbonate 3.0g/L；6.8 pH before disappearing.Loading amount 25ml/250ml, 121 DEG C sterilize 30 minutes.

Two, bacterial strain NRRL8112 is seeded in the seed culture medium of shaking flask and is cultivated, 25 DEG C of cultivation temperature, cultivated wet Spend 40-60%, shaking table amplitude 5cm, shaking speed 250rpm, cultivation cycle 63h.

Three, the preparation of fermentation medium

Fermentation medium: glucose 15.0g/L, maltodextrin 10.0g/L, soya-bean oil 50.0g/L, cottonseed meal 20.0g/L, Soybean cake powder 10.0g/L, extraction from yeast powder 3.0g/L, ammonium sulfate 1.0g/L, calcium carbonate 5.0g/L, four hydrated manganese sulfate 0.02g/ L, cobalt chloride hexahydrate 0.04g/L and defoaming agent 0.6g/L；6.3 pH before disappearing.Loading amount 25ml/250ml, 121 DEG C sterilize 30 points Clock.

Four, by seed liquor that step 2 obtains according to culture transferring amount 10% (volumn concentration) move in fermentation medium into Row culture.Shake flask fermentation technique:, cultivating humidity 40-60%, shaking table amplitude 5cm, shaking speed 250rpm by 25 DEG C of cultivation temperature, Cultivation cycle 212h.

Five, HPLC is detected

Using echinocandin B standard items as control, the fermentation of the echinocandin B for the fermentation liquid that HPLC detecting step four obtains Potency.

HPLC testing conditions:

Chromatographic column: ODS, C18 (250 × 4.6mm, 5 μm)；

Detection wavelength: 225nm；

Mobile phase: acetonitrile: water volume ratio 50: 50；

Flow velocity: 1ml/min.

The result shows that the fermentation titer of echinocandin B reaches 598mg/L in the fermentation liquid that step 4 obtains.

The preparation of embodiment 2, bacterial strain 709-N5253

One, the NRRL8112 strain biography inclined-plane ISP2 is cultivated, 28 DEG C of cultivation temperature, is cultivated 12 days, with sterile washing Lower inclined plane thallus is put into the small test tube equipped with 8 beades and vibrates, then filtered with lens wiping paper, and it is outstanding to obtain NRRL8112 bacterium Liquid, microscopy cell dispersion degree reach 95% or more, with ensure it is most of be it is unicellular, subsequent mutagenic treatment can be used for.

Two, the NTG (N- methyl-N '-nitro-N nitrosoguanidine) for accurately weighing 10.0mg is dissolved in l mL acetone, is added NRRL8112 bacteria suspension makes its final concentration reach 1.0mg/L.4 DEG C slowly shake processing 30min, and supernatant is abandoned in centrifugation, with sterile life Reason salt water breaks up precipitating, washes repeatedly 2 times, it is made to terminate reaction.

Three, it draws in the bacteria suspension containing mutagenesis to the culture dish containing ISP2 culture medium and is coated culture.Culture 26 DEG C of temperature, cultivation cycle is 12 days.Calculated result shows that the lethality of the NTG mutagenesis reaches 98%.

Four, from picking single colonie on the plate after mutagenesis, into seed bottle culture 3 days (except cultivation cycle and by bacterial strain NRRL8112 is replaced with other than the single colonie after the mutagenesis picked from the plate, and condition of culture is with step 2 in embodiment 1), by One carries out 250ml fermentation shake flask culture 11 days, and (in addition to cultivation cycle and seed liquor are different, condition of culture in embodiment 1 the same as walking It is rapid four), according to step 5 in embodiment 1, HPLC detects echinocandin B content.

The HPLC map of echinocandin B standard items is as shown in Figure 2.Fig. 2 shows the retention time of echinocandin B standard items For 11.353min.

The HPLC map for finally screening to obtain the fermentation liquid of plant mutant strain a 709-N5253,709-N5253 is as shown in Figure 3. Fig. 3 shows that the retention time of echinocandin B in fermentation liquid that step 4 obtains is 11.376min.

ESI/MS analysis is carried out for the characteristic peak of echinocandin B in 709-N5253 fermentation liquid, as a result as shown in Figure 4.Figure 4 show that the molecular weight of substance contained in this feature peak is 1060, further determine that it for echinocandin B.

The shake flask fermentation potency of mutant strain 709-N5253 is 4325mg/L.From the point of view of fermentation results, mutant strain 709- Fermentation titer of the fermentation titer of N5253 than starting strain NRRL8112 improves more than 7 times.

Feature is learned in embodiment 3, the morphology of bacterial strain 709-N5253 and culture

Referring to " common mycology ", " common bacteria system identification handbook ", " Molecular Cloning:A Laboratory guide " and " middle traditional Chinese medicines Allusion quotation " the related content in the books such as (2010 editions) tested as follows；Wherein color of the color judgement referring to RAL K7 colour atla series It is compareed.

Cultural characteristic: ISP1, ISP2, ISP3, ISP4, ISP5, YMS, Gause I, calcium malate, nutrient agar are used With ten kinds of culture mediums of Cha Shi, behind 709-N5253 5~7 days that 28 DEG C of culture embodiments 2 obtain, mycelial color and color are observed Plain situation, the results are shown in Table 2.

Cultural characteristic of the 2 bacterial strain 709-N5253 of table on 10 kinds of culture mediums

* note: 0, no growth；1, it grows very weak；2, it can grow, there is a small amount of spore；3, well-grown has a large amount of spores；4, It grows best, there is abundant spore.

Table 2 show bacterial strain 709-N5253 can ISP1, ISP2, ISP3, ISP4, ISP5, Gause I, nutrient agar, Well-grown in YMS and czapek's medium, and the aerial hyphae of white is all generated, no soluble pigment generates, but in apple It is not grown on sour calcium solid medium.

The physiological and biochemical property of embodiment 4, bacterial strain 709-N5253

Physiological and biochemical test: in addition to temperature experiment, 709-N52535~7 day that embodiment 2 obtains are cultivated with 28 DEG C.Carbon Source using: using ISP9 as basis culture medium, the final concentration of various carbon sources is 1.0% (weight percentage).It is inorganic Nitrogen source using: using ISP9 as basis culture medium, the concentration of potassium nitrate and ammonium sulfate is that 0.1% (weight percent contains Amount).Degrading experiment and NaCl tolerance test use basal medium for GYEA (pH 6.8), the concentration of various degradation products such as table 4 Shown, wherein % represents weight percentage.Oxidizing ferment and catalase test, pH test and humid test are all made of PDA Agar medium.Partial results are as shown in table 3- table 5.

The carbon source of 3 bacterial strain 709-N5253 of table and the utilization power of nitrogen source

* note: 0, no growth；1, it grows very weak；2, it can grow, there is a small amount of spore；3, well-grown has a large amount of spores；4, It grows best, there is abundant spore；+, it is positive；, negative.

Table 3 shows that bacterial strain 709-N5253 can utilize D-Glucose, D- gossypose, D- xylose, D- sucrose to make well For carbon source, but do not utilize xylan.In addition ammonium salt can be effectively utilized and Nitrates are inorganic nitrogen-sourced.

The Degrading experiment result of 4 bacterial strain 709-N5253 of table

* note: 0, no growth；1, it grows very weak；2, it can grow, there is a small amount of spore；3, well-grown has a large amount of spores；4, It grows best, there is abundant spore；+, it is positive；, negative.

Table 4 shows that bacterial strain 709-N5253 can be grown on the substances such as the purines such as adenine, guanine and tyrosine Well, but this series material that can not degrade.

The major physiological biochemical character of 5 bacterial strain 709-N5253 of table

* note :+, it is positive；, negative.

Table 5 shows that bacterial strain 709-N5253 is able to carry out gelatin liquefaction and nitrate reduction.

Comprehensive Experiment is as a result, bacterial strain 709-N5253 has following Microbiological Characteristics:

1, morphologic characteristic

It is observed under the microscope when solid medium turns out the mycelium come, for cell in the filiform separated, cell is straight Diameter is 5-8 μm.

2, the characteristic that culture is learned

In 14~45 DEG C of temperature, 3.5~7.5 pH, bacterium colony well-grown is more than the range it is possible that growth Poor or non-growing situation.

3, physiological property

Can xylan not be utilized using D-Glucose, D- gossypose, D- xylose, D- sucrose as carbon source well. Ammonium salt can be effectively utilized and Nitrates are inorganic nitrogen-sourced.It can be in the objects such as the purines such as adenine, guanine and tyrosine Well-grown in matter, and it is able to carry out gelatin liquefaction and nitrate reduction.

Embodiment 5, the analysis of the 26S rDNA sequence of bacterial strain 709-N5253

The new fresh thalli for collecting the 709-N5253 of the acquisition of embodiment 2 of PDA culture, is extracted total using liquid nitrogen wall-breaking method DNA, using it as template, using Fungi Identification PCR Kit (TaKaRa product, catalog number D317) The amplification of the region gene D1/D2 26S rDNA is carried out, pcr amplification product is obtained.Pcr amplification product is examined through agarose gel electrophoresis After survey, directly it is sequenced.

The partial sequence of the 26S rDNA of bacterial strain 709-N5253 is as follows.

5’-AGCTTGCATGCCTGCAGGTCGACGATTCGCCAGGGTTTTCCCAGTCAGGACGCAAATCAATAAGC GGAGGAAAAGAAACCAACCGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAAGAGCTCAAATTTGAAAGCTGGCC CCTTCGGGGTCCGCGTTGTAATTTGCAGAGGATGCTTCGGGTGCGGCCCCTGTCTAAGTGCCCTGGAACGGGCCGT CAGAGAGGGTGAGAATCCCGTCTTGGGCAGGGTGCCCGTGCCCGTGTGAAGCTCCTTCGACGAGTCGAGTTGTTTG GGAATGCAGCTCTAAATGGGTGGTAAATTTCATCTAAAGCTAAATACCGGCCGGAGACCGATAGCGCACAAGTAGA GTGATCGAAAGATGAAAAGCACTTTGAAAAGAGAGTTAAACAGCACGTGAAATTGTTGAAAGGGAAGCGCTTGCGA CCAGACTCGGCCCCGGGGTTCAGCCAGCACTCGTGCTGGTGTACTTCCCCGGGGGCGGGCCAGCGTCGGTTTGGGC GGCCGGTCAAAGGCCCCAGGAATGTATCGCCCTCCGGGGTTGTCTTATAGCCTGGGGTGCAATGCGGCCAGCCCGG ACCGAGGAACGCGCTTCGGCACGGACGCTGGCGTAATGGTCGCAAACGACCCGTCTTGAAACACGGACCCCTGTGT GAAATTGTTATCCGCTCAATCTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGT CGTGACT-3’(SEQ ID No.1)

26S rDNA sequence analysis: 26S rDNAD1/D2 region sequence sequence to related GenBank of bacterial strain 709-N5253 Column carry out BLAST comparison result as shown in table 6 (the higher bacterial strain of homology is only listed in table 6).

The homology of table 6 bacterial strain 709-N5253 and related strain

The bacterial strain and the naked born of the same parents' shell (Emericella of structure nest are found by the experiment to bacterial strain 709-N5253 appearance features Nidulans) classification relevant parameter is very close, while to the area the 26S rDNA D1/D2 sequencing result ratio of bacterial strain 709-N5253 Homology compared with discovery, and Emericella nidulans strain ATCC 10074 has been up to 100%, therefore Bacterial strain 709-N5253 is accredited as the naked born of the same parents' shell of structure nest (Emericella nidulans).

It is common that bacterial strain 709-N5253 has been preserved in China Committee for Culture Collection of Microorganisms on June 17th, 2015 Microorganism center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number CGMCC No.10988。

The fermentation lab scale craft research of embodiment 6, bacterial strain 709-N5253 on 50L tank

Based on the technological parameter that 250ml shaking flask obtains, the characteristic of reference strain 709-N5253, design 50L tank fermentation Technique simultaneously optimizes.Investigate the plain ability of best production of the growth metabolism situation and echinocandin B of bacterial strain 709-N5253.

One, the preparation of seed culture medium

The preparation of seed culture medium is the same as the step of embodiment 1 one.

Two, bacterial strain 709-N5253 is seeded in the seed culture medium of seeding tank and carries out seed culture.Seeding tank controls work Skill: 25 DEG C of cultivation temperature, speed of agitator 200-600rpm, dissolved oxygen 30-50%, air mass flow 0.5-1.5vvm, cultivation cycle 43h。

Three, the preparation of fermentation medium

Fermentation medium: glucose 15.0g/L, maltodextrin 10.0g/L, soya-bean oil 80.0g/L, cottonseed meal 25.0g/L, Soybean cake powder 18.0g/L, extraction from yeast powder 3.0g/L, ammonium sulfate 2.5g/L, calcium carbonate 5.0g/L, four hydrated manganese sulfate 0.02g/ L, cobalt chloride hexahydrate 0.04g/L and defoaming agent 0.6g/L, pH 6.3 before sterilizing.

Four, the seed liquor for obtaining step 2 is according to culture transferring amount 10% (volumn concentration) culture transferring into fermentation medium It is cultivated.Zymotechnique control: 26 DEG C of cultivation temperature, speed of agitator 200-500rpm, dissolved oxygen 20-40%, air mass flow 0.5-1.0vvm, cultivation cycle 221h.The pH detected in fermentation process is 4.5-6.5.

Fermentation process carries out the detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc..Wherein, according to reality Step 5 in example 1 is applied, HPLC detects the content of echinocandin B in fermentation liquid.The result shows that tank effect is put in the fermentation of echinocandin B Valence reaches 3789mg/L.

The fermentation scale-up culture of embodiment 7, bacterial strain 709-N5253 on 5000L tank

The characteristic of the technological parameter and bacterial strain 709-N5253 that are obtained according to the 50L fermentor of embodiment 6 designs in 5000L Examination tank fermentation technology simultaneously optimizes.Investigate growth metabolism situation and echinocandin B of the bacterial strain 709-N5253 under pilot-scale The plain ability of best production.

One, the preparation of seed culture medium

The preparation of seed culture medium is the same as the step of embodiment 1 one.

Two, seeding tank loading amount is 350L/500L.By bacterial strain 709-N5253 be seeded in the seed culture medium of seeding tank into Row seed culture.Seeding tank control technique: 25 DEG C of cultivation temperature, speed of agitator 50-200rpm, dissolved oxygen 30-40%, air mass flow 0.3-1.0vvm, cultivation cycle 62h.

Three, the preparation of fermentation medium

Fermentation medium: glucose 15.0g/L, maltodextrin 10.0g/L, soya-bean oil 100.0g/L, cottonseed meal 25.0g/ L, soybean cake powder 18.0g/L, extraction from yeast powder 3.0g/L, ammonium sulfate 2.5g/L, calcium carbonate 5.0g/L, four hydrated manganese sulfates 0.02g/L, cobalt chloride hexahydrate 0.04g/L and defoaming agent 0.8g/L, pH 6.5 before sterilizing.

Four, fermentor loading amount is 3500L/5000L.The seed liquor that step 2 is obtained is according to (the volume hundred of culture transferring amount 10% Point content) it moves to and carries out fermented and cultured in fermentation medium.Zymotechnique control: fermentor technology controlling and process: 26 DEG C of cultivation temperature, Speed of agitator 30-150rpm, dissolved oxygen 20-30%, air mass flow 0.2-1.0vvm, cultivation cycle 283h are detected in fermentation process It is 4.5-6.5 to pH.

Fermentation process carries out the detection of total reducing sugar, reduced sugar, ammonia nitrogen, mycelial concentration and fermentation titer etc..Wherein, according to reality Step 5 in example 1 is applied, HPLC detects the content of echinocandin B in fermentation liquid.The result shows that tank potency is put in echinocandin B fermentation Reach 4016mg/L.

Claims (10)

1. the naked born of the same parents' shell of one plant of structure nest (Emericella nidulans), deposit number is CGMCC No.10988.

2. a kind of method for preparing echinocandin B, including by the naked born of the same parents' shell of structure nest described in claim 1 in the fermentation medium into Row fermented and cultured.

3. according to the method described in claim 2, it is characterized by: the condition of the fermented and cultured are as follows: cultivation temperature 23-27 DEG C, speed of agitator 30-600rpm, dissolved oxygen is greater than 10%, air mass flow 0.2-1.5vvm, pH 4.5-6.5.

4. according to the method described in claim 2, it is characterized by: organic carbon source is glucose, cream in the fermentation medium One of sugar, sucrose, dextrin, cornstarch, soya-bean oil, rapeseed oil and methyl oleate are a variety of, organic nitrogen source be soybean cake powder, One of peptone, cottonseed meal and yeast powder are a variety of.

5. according to any method of claim 2-4, it is characterised in that: the fermentation medium contains glucose 10.0- 30.0g/L, maltodextrin 5.0-15.0g/L, soya-bean oil 50.0-100.0g/L, cottonseed meal 20.0-40.0g/L, soybean cake powder 10.0-30.0g/L, extraction from yeast powder 1.0-6.0g/L, ammonium sulfate 1.0-5.0g/L, calcium carbonate 2.0-8.0g/L, four hydration sulphur Sour manganese 0.01-0.03g/L, cobalt chloride hexahydrate 0.03-0.06g/L, defoaming agent 0.2-0.8g/L, pH5.8-6.8.

6. according to any method of claim 2-4, it is characterised in that: the naked born of the same parents' shell of structure nest is by seed liquor culture transferring Extremely the fermented and cultured is carried out in the fermentation medium；

The seed liquor is that the naked born of the same parents' shell of structure nest described in claim 1 is carried out seed culture to obtain in seed culture medium；

The condition of the seed culture are as follows: 23-27 DEG C of cultivation temperature, speed of agitator 30-600rpm, dissolved oxygen is greater than 10%, air Flow 0.2-1.5vvm.

7. according to the method described in claim 6, it is characterized by: the seed culture medium contain glucose 8.0-12.0g/L, Soluble starch 18.0-22.0g/L, hot moulding soybean cake powder 18.0-22.0g/L, cottonseed meal 12.0-17.0g/L, extraction from yeast Powder 2.0-4.0g/L, dipotassium hydrogen phosphate 3.0-4.0g/L, calcium carbonate 2.0-4.0g/L, pH 6.5-7.0.

8. according to the method described in claim 6, it is characterized by: the seed liquor is moved according to volumn concentration 8-12% Kind is into the fermentation medium.

9. according to the method described in claim 6, it is characterized by: the seed liquor is according to 10% culture transferring of volumn concentration To in the fermentation medium.

10. application of the naked born of the same parents' shell of structure nest described in claim 1 in preparation echinocandin B.