Study On The Breeding Of Echinocandin B High-producing Strain And Optimization Of Fermentation Conditions

Anidulafungin is the drug of the third generation that is used in the treatment of fungus invasive infections.Owing to its good curative effect,low toxicity and good resistance for other antifungal drugs,anidulafungin is called as "trump card" among the antifungal drugs.Echinocandin B(ECB),as the precursor of anidulafungin,is fermentationed by Aspergillus nidulans.In order to obtain high-producing and stable strain,in this paper,breeding high-producing strains with Aspergillus nidulans ZJB0817 and optimizing culture conditions are the research that focus in the field of increasing the productivity of Echinocandin B.Results are as follows.Combination of sequential mutagenesis and optimization of fermentation conditions to isolate helicosporous hyphomycete fungus was studied to maximize echinocandin B production from Aspergillus nidulans ZJB0817.The mutants using ultraviolet radiations,lithium chloride,and sodium nitrite were evaluated for their highest titre,morphology and stability.Among these mutants,the most promised mutant ULN-59 induced by sodium nitrite produced maximize ECB(.i.e.1583.1 mg/1),which was 2.1 times higher than the wild-type strain(.i.e.750.8 mg/1)and had stable genetic traits for five generations.Furthermore,in order to obtain high-production of ECB,the effects of culture conditions and culture medium on ECB production with ULN-59 were studied.The results indicated that the optimal culture conditions were consisted of 13 to 15 day of slant culture time,48 h of seed culture time,24℃ of seed culture temperature,complex carbon source(10 g/1 glycerin and 90 g/1 manntol)and complex nitrogen source(20 g/1 peptone and 50 g/l soylbean).According to this condition,the initial culture conditions were consisted of 0.5 g/1 calcium chloride and 0.2 g/l manganese sulfate,1.5 g/1 tomato meal and 4 g/1 L-ornithine,and 20 g/1 dipotassium hydrogen phosphate.Under this optimal conditions,the production of ECB was 1952.6 mg/l,which was 1.2 times higher than that of parental strain ZJB0817.Response surface methodology was used to optimize the initial medium composition for ECB production by ULN-59 on the optimal culture conditions.It showed that peptone,manntol,L-Ornithine and dipotassium hydrogen phosphate notably influenced ECB yield(p<0.01),and the optimum concentration of them were confirmed by central composite design.Results showed that it was notable for the interaction between peptone and manntol,peptone and dipotassium hydrogen phosphate,and peptone and L-Ornithine(p<0.01).Under the optimal conditions which consisted of 8.6 g/1 peptone,97.3 g/1 manntol,8.4 g/l dipotassium hydrogen phosphate and 5.3 g/1 L-Ornithine,the production of ECB reached 2277.2 mg/1.