Optimization Of Fermentation Conditions For Echinocandin B Production By Aspergillus Nidulans

Echinocandin B(ECB)is a novel lipopeptide compounds which contains a hexapeptide ring and fatty acid side chain,it is the precursor of an echinocandin drug anidulafungin.At present,there was no domestic production of these drugs because of the patent limitation,which leading to its market monopoly and high prices.But this patent will be expired in 2014,therefore,it is important to develop the efficient production technology to achieve its localization which will help us to break the monopoly of international pharmaceutical companies and provide cheap but high quality antifungal drugs to patient.The study has important economic and social benefits.Currently the ECB fermentation production was still low,thus it is urgent to make a breakthrough in the production capacity of strains and the fermentation metabolic regulation.In this work,the Aspergillus nidulans ZJB0817 strain was selected for ECB production.The main work was carried out to optimize the fermentation conditions and study on the influence of adding key precursors.In addition,the effects of micro-particles and shear stress on cell morphology and ECB production,and the fermentation process optimization in bioreactor were explored.The major research contents and results are as follows:Firstly,the cultivation conditions of ZJB0817 in shaking flask were optimized and results were as follows:seed age 56 h,inoculum size 12%,loading volume 50 ml per 250 ml,initial pH 7.0,cultured at 37? for the first 3 days,then at 25? for the last 9 days,and the shaking speed 220 rpm.The optimal metal ion concentration in the medium was(g/L):MnSO4·HZO 0.2,CaCl2 0.4,CuSO4·5H2O 0.6.Under the above conditions,the maximum ECB production reached to 1071 mg/L,which was 47.9%higher than that of the initial conditions(724 mg/L).Secondly,the addition of precursors and micro-particles,as well as the shear stress on cell morphology and ECB production were studied.It was found that tyrosine,L-threonine,ornithine and L-proline could affect the production of ECB greatly.The best adding strategies were as follows:5 g/L tyrosine or 1 g/L L-threonine was added at the early stage of fermentation,2 g/L ornithine and L-proline were added on the third day and the sixth day,respectively.The ECB production could reach to 1426 mg/L and 1444 mg/L under the above conditions,which improved by 33.1%and 34.6%,respectively.In addition,micro-particles and shear stress had great impact on mycelia morphology.ECB production reached to 1262 mg/L by adding 20 g/L talcum power(325 mesh,14.2 ?m),which was 33.1%higher than the blank contrast.The ECB production could also be improved by increasing shearing stress on logarithmic growth phase and reducing it on the decline phase.Finally,the effects of ventilation and stirring speed on the ECB production were studied in the fermenter.The optimum ventilation was 1.7 VVM;the agitation speed was controlled at 600 rpm at the first 6 days,and then decreased to 400 rpm at the last days.Under the above conditions,the maximum production of ECB was 608 mg/L,which was 193%higher than before.