Construction Of Echinocandin B Deacylase Recombinant Engineering Strain And Biotransformation Of Echinocandin B

Echinocandin B?ECB?deacylase can catalyze echinocandin B.The side chain amide bond was removed.In this way,ECB nucleus was acquired.In the new antifungal,the chemical-enzymatic synthesis has important applications in preparation of anidulafungin.Results are as follows.Clone of ECB dealyse gene from pUC18-ecbbD,The size of 4.3 kb ECB deacylase gene was getted.The fragment was linked into the overexpression vector pSET152 by T4 DNA ligase.Then the plasmid was transformed into Streptomyces coelicolor A3 by the E.coli ET12567/pUZ8002-Streptomycesintergenericconjugaltransfersystem.Construction of genetically engineered strain,named as S.coelicolor A3/pSET152-ecbD.This genetically engineered strain could transform ECB into ECB nucleusand.In order to obtain high-production of ECB deacylase,the effects of culture conditions and culture medium on ECB deacylase production with S.coelicolor A3/pSET152-ecbD were studied.The results indicated that the optimal culture conditions were consisted of 10 day of slant culture time,72 h of seed culture time,30?of seed culture temperature.The fermentation medium was sourse 6%,peanut powder 1.2%,CaCO₃ 0.8%,yeast extract 0.30%.The optimal culture conditions were consisted of 3 d day of fermentation culture time,28?of fermentation culture temperature,pH=8.0.According to this condition,the production of ECB deacylase was 544 U/L,which was 1.75 times higher than that of parental strain S.coelicolor A3/pSET152-ecbD.The biotransformation of ECB deacylase was investigated.The optimal reaction conditions are as follows.The optimum reaction temperature was 45?,the optimum reaction pH was 7.5.Zn²⁺,Mg²⁺,Mn²⁺,Fe²⁺have a significant activation on the ECB deacylase,but the Cu²⁺and Co²⁺have a inhibitory effect on ECB deacylase.The substrate concentration was 1.5 g/L,the best reaction time was 28 h.At this time,the conversion rate was up to 39.1%.