Improving The Acarbose Production Of Actinoplanes Utahensis ZJB-08196 By Mutagensis And Fermentation Process Optimization

Acarbose is a potent α-glucosidase inhibitor produced by Actinomycetes such as Actinoplanes sp,with excellent pharmacokinetic and safety,It has been widely applied in treatment of type II diabetes.Recently,as the patients of diabetes increased sharply in our country,the demand for acarbose is growing quickly,which evident its promising market prospects.However,the production of acarbose by fermentation suffers the drawback of low productivity and high production costs.Therefore,improving the acarbose production of acarbose producing strains by mutagensis and/or fermentation processes optimization is one of the central aims for industrial production.In this paper,mutant ZRP4 with acarbose production of 3271.33 mg/1 was selected from Actinoplanes utahensis ZJB-08196.By UV and microwave irradiation,UV-3 and MI-24,two mutants with acarbose production of 3658.22 mg/1 and 3803.07 mg/1 were selected,respectively.Then MU-12,a mutant with acarbose production of 4023.35mg/l by combined UV and microwave mutangensis,increased by 22.99%on the relative to the original strains A.utahensis ZRP4.The mutant A.utahensis ZRP4 was further improved by ribosomes engineering.Treated with rifampicin,streptomycin,gentamycin,and neomycin,four mutants with improved acarbose production were obtained.The acarbose production of R10-9(resistant to rifampicin),S20-9(resistant to streptomycin),G5-11(resistant to entamycin),and N5-22(resistant to neomycin)reached 4370.02,4370.93,4294.89 and 4067.24 mg/1,respectively.The mutants MU-12,R10-9,S20-9,G5-11 and N5-22 were further improved by combination with rifampicin,streptomycin,gentamycin,and neomycin,GNSR-9 and GNSR-15,two mutants with multiple antibiotics resistance and high yield were obtained.The acarbose production of GNSR-9 and GNSR-15 were raised to 4681.71 and 4725.13 mg/1,which increase by 43.11%and 44.44%on compared with A.utahensis ZRP4,respectively.With better genetic stability,GNSR-9 was chosen for furhter study.The optimal fermentation conditions were as follows:medium loading 30ml per 250ml shaking flask,seed cultivation time 60 h,inoculum size of 10%(v/v),initial pH 7.0,28℃,200 rpm,under these conditions,the production of acarbose was raise to 4835.21 mg/l.By single factor experiments,glucose,maltose,and soybean meal were chosen as the best carbon source,nitrogen source.The medium components were further optimized by Uniform Design optimization.The optimized medium(g/1)were as follow:glucose 30.0,saccharified liquid 312.5 ml(maltose 50.0),glycerin 10.0,soybean meal 15.0,sodium glutamate 6.0,CaC03 2.0,CaCl23.0,FeCl3 1.2,K2HPO4 0.8.Under the optimum conditions,the production of acarbose was reached to 5250.40mg/l.Finally,fed-batch fermentation was carried out,8 ml fermentation medium were added at 24,48,72 and 96 h of the fermentation,2 ml per time.The optimal glucose and maltose concentrations were 30 g/l and 70 g/1,respectively.Under the optimization of fermentaion process,maximum acarbose production reached 6632.03 mg/1,which increase the output of acarbose significantly.