Selection Of The Strain And Optimization Of Fermentation Conditions For Acarbose Production

Acarbose is an α-glucosidase inhibitor,firstly isolated from the fermentation broth of the genus of Actinoplanes as a secondary metabolite,which has a strong inhibitory effect towards intestinal sucrase,maltase,amylase,dextrin enzymes.It possesses good pharmacokinetic properties and low toxicity,and can reduce the human intestinal decomposition of polysaccharides and oligosaccharides,thereby reducing blood glucose levels.Today,it is clinically used in the treatment of type Ⅱdiabetes.We isolated a strain ZJB-08196 with high acarbose synthesizing capacity.It was identified as Actinoplanes utahensis according to morphological characteristics and 16S rDNA gene sequencing.A precise and stable HPLC detection method for acarbose was established.Single factor experimental design was firstly adopted to optimize conditions for A.utahensis ZJB-08196 biosynthesizing acarbose.Cultivation outcome indicated that the optimized culture media were composed of（g/1）:maltose 50.0,glucose 30.0,soybean meal 16.0,monosodium glutamate 5.0,CaC03 2.5,CaCl2 2.0,FeCl3 0.5,K2HPO42.0,glycerol 5.0;the optimal fermentation conditions were as follows:shaking the flask selected without baffle,initial medium pH 7.0,loading volume of 50ml per 500ml,shaker speed 200 rpm,inoculum size of 9%（v/v）,culture temperature 27℃,and fermentation time 156 h.Under these optimal conditions,the maximal acarbose titer peaked at 4210 mg/1.Results showed that addition of 0.24 g/1 validamycin A increased acarbose yield by 47.5%.It was found that glycerol in the medium as substances of regulating Osmolyte,can increase the yield of acarbose.Impact of the initial Osmolality was further investigated.The capacity of A.utahensis ZJB-08196 to synthesize acarbose peaked at 591 mOsm/kg.Fed-batch fermentation optimization of A.utahensis ZJB-08196 showed that the ideal feeding protocol was as follows:the initial working volume of 30 ml media per 500 ml,feeding time from 48h,5ml of feeding stuff each 24h for four times.The optimized feed was composed of（g/1）:maltose 14.0,glucose 6.0,soybean meal 9.0.Under these optimal fed batch fermentation conditions,peak acarbose titer of 4877.75 mg/1 was reached.Using Logistic,Luedeking-Piret equation and data collected from the batch fermentation of A.utahensis ZJB-08196 in 500ml shaking-flaskes,we developed mathematical models describing the kinetics of A.utahensis ZJB-08196 growth and acarbose formation.Besides,the parameters of the models were obtained through nonlinear curve fitting the established model into experimental data by OriginPro 8 software.The regressed models were presented.A.utahensis ZJB-08196 growth model:dX/dt=7.9×10-2X（1-X/29.05）Acarbose formation model:dP =-11.59×dX/dt+1.13XSubstrate depletion model:-dS/dt=1.2dX/dt+ 7.2×10^-3 dP/dt...