Protoplast Mutagenesis Of Saccharomyces Cerevisiae With High Inulin Activity And Its Ethanol Fermentation Research

Jerusalem artichoke is a perennial herbaceous plant, and is one of the ideal materialsthat can produce in large-scale fuel ethanol economicly in China. The major componentsof dry matter of jerusalem artichoke is inulin. Inulin is a kind of linear fructan which ispolymerized by fructose via β-2,1glycosidic bond. Under the catalytic action of inulinase,inulin is hydrolyzed to fructose which is easy to be fermented to produce ethanol.The ethanol fermentation performance of S.cerevisiae used in ethanol industry isperfect, but its inulinase activity is very low. Although strain improvments such as UVmutagenesis and microwave radiation have been carried out, the inulinase activity ofS.cerevisiae is still low, which is one of the biggest bottleneck in ethanol industrialproduction using Jerusalem artichoke as raw material. In this study, S.cerevisiae Y05screened in our laboratory was used as starting strain, a strain of S.cerevisiae with higherinulinase activity was screened by using protoplast mutagenesis breeding technology, andits performance of ethanol fermentation was investigated.The premise of protoplast mutagenesis is preparation of protoplast. In this study,snail enzyme was used to hydrolyze the cell wall of S.cerevisiae Y05, then protoplasts wreregenerated in solid regeneration culture media to obtian new cell colonies. After thesingle factor experiments and orthogonal optimization tests, the optimum conditionsobtained for protoplast preparation were: cell age10h, snail enzyme concentration4%,enzymolysis time2.5h, hydrolysis temperature37℃, pretreatment time10min(pretreatment agent was mixture of0.2%mercaptoethanol and0.1mol/L EDTA-Na2). Cellage, enzyme concentration and enzymolysis time had very important effect on protoplastpreparation. Under the optimum conditions, the protoplast formation rate was76.2%andthe regeneration rate was15.4%. it was found that snail enzyme could hydrolyze yeast cellwalls effectively. The protoplast formation rate was97.0%, but the regeneration rate wasvery low. The reason was not clear. How to increase the regeneration rate and to improveprotoplast mutagenesis efficiency needs to be further investigated.The protoplast of S.cerevisiae Y05obtained undergone mutagenesis of UV andmicrowave together in this study. After preliminary screening and further screening,6mutant strains (Z1, Z4, Z6, Z7, Z10, Z15) were got, and their inulinase activities andethanol production ability were improved. Among them, Z10mutant strain was excellentand its inulinase activity was up to130.60U/ml, which was about as1.88times as that of its parent yeast strain. The ethanol concentration, ethanol yield and total sugar utilizationrate achieved were54.79g/L,0.445g/g and64.81%respectively，which increased by65.58%、19.95%、37.98%respectively comparing with its parent yeast strain.After the strain improvement, the effects of dissolved oxygen on ethanol fermentaionof S.cerevisiae Z10was carried out. Although ethanol fermentation is a anaerobic process,provide limited oxygen could improve ethanol fermentaion of S.cerevisiae Z10greatlybecause production of inulinase was a oxygen consumption process. It was found thatinulinase activity increased as the ventilation rate raises. At the same time, total sugarconsumption and the rate of cell growth increase. When the ventilation rate reaches0.4VVM, ethanol concentration was70g/L, and the ethanol yield was0.45, which was almost90%of theoretical ethanol yield.