Studies Of Enzyme Immobilization By Using Saccharomyces Cerevisiae Spores

Most of the enzyme immobilization is performed through either physical or chemicalmethods so far. In the research, we report a novel method to produce immobilized enzymesusing Saccharomyces cerevisiae spores. Since yeast spore wall has a unique stratifiedstructure, in order to test whether spores can be used as a vectorof enzyme immobilization, wetry to express a secreted enzyme in spores. When the enzymes are held around the spore wall,they acquired some beneficial characters which need to be verified, then we knock out somegenes involved in the assembling of spore wall, thus creating the spore wall mutants whichcan improve the ability of enzyme immobilization.First we tried to express the green fluorescent protein (Gfp) with signal peptide in wildtype spore and dit1Δ mutant spore. Since the wild type spores have the outermost layerdityrosine which can prevent the Gfp from diffusing. However, dit1Δ mutant spore can nothold Gfp due to the absence of dityrosine, most of the Gfp diffused in the ascus. Plasmidswhich carry the GFP and a special tail EDEDE (three glutamic acids and two aspartic acidslinked alternately) or YYYYY (five tyrosines linked in tandem) were expressed in the dit1mutant. But still can not improve the immobilization.A red fluorescent protein(Rfp) fusion of the α-galactosidase, Mel1, expressed in sporesand vegetative cells were observed, the result showed that spores wall can hold Mel1-Rfpfusion, while that of vegetative cells can not. Besides the Rfp localization, we made anenzyme activity assay which illustrated that the spores have a relative higher activity. Byencapsulated in the spore wall, the enzymes acquire some beneficial characters, such as,resistance of digestive enzyme, high temperature, and salty washing. The osw2Δ mutant thatwas achieved by gene disruption, can improve the ability of immobilization significantly.Besides the Mel1, we also show that spores can retain the invertase. Invertase wasexpressed in four different mutant spores osw2,dit1,ydr326c,ydl186w.Interestingly theencapsulated invertase has significantly low activity towards raffinose compared to sucrose.This suggests that substrate selectivity could be altered by the encapsulation.In all,we have demonstrated that the yeast spores which have some special characters canbe used for enzyme immobilization...