Introduction

Extensive studies have been conducted on various methods of immobilization of enzymes. Conventional methods for immobilization of enzymes through covalent bonds have some merits; the enzymes are immobilized through strong bondage, and dissociation is low in substrate or salt solution even at high concentrations. However, change in structure of the immobilized protein or in their characteristics often occurs owing to severe treatment and there are many difficulties in the determination of the immobilization reaction conditions. On the other hand, immobilization through ionic bonds does not share these disadvantages, but the enzymes are easily dissociated.

The genetic engineering method, combined with the immobilization method using cell surface as a carrier for immobilization of enzymes, overcomes the disadvantages of the above methods; enzymes are immobilized by a strong covalent bondage. They can be regenerated according to the activation of the promoter, and they are "naturally" immobilized on the cell surface. Immobilization of enzymes on cell surface saves tedious purification process of enzymes to be used in conventional immobilization. Expression of proteins on the cell surface of S. cere-visiae would offer more advantages than other microbial cells. First, since S. cerevisiae is widely used in industrial production of proteins and chemicals, enzyme-coated yeast cells could be used as novel whole-cell biocatalysts, because surface-immobilized proteins are covalently linked to glucan in the cell wall, rendering them resistant to extraction. Second, as S. cerevisiae is generally regarded as safe (GRAS) for oral use, it can be used in food and pharmaceutical products.

The immobilized enzyme displayed on the cell surface is also regarded as a kind of a self-immobilized enzyme on the cell surface; this phenomenon is passed on to daughter cells as long as the plasmid or the integrated gene is retained by the cells. This immobilization system could turn or remake S. cerevisiae into a novel and attractive microorganism as a whole-cell biocatalyst by surface expression of various enzymes, especially when target substrates are not able to be taken up by the cells.

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