Development of Revolutionary Enzymatic Reactions in Organic Solvents with Molecular Display

Abstract

We have seen increasing use of the term "White biotechnology". White biotechnology involves the use of microbial cells and enzymes in the production of bulk and fine chemicals such as amino acids and polymers. This generally results in cleaner processes with minimum waste generation and energy use. Most of the organic syntheses using enzymes are carried out in nearly anhydrous organic solvents or solvent-free media. Ionic liquids have more recently emerged as another nonaqueous media, which, in view of their low vapor pressure, are viewed as "green solvents". Organic solvents may alter the structure and activity of enzymes that usually function in an aqueous environment. One alternative is to immobilize the enzymes on solid supports to increase their function and stability in response to organic solvents or increased temperatures. Enzymes may be stabilized by chemical and physical processes. With chemical methods, enzymes are immobilized by strong covalent bonding, but changes in protein structure often result. In physical stabilization processes, the interactions between enzymes and solids usually are weaker, resulting in fewer changes in the enzyme's structure. Yeast cell surface engineering is an alternative approach that immobilizes enzymes on the yeast cell surface. Proteins are immobilized by using an outer shell cell-wall protein, the C-terminal half of alpha-agglutinin. Display of enzymes on the yeast cell surface has at least two advantages relative to other physical immobilization methods. First, the displayed enzymes can be readily produced in a standard fermentation. No further work is required to either purify or immobilize the enzymes. Second, enzyme displayed on the yeast cell surface can be modified directly by conventional genetic engineering, which enables error-prone PCR, DNA shuffling, and combinatorial mutagenesis to be used quickly and efficiently to create strains (whole-cell biocatalysts) with enhanced enzyme activity.