Olivera Prodanovic

Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases

Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had kcat values of 33.3 and 61.3s(-1) and Km values for glucose of 33.4 and 27.9mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.

Activity and stability of soluble and immobilized α-glucosidase from baker's yeast in cosolvent systems

The activity of α-glucosidase from bakers yeast was determined in various concentrations of dioxan, tetrahydrofuran, tert-butanol, dimethylformamide, methanol and dimethylsulfoxide (DMSO). Higher activities were observed with sucrose than with nitrophenylglucoside as substrate in cosolvent mixtures. In 30% (v/v) DMSO, 25% of the activity obtained in pure water was detected, and in 30% (v/v) methanol 12.5% of the activity in pure water was detected, while in other cosolvents there was almost no activity under these conditions. α-glucosidase was immobilized onto a macroporous copolymer of ethylene glycol dimethacrylate and glycidyl methacrylate, poly(GMA-co-EGDMA), by the glutaraldehyde method. By immobilization, the half-life of the enzyme in 35% (v/v) methanol was increased from 6 to 60 min and from 4 to 15 min in 45% (v/v) DMSO. The activity of the immobilized enzyme in 30% (v/v) DMSO and 30% (v/v) methanol was 22% and 18% of the activity in pure water, respectively.