Development of high throughput screening systems of mutant libraries for sugar modifying enzymes (glucose oxidase and cellulases)

The enzyme glucose oxidase (GOx) has numerous applications, including the removal of oxygen from foods and beverages, the generation of hydrogen peroxide for food preservation, the measurement of free glucose in clinical samples and bioreactors, and the development of miniature biofuel cells. Many industrial processes could therefore benefit from improved versions of GOx, but three enzyme properties are particularly relevant targets in the context of biofuel cells: (1) the rate of electron transfer from the electrode to the enzyme (the intrinsic enzyme activity); (2) the activity of the enzyme under physiological conditins ( pH 7.4 and 5 mM glucose); and (3) thermal stability. We have developed an ultra-high-throughput screening system allowing the quantitative selection of improved enzymes (rather than the plus/minus screening capabilities of other systems) based on our previously described tyramide-fluorescein assay. We improved the performance of the system by using yeast surface display for GOx expression, by estimating the abundance of the enzyme using a c-myc epitope tag and antibody labeling, and by delivering exogenous glucose using octylglucoside in the oil phase and glucosidase in the water microdroplets. Five mutants with greater activity than wild type GOx were isolated from a gene library containing 105 mutants created using the consensus approach. These include mutant A2, which has a 5.8-fold higher activity than wild-type GOx at pH 7.4, and thermostable mutant F9-1 whose half-life at 60°C in the absence of substrate is twice that of the wild-type enzyme. In addition we have developed a novel, ultra-high-throughput screening assay for the detection of cellulase activity based on fluorescence activated cell sorting (FACS) and double emulsion technology. Cellulase activity is detected using a series of coupled enzymes including hexose oxidase (HOx), which generates hydrogen peroxide from the reducing sugars released by cellulases in the presence of any natural or artificial substrate. The assay can be adapted to suit a microtiter plate format, but the highest throughput is achieved by using FACS to screen high-complexity cellulose clone libraries. Using this approach, we achieved a 12-fold enrichment of positive (cellulose-expressing) cells in cellulase reference libraries after just one sorting round