Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants

The bacterial surface display method was used to selectively screen for imp roved variants of carboxymethyl cellulase (CMCase). A library of mutated CM Case genes generated by DNA shuffling was fused to the ice nucleation prote in (Inp) gene so that the resulting fusion proteins would be displayed on t he bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogene ous colonies. In contrast, cells displaying the nonmutated parent CMCase fo rmed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the a ctivity of variant CMCases at the cell surface. Staining assays indicate th at large, rapidly growing colonies have increased CMCase activity. Increase d CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increas e in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occ urred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct wa y to correlate evolved enzyme activity with cell growth rates. This techniq ue will provide a useful technology platform for directed evolution and hig h-throughput screening of industrial enzymes, including hydrolases.