DIRECTED EVOLUTION OF A FUCOSIDASE FROM A GALACTOSIDASE BY DNA SHUFFLING AND SCREENING

An efficient beta-fucosidase was evolved by DNA shuffling from the Esc herichia coli lacZ beta-galactosidase, Seven rounds of DNA shuffling a nd colony screening on chromogenic fucose substrates were performed, u sing 10,000 colonies per round, Compared with native beta-galactosidas e, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity For o-nitrophenyl fucopyr anoside versus o-nitrophenyl galactopyranoside and a 300-fold increase d substrate specificity for p-nitrophenyl fucopyranoside versus p-nitr ophenyl galactopyranoside. The evolved cell line showed a tig-fold inc rease in p-nitrophenyl fucosidase specific activity, The evolved fucos idase has a 10- to 20-fold increased k(cat)/K-m for the fucose substra tes compared with the native enzyme. The DNA sequence of the evolved f ucosidase gene showed 13 base changes, resulting in six amino acid cha nges from the native enzyme, This effort shows that the library size t hat is required to obtain significant enhancements in specificity and activity by reiterative DNA shuffling and screening, even for an enzym e of 109 kDa, is within range of existing high-throughput technology, Reiterative generation of libraries and stepwise accumulation of impro vements based on addition of beneficial mutations appears to be a prom ising alternative to rational design.