Jh. Zhang et al., DIRECTED EVOLUTION OF A FUCOSIDASE FROM A GALACTOSIDASE BY DNA SHUFFLING AND SCREENING, Proceedings of the National Academy of Sciences of the United Statesof America, 94(9), 1997, pp. 4504-4509
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.