Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening

被引:210
作者
Zhang, JH
Dawes, G
Stemmer, WPC
机构
[1] MAXYGEN INC,SANTA CLARA,CA 95051
[2] AFFYMAX RES INST,SANTA CLARA,CA 95051
关键词
D O I
10.1073/pnas.94.9.4504
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
An efficient beta-fucosidase was evolved by DNA shuffling from the Escherichia coli lacZ beta-galactosidase, Seven rounds of DNA shuffling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round, Compared with native beta-galactosidase, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity For o-nitrophenyl fucopyranoside versus o-nitrophenyl galactopyranoside and a 300-fold increased substrate specificity for p-nitrophenyl fucopyranoside versus p-nitrophenyl galactopyranoside. The evolved cell line showed a tig-fold increase in p-nitrophenyl fucosidase specific activity, The evolved fucosidase has a 10- to 20-fold increased k(cat)/K-m for the fucose substrates compared with the native enzyme. The DNA sequence of the evolved fucosidase gene showed 13 base changes, resulting in six amino acid changes from the native enzyme, This effort shows that the library size that is required to obtain significant enhancements in specificity and activity by reiterative DNA shuffling and screening, even for an enzyme of 109 kDa, is within range of existing high-throughput technology, Reiterative generation of libraries and stepwise accumulation of improvements based on addition of beneficial mutations appears to be a promising alternative to rational design.
引用
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页码:4504 / 4509
页数:6
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