원문정보
초록
영어
We have developed a novel strategy for rapid, combinatorial optimization of hot spots using the lipase B from Candida antartica (CalB) as a model enzyme. After combinatorial randomization of target locations that are known to affect the enzymatic activity of CalB, individual variant genes isolated in the E.coli cells were PCRamplified and expressed in a cell-free protein synthesis system to analyze the enzymatic activity of the CalB variants. Through an extensive expression screening, we were able to find a series of variant CalB enzymes whose activity was improved as much as 20 folds compared to that of wild-type enzyme. Furthermore, similar extent of increase in enzymatic activity was observed when the selected clones were expressed in Pichia
pastoris indicating that amino acid substitutions give similar effects to the variant enzymes either in vivo or in vitro. Based on these results, we expect that the proposed strategy can be used as a powerful platform for engineering the enzymes of industrial importance.