We have developed a strategy for rapid and combinatorial optimization of the hot spot residues of enzymes. After combinatorial randomization of target locations in the Candida antarctica lipase B (CalB) gene, the individual variant genes isolated in the E. coli cells were expressed in the cell-free protein synthesis system to analyze different parameters of the resulting CalB variants. The enzymatic assays for the hydrolysis of para-nitrophenylester (pNP-ester) and triglyceride, synthesis of wax ester, and thermal stability of the variant enzymes were carried out simultaneously in 96-well microtiter plates. From the 1,000 variant genes tested in each assay, we were able to identify a series of the variant enzymes having markedly improved hydrolytic, synthetic activity, or thermal stability. The improved traits of the cell-free selected CalB variants were well reproduced when the corresponding genes were expressed in Pichia pastoris. Therefore, we expect that the proposed strategy of cell-free expression screening can serve as a viable option for rapid and precise tuning of enzyme molecules, not only for analytical purposes but also for industrial applications through large scale production using microbial cells transformed with variant genes selected from the cell-free expression screening. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 27: 47-53, 2011