Wednesday, July 29, 2009 - 10:00 AM
S111
New biocatalysts by circular permutation
Ying Yu, Zhen Qian, Patrick Baldwin, and Stefan Lutz. Department of Chemistry, Emory University, Emerson 205, Atlanta, GA 30322
Circular permutation of enzymes offers a new approach for altering and improving catalytic performance, in particular in connection with the enantioselective synthesis and hydrolysis. Working on lipase B from Candida antarctica (CALB), we have hypothesized that circular permutation changes the structural integrity of the protein and alters active site accessibility and backbone flexibility, all factors affecting an enzyme’s substrate recognition and turnover. Using a combination of biochemical and biophysical methods, we now have unraveled the consequences of circular permutation on the structure and function of CALB. Going beyond the static image of the crystallized permutant cp283D7, our attention has shifted towards the newly created termini in close proximity to the active site which lack electron density in the x-ray structure. A series of truncation and mutagenesis experiments, as well as time-resolved fluorescence anisotropy measurements have revealed the critical functional role of this region, particularly the C-terminus. The results from these studies not only provide a better understanding of the impact of circular permutation of CALB but, equally important, shed light on the region’s potential role as a lid in the wild type enzyme.
In respect to the practical performance of these engineered enzymes, results from transesterification experiments with a variety of pure triglycerides, as well as vegetable oils and spent cooking oil will presented.
Finally, our findings have been helpful in guiding the design and interpretation of results from circular permutation experiments on other enzymes, namely members of the glycosyl hydrolase family and flavin-dependent oxidoreductases.
See more of Advances in biocatalyst development (emphasis on novel biocatalysts)
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See more of The Annual Meeting and Exhibition 2009 (July 26 - 30, 2009)
See more of Invited Oral Papers
See more of The Annual Meeting and Exhibition 2009 (July 26 - 30, 2009)