Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1177250 | Analytical Biochemistry | 2008 | 7 Pages |
Abstract
For the rapid identification of functional activity of unknown genes from a sequence database, a new method based on in vitro protein synthesis combined with mass spectrometry was developed. To discriminate their subtle enzymatic activity, in vitro synthesized and one-step purified lipolytic enzymes, such as lipA and lipB from Bacillus subtilis and an unknown protein ybfF from Escherichia coli, were reacted with a mixture of triglycerides with different carbon chain lengths. Using direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of reaction product, all three enzymes were revealed to have strong esterase activity rather than true lipase activity, which has no reactivity on long-chain fatty acids such as triolein. These results were also confirmed by classical color assay using p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as representative lipolytic substrates.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
June-Hyung Kim, Kyoung-Soon Jang, Yung-Hun Yang, Yun-Gon Kim, Ji-Hye Lee, Min-Kyu Oh, Byung-Gee Kim, Chang-Soo Lee,