Chengdu Institute of Biology Prof.WU Zhongliu's team report for the first time, the identification of critical amino acid residues responsible for the thermostability of endogluca nase CelA. Due to the well-known complicated nature of proteins, rational prediction of stability-related residues remains difficult, even though quite a few factors, intramolecular interactions in particular, such as hydrogen bondings, ion pairs, hydrophobic interactions have been identified as of general significance in the stability of proteins.
In WU's study on cellulase CelC from C. thermocellum, carefully designed mutations with additional electrostatic interactions for enhanced thermostability returned unsuccessful results, and lost of activity was also observed. Therefore, in this work, researchers applied a random library approach instead, which was constructed using family shuffling method with two parental enzymes, CelA and CelB from Clostridium josui with a temperature optimum at 60°C.
Finally, by analyzing the sequences of randomly picked mutants with varied thermostability, four amino acid substitutions were identified as having significant impact on the thermostability of CelA, while enzymatic activities remained unchanged. Their effects were discussed in the context of the X-ray crystal structure.
The research has been published in BIOTECHNOLOGY LETTERS, 32(12),Dec,2010. and received grants from Chinese Academy of Sciences (KSCX1-YW-11B2) and Province Science Foundation of Sichuan, China (08ZQ026-023 2010SZ0128 ).