Thermostable Bacillus subtilis Lipases: In Vitro Evolution and Structural Insight Shoeb Ahmad, Md. Zahid Kamal†, Rajan Sankaranarayanan⁎ and N. Madhusudhana Rao⁎ Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad-500007, India Received 28 February 2008; received in revised form 9 May 2008; accepted 16 May 2008 Available online 2 July 2008 In vitro evolution methods are now being routinely used to identify protein variants with novel and enhanced properties that are difficult to achieve using rational design. However, one of the limitations is in screening for beneficial mutants through several generations due to the occurrence of neutral/negative mutations occurring in the background of positive ones. While evolving a lipase in vitro from mesophilic Bacillus subtilis to generate thermostable variants, we have designed protocols that combine stringent three-tier testing, sequencing and stability assessments on the protein at the end of each generation. This strategy resulted in a total of six stabilizing mutations in just two generations with three mutations per generation. Each of the six mutants when evaluated individually contributed additively to thermostability. A combination of all of them resulted in the best variant that shows a remarkable 15 °C shift in melting temperature and a millionfold decrease in the thermal inactivation rate with only a marginal increase of 3 kcal mol −1 in free energy of stabilization. Notably, in addition to the dramatic shift in optimum temperature by 20 °C, the activity has increased two- to fivefold in the temperature range 25–65 °C. High-resolution crystal structures of three of the mutants, each with 5° increments in melting temperature, reveal the structural basis of these mutations in attaining higher thermostability. The structures highlight the importance of water-mediated ionic networks on the protein surface in imparting thermostability. Saturation mutagenesis at each of the six positions did not result in enhanced thermostability in almost all the cases, confirming the crucial role played by each mutation as revealed through the structural study. Overall, our study presents an efficient strategy that can be employed in directed evolution approaches employed for obtaining improved properties of proteins. © 2008 Elsevier Ltd. All rights reserved. Edited by F. Schmid Keywords: lipase; thermostability; directed evolution; X-ray structure; water bridges Introduction The stability of a protein is measured by the energy required for its disruption. The energies associated with stability are known to be small in proteins, since the difference between native and denatured state is a sum of only a few weak interactions. 1–3 The small difference between native and denatured states of proteins has a critical bearing on protein turnover within a cell. Several lines of investigation including structural properties of homologous proteins from extremophiles and contributions of mutations on stability of proteins point to the fact that knowledge-based design for stable proteins is still a challenge. 3,4 Site-directed approaches to improve protein stability have limited success, since the prediction of weak but profound interactions in a protein are not trivial. We need to improve our understanding of the stability of proteins also because biotechnological applications demand stabilized proteins. 5 *Corresponding authors. E-mail addresses: sankar@ccmb.res.in; madhu@ccmb.res.in. † M.Z. Kamal has done crystallization and solved the structure of lipase mutants. Abbreviations used: EP-PCR, error-prone PCR; Lip A, Bacillus subtilis lipase A; PNPA, p-nitrophenyl acetate; PNPB, p-nitrophenyl butyrate; PNPO, p-nitrophenyl oleate; TM, triple mutant. doi:10.1016/j.jmb.2008.05.063 J. Mol. Biol. (2008) 381, 324–340 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.