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Volume 2016, Article ID 4902450, 7 pages
Research Article

Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase

1Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey
2Istanbul Technical University Molecular Biology-Biotechnology & Genetics Research Center, 34469 Istanbul, Turkey
3Department of Molecular Biology and Genetics, Faculty of Science and Letters, Yıldız Technical University, Istanbul, Turkey
4Department of Biochemistry, University of Bristol, Bristol, Avon BS8 1TD, UK

Received 16 December 2015; Revised 25 June 2016; Accepted 4 August 2016

Academic Editor: Carsten Wrenger

Copyright © 2016 Gülşah P. Özgün et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to -fold and -fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH.