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Journal of Biomedicine and Biotechnology
Volume 2009, Article ID 201075, 7 pages
http://dx.doi.org/10.1155/2009/201075
Research Article

Engineering and Directed Evolution of a Ca2+ Binding Site A-Deficient AprE Mutant Reveal an Essential Contribution of the Loop Leu75–Leu82 to Enzyme Activity

1Departamento de Biología, Universidad de Guanajuato, Colonia Noria Alta S/N, Guanajuato, 36050 Guanajuato, Mexico
2Centro de Investigación en Alimentos y Nutrición, Facultad de Medicina, Universidad Juárez del Estado de Durango, Avenida Universidad y Anitúa S/N, 34000 Durango, Mexico
3Departamento de Ciencias Naturales, Universidad Autónoma Metropolitana Unidad Cuajimalpa, Pedro Antonio de los Santos 84, San Miguel Chapultepec 11850, Mexico

Received 12 February 2009; Revised 17 May 2009; Accepted 15 June 2009

Academic Editor: George Makhatadze

Copyright © 2009 Eliel R. Romero-García 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.

Abstract

An aprE mutant from B. subtilis 168 lacking the connecting loop which is predicted to encode a binding site was constructed. Expression of the mutant gene (aprE) produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprE. An AprE variant with reestablished enzyme activity was selected by directed evolution. The novel mutations Met/Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprE. These results support the proposal that in addition to function as a calcium binding site, the loop that connects -sheet e3 with -helix c plays a structural role on enzyme activity of AprE from B. subtilis 168.