SAGE-Hindawi Access to Research Enzyme Research Volume 2011, Article ID 157294, 7 pages doi:10.4061/2011/157294 Research Article Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae Alexsandro Sobreira Galdino, 1 Roberto Nascimento Silva, 2 Muriele Taborda Lottermann, 3 Alice Cunha Morales ´ Alvares, 3 L´ ıdia Maria Pepe de Moraes, 4 Fernando Araripe Gonc ¸alves Torres, 4 Sonia Maria de Freitas, 3 and Cirano Jos´ e Ulhoa 5 1 Laborat´ orio de Biotecnologia, Universidade Federal de S˜ ao Jo˜ ao del-Rei, 35501-296 Divin´ opolis, MG, Brazil 2 Departamento de Bioqu´ ımica e Imunologia, Escola de Medicina, Universidade de S˜ ao Paulo, 14049-900 Ribeir˜ ao Preto, SP, Brazil 3 Laborat´ orio de Biof´ ısica, Instituto de Biologia, Universidade de Bras´ ılia, 70910-900 Bras´ ılia, DF, Brazil 4 Laborat´ orio de Biologia Molecular, Instituto de Biologia, Universidade de Bras´ ılia, 70910-900 Bras´ ılia, DF, Brazil 5 Laborat´ orio de Enzimologia, Departamento de Ciˆ encias Fisiol´ ogicas (ICB), Universidade Federal de Goi´ as, 74001-970 Goiˆ ania, GO, Brazil Correspondence should be addressed to Cirano Jos´ e Ulhoa, ulhoa@icb.ufg.br Received 31 October 2010; Accepted 18 December 2010 Academic Editor: Alane Beatriz Vermelho Copyright © 2011 Alexsandro Sobreira Galdino 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. An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH 4 + and inhibited by Cu +2 and Hg +2 . Significant biochemical and structural discrepancies between wild-type and recombinant α- amylase with respect to K m values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1 w , points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels. 1. Introduction Starch is a major storage product of many economi- cally important crops such as wheat, rice, cassava, and potato [1]. A large variety of microorganisms employ extracellular or intracellular enzymes to hydrolyze starch thus enabling its utilization as a source of energy. One of the most important groups of enzymes that process starch is represented by the α-amylase family or family 13 glycosyl hydrolases [2, 3]. Amylases (EC 3.2.1.1, α-1,4- glucan-glucanohydrolase) are enzymes that hydrolyze starch polymers yielding diverse products including dextrins and smaller polymers of glucose. These enzymes are of great biotechnological interest with many applications ranging from food industry to the production of biofuels. Since each different application requires amylases with unique properties it is often necessary to search the biodiversity for new sources of these enzymes [4]. Several amylases isolated from yeasts such as Candida antarctica, Candida japonica [4], Lipomyces kononenkoae, Saccharomycopsis fibuligera, Schwanniomyces alluvius [5], Trichosporon pullulans, and Filobasidium capsuligenum [6] have been described. We have previously reported the characterization of an α-amylase (Amy1) from the basidiomycetous yeast Cryptococcus flavus