title: Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae.
abstract: An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) 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.
authors.affiliation: Laboratório de Biotecnologia, Universidade Federal de São João del-Rei, 35501-296 Divinópolis, MG, Brazil.
authors.forename: Alexsandro Sobreira
authors.initials: AS
authors.lastname: Galdino
authors.forename: Roberto Nascimento
authors.initials: RN
authors.lastname: Silva
authors.forename: Muriele Taborda
authors.initials: MT
authors.lastname: Lottermann
authors.forename: Alice Cunha Morales
authors.initials: AC
authors.lastname: Alvares
authors.forename: Lídia Maria Pepe
authors.initials: LM
authors.lastname: de Moraes
authors.forename: Fernando Araripe Gonçalves
authors.initials: FA
authors.lastname: Torres
authors.forename: Sonia Maria
authors.initials: SM
authors.lastname: de Freitas
authors.forename: Cirano José
authors.initials: CJ
authors.lastname: Ulhoa