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Biotechnol Lett (2010) 32:14491455 DOI 10.1007/s10529-010-0304-y
ORIGINAL RESEARCH PAPER
Tunicamycin inhibition of N-glycosylation of a-glucosidase from Aspergillus niveus: partial inuence on biochemical properties
Tony Mrcio Silva Fausto Bruno Dos Reis Almeida
Andr Ricardo de Lima Damsio Alexandre Maller Michele Michelin
Joo Atlio Jorge Ebert Seixas Hanna Maria Cristina Roque-Barreira
Hctor F. Terenzi Maria de Lourdes Teixeira de Moraes Polizeli
Received: 2 March 2010 / Accepted: 10 May 2010 / Published online: 21 May 2010 Springer Science+Business Media B.V. 2010
Abstract Treatment of Aspergillus niveus with 30 lg tunicamycin/ml did not interfere with a-glucosidase production, secretion, or its catalytic properties. Fully- and under-glycosylated forms of the enzyme
had similar molecular masses, *56 kDa. Moreover, the absence of N-glycans did not affect either pH optimum (6.0) or temperature optimum (65C). The
Km and Vmax values of under- and fully-glycosylated forms of a-glucosidase were similar when assessed for hydrolysis of starch (*0.6 mg/ml, *350 lmol glucose per min per ml), maltose (*0.54 lmol, *330 lmol glucose per min per ml) and p-nitrophenyl-a-D-glucopyranoside (*0.54 lmol, *8.28 lmol p-nitrophenol per min per ml). However, the under-glycosylated form was sensitive to high temperatures probably because, in addition to stabilizing the protein conformation, glycosylation may also prevent unfolded or partially folded proteins from aggregating. Binding assays clearly showed that the under-glycosylated protein did not bind to concanavalin A but has conserve its jacalin-binding property, suggesting that only O-glycans might be intact on the tunicamycin treated form of the enzyme.
Keywords Aspergillus niveus a-glucosidase
N-glycans O-glycans Tunicamycin
Introduction
a-Glucosidase (a-D-glucoside glucohydrolase; EC3.2.1.20) hydrolyses the a-glycosidic bounds from the non-reducing end of oligosaccharides and polysaccharides with the release of glucose (Chiba et al. 1979; Silva et al. 2009). The hydrolysis of starch to
Purpose of work We have shown how the inhibition of N-glycosylation by tunicamycin reects on the secretion, activity, binding afnity, substrate specicity and stability of an a-glucosidase produced by Aspergillus niveus.
T. M. Silva and F. B. Dos Reis Almeida both contributed equally to this work.
T. M. Silva M. Michelin J. A. Jorge
H. F. Terenzi M. de Lourdes Teixeira de Moraes
Polizeli (&)
Departamento de Biologia, Faculdade de Filosoa, Cincias e Letras de Ribeiro Preto, Universidade de So Paulo, Avenida Bandeirantes 3900, Monte Alegre, 14040-901 Ribeiro Preto, SP, Brazile-mail:...