The glucoamylase from <i>Aspergillus wentii</i>: Purification and characterization
Munira C. Lago, Fabiane Cristina dos Santos, Paulo Sérgio Alves Bueno, Marco Aurélio Schüler de Oliveira, Ione Parra Barbosa‐Tessmann
Abstract
Abstract This study describes for the first time the purification and characterization of a glucoamylase from Aspergillus wentii (strain PG18), a species of the Aspergillus genus Cremei section. Maximum enzyme production (∼3.5 U/ml) was obtained in submerged culture (72 h) with starch as the carbon source, at 25°C, and with orbital agitation (100 rpm). The enzyme was purified with one‐step molecular exclusion chromatography. The 86 kDa purified enzyme hydrolyzed starch in a zymogram and had activity against p ‐nitrophenyl α‐ d ‐glucopyranoside. The optimal enzyme pH and temperature were 5.0 and 60°C (at pH 5.0), respectively. The T m of the purified enzyme was 60°C, at pH 7.0. The purified glucoamylase had a K M for starch of 1.4 mg/ml and a V max of 0.057 mg/min of hydrolyzed starch. Molybdenum activated the purified enzyme, and sodium dodecyl sulfate inhibited it. A thin layer chromatography analysis revealed glucose as the enzyme's main starch hydrolysis product. An enzyme's peptide sequence was obtained by mass spectrometry and used to retrieve a glucoamylase within the annotated genome of A. wentii v1.0. An in silico structural model revealed a N‐terminal glycosyl hydrolases family 15 (GH15) domain, which is ligated by a linker to a C‐terminal carbohydrate‐binding module (CBM) from the CBM20 family.