Purification and functional properties of a novel glucoamylase activated by manganese and lead produced by Aspergillus japonicus

• Published in: International journal of biological macromolecules 2017-09, Vol.102, p.779-788
• Main Authors: Pasin, Thiago Machado, Benassi, Vivian Machado, Heinen, Paulo Ricardo, Damasio, André Ricardo de Lima, Cereia, Mariana, Jorge, João Atílio, Polizeli, Maria de Lourdes Teixeira de Moraes
• Format: Article
• Language: English
• Subjects: Amylose - metabolism; Aspergillus - enzymology; Aspergillus japonicus; Dose-Response Relationship, Drug; Edetic Acid - pharmacology; Enzyme Activation - drug effects; Glucan 1,4-alpha-Glucosidase - chemistry; Glucan 1,4-alpha-Glucosidase - isolation & purification; Glucan 1,4-alpha-Glucosidase - metabolism; Glucoamylase; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lead - pharmacology; Maltose - pharmacology; Manganese - pharmacology; Mercaptoethanol - pharmacology; Molecular Weight; Phylogeny; Purification; Temperature
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2017.04.016

•Amylase produced by A. japonicus was purified in a unique step using a DEAE-cellulose ion exchange chromatography column and it showed 72kDa of molecular mass.•Amylase was identified as glucoamylase by thin layer chromatography on silica and it was biochemically characterized.•MnCl2 and Pb(C2H3O2)2.3H2O showed to be a good glucoamylase activators.•Glucoamylase carbohydrate content was 5.5%, Km and Vmax values were estimated as 0.59mg/mL and 308.01U/mg.•Glucoamylase showed to be phylogenetically located closer to A. ficuum and A. niger glucoamylases. Microbial amylases are used to produce ethanol, glucose and can be applied in textiles products, detergents and other industries. This study aimed to determine the best carbon source concentration to induce the amylase production by A. japonicus, and its purification and biochemical characterization. For that, this fungus was cultivated in Khanna medium, pH 5.5, for 4 days, at 25°C, in static condition, supplemented with potato starch and maltose in different concentrations. The fungal crude enzymatic extract was purified in a unique elution in DEAE-cellulose column and the molecular mass was determined as 72kDa. The optimum temperature and pH was 65°C and 5.0, respectively. Amylase remained 75% of its activity after one hour at 50°C and was stable in the pH range 3.0–7.0. The analysis of the end-products by thin layer chromatography showed only glucose formation, which characterizes the purified enzyme as a glucoamylase. Amylopectin was the best substrate for the enzyme assay and Mn+2 and Pb+2 were good glucoamylase activators. This activation, in addition to the biochemical characteristics are important results for future biotechnological applications of this glucoamylase in the recycling and deinking process by the paper industries.