Properties of a purified thermostable glucoamylase from Aspergillus niveus

Abstract

A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40 degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65 degrees C and retained 100% activity after 240 min at 60 degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The K(m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.

Highly Potent Saccharification of Arthrospira maxima Glycogen by Fungal Amylolytic Enzyme from Trichoderma Species J113

“…Earlier studies have shown that the addition of various metal ions and chemicals can have a positive effect on hydrolytic [34][35][36]. Here, we questioned whether the application of select ions or chelators at two different concentrations (2.5 and 5 mM) could enhance the activity of Ayt40 and Ayt70 (Fig.…”

Gel Electrophoresis for Investigating Enzymes with Biotechnological Application

“…One sector that has been widely economically explored is the application of amylases in processes of starch saccharification (Silva et al, 2009a(Silva et al, , 2009b. For so, there is the need of several enzymes such as -amylase, which forms maltooligosaccharides; glucoamylases and -amylases, which hydrolyze starch to glucose and maltose and the glucosyltransferases with the production of cyclodextrins.…”

“…Fig. 2A illustrates the activity of -glucosidase, one of the enzymes of the amylolytic system, which leads to the formation of glucose as end product (Aquino et al, 2001;Silva et al, 2009aSilva et al, , 2009b.…”

Characterization of Sporulation-Specific Glucoamylase of Saccharomyces diastaticus

“…The reaction mixture was dissolved in 50 μl sodium phosphate buffer (pH 5.5). Fifty mUnit of Endoglycosidase H (Genzyme, Cambridge, MA) was added to the reaction mixture and incubated at 37 o C for 24 hr followed by 9% SDS-PAGE analysis [2]. Effect of temperature on enzyme activity…”