Purification and characterization of two thermostable glucoamylases produced from Aspergillus niger B-30

Abstract

Two thermostable glucoamylases were produced from Aspergillus niger B-30 by submerged fermentation. The two glucoamylases GAM-1 and GAM-2 were purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose fast flow(DEAE FF) and Superdex G-75 gel filtration columns. The molecular weights of GAM-1 and GAM-2 were determined as 9.72×10⁴ and 7.83×10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the molecular weights of GAM-1 and GAM-2 were determined to be 8.05×10⁴ and 7.04×10⁴ by matrix assisted laser desorption ionizationtime-of-flight(MALDI-TOF) mass spectrometry, respectively. Both the enzymes were glycosylated, with 10.4% and 11.4% carbohydrate content, respectively. The optimal pH and temperature were 4.0–4.6 and 70 °C for both. The two glucoamylases were maintained 100% relative activity after incubation at 60 °C for 120 min. After the hydrolysis of starch for 120 min, glucose was the only product, confirming that the two enzymes were of high efficiency towards starch. The GAM-2 exhibited higher catalytic activity towards oligosaccharides such as maltose than GAM-1, and the kinetic analysis shows that the affinity of GAM-2 to starch was lower than that of GAM-1. The high thermostability and effectiveness make the two glucoamylases potentially attractive for biotechnological application.

Keywords

Glucoamylase / Thermostability / Kinetic analysis / Glycosylation

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