Purification and characterization of glucoamylase from endophytic fungus EF6

Abstract

Selective 25 endophytic fungi from stock culture of RCBC found that EF6 has maximum amylase activity was obtained after 8 days of incubation. Amylase from EF6 was extracted with 90% (NH[subscript 4])[subscript 2]SO[subscript 4] and purified by ion exchange chromatography using Q sepharose and gel filtration using Superdex75, respectively. The purity of amylase was 14.93 fold over the original culture filtrate with a 10.93% recovery activity. The estimated molecular mass of purified amylase is about 62 kDa. Optimum conditions for this amylase are 50-60 degree celsius, pH 5.0-6.0. Amylase still functions with the temperature below 60 degree celsius. At this point the activity was decreased to 43%. The activity of this enzyme can be increased with the present of Ba[superscript 2+] , Ca[superscript 2+] and Mg[superscript 2+] whereas AI[superscript 3+] , Ag[superscript 2+] and Cu[superscript 2+] acted as enzyme inhibitors. For various type of starch, tapioca starch is the easiest material for the digestion process. The K[subscript m] and V[subscript max] value were 2.63 mg and 1.25 mM/min, respectively. The purified amylase was indentified by mass spectrometry. The result shown that purified amylase may be glucoamylase (3.2.1.3) because partial amino acid sequences of that enzyme similar to glucoamylase (3.2.1.3) from Amorphotheca resinae.