AN EXTRACELLULAR GLUCOAMYLASE PRODUCED BY ENDOPHYTIC FUNGUS EF6

摘要

A strain of endophytic fungus EF6 isolated from Thai medicinal plants was found to produce higher levels of extracellular glucoamylase. This strain produced glucoamylase of culture filtrate when grown on 1 % soluble starch. The enzyme was purified andcharacterized. Purification steps involved (NH_4)_2SO_4 precipitation, anion exchange, and gel filtration chromatography. Final purification fold was 14.49 and the yield obtained was 9.15%. The enzyme is monomelic with a molecular mass of 62.2 kDa as estimated by SDS-PAGE, and with a molecular mass of 62.031 kDa estimated by MALDI-TOF spectrometry. The temperature for maximum activity was 60°C. After 30 min for incubation, glucoamylase was found to be stable lower than 50°C. The activity decrease rapidly when residual activity was retained about 45% at 55°C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0—7.0 at 50°C. The activity of glucoamylase was stimulated by Ca~(2+), Co~(2+), Mg~(2+), Mn~(2+), glycerol, DMSO, DTT and EDTA, and strongly inhibited by Hg~(2+). Various types of starch were test, soluble starch proved to be the best substrate for digestion process. The enzyme catalyzes the hydrolysis of soluble starch and maltose as the substrate, the enzyme had K_m values of 2.63, and 1.88 mg/ml and V_(max), values of 1.25, and 2.54 U/min/mg protein, and V_(max)/K_m values of 0.48 and 1.35, respectively. The internal amino acid sequences of endophytic fungus EF6 glucoamylase; RALAN HKQW DSFRS have similarity to the sequence of the glucoamylase purified form Thermomyces lanuginosus. From all results indicated that this enzyme is a glucoamylase (1,4-a-D-glucan glucanohydrolase).