Purification and kinetics of a raw starch-hydrolyzing, thermostable, and neutral glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae

The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae had a molecular mass of 42 kDa with a pI of 8.2. It is a glycoprotein with 9-10.5% carbohydrate content, which acted optimally at 60 degreesC and pH 7.0, with a t(1/2) of 12 h at 60 degreesC and 7 h at 80 degreesC. Its experimental activation energy was 43 KJ mol(-1) with temperature quotient (Q(10)) of 1.35, while the values predicted by response surface methodology (RSM) were 43 KJ mol(-1) and 1.28, respectively. The enzyme hydrolyzed soluble starch at 50 degreesC (K-m 0.50 mg mL(-1) and V-max 109 mumol mg(-1) protein min(-1)) and at 60 degreesC (K-m 0.40 and V-max 143 mumol mg(-1) protein min(-1)). The experimental K-m and V-max values are in agreement with the predicted values at 50 degreesC (K-m 0.45 mg mL(-1) and V-max 111.11 mumol mg(-1) protein min(-1)) and at 60 degreesC (K-m 0.36 mg mL(-1) and V-max 142.85 mumol mg(-1) protein min(-1)). An Arrhenius plot indicated thermal activation up to 60 degreesC, and thereafter, inactivation. The enzyme was strongly stimulated by Co2+, Fe2+ Ag2+, and Ca2+, slightly stimulated by Cu2+ and Mg2+, and inhibited by Hg2+, Zn2+, Ni2+, and Mn2+. Among additives, dextran and trehalose slightly enhanced the activity. Glucoamylase activity was inhibited by EDTA, beta-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activity and the critical role of disulfide linkages in maintaining the conformation of the enzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch (K-m 2.4 mg mL(-1), V-max 50 mumol mg(-1) protein min(-1)), and it was remarkably insensitive to glucose, suggesting its applicability in starch saccharification.