Purification and Kinetics of a Raw Starch-Hydrolyzing, Thermostable, and Neutral Glucoamylase of the Thermophilic Mold Thermomucor indicae-seudaticae
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- 作者：Sanjeev Kumar and T. Satyanarayana
- 刊名：Biotechnology Progress
- 出版年：2003
- 出版时间：June 2003
- 年：2003
- 卷：19
- 期：3
- 页码：936 - 944
- 全文大小：427K
- 年卷期：v.19,no.3(June 2003)
- ISSN：1520-6033
文摘
The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticaehad 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 
C and pH 7.0, with a t1/2 of 12 hat 60 C and 7 h at 80 C. Its experimental activation energy was 43 KJ mol-1 withtemperature quotient (Q10) of 1.35, while the values predicted by response surfacemethodology (RSM) were 43 KJ mol-1 and 1.28, respectively. The enzyme hydrolyzedsoluble starch at 50 C (Km 0.50 mg mL-1 and Vmax 109 
mol mg-1 protein min-1) andat 60 C (Km 0.40 and Vmax 143 mol mg-1 protein min-1). The experimental Km andVmax values are in agreement with the predicted values at 50 C (Km 0.45 mg mL-1and Vmax 111.11 mol mg-1 protein min-1) and at 60 C (Km 0.36 mg mL-1and Vmax142.85 mol mg-1 protein min-1). An Arrhenius plot indicated thermal activation upto 60 C, 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 theactivity. Glucoamylase activity was inhibited by EDTA, 
hars/beta2.gif" BORDER=0 ALIGN="middle">-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activityand the critical role of disulfide linkages in maintaining the conformation of theenzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch(Km 2.4 mg mL-1, Vmax 50 mol mg-1 protein min-1), and it was remarkably insensitiveto glucose, suggesting its applicability in starch saccharification.
C and pH 7.0, with a t1/2 of 12 hat 60 C and 7 h at 80 C. Its experimental activation energy was 43 KJ mol-1 withtemperature quotient (Q10) of 1.35, while the values predicted by response surfacemethodology (RSM) were 43 KJ mol-1 and 1.28, respectively. The enzyme hydrolyzedsoluble starch at 50 C (Km 0.50 mg mL-1 and Vmax 109 mol mg-1 protein min-1) andat 60 C (Km 0.40 and Vmax 143 mol mg-1 protein min-1). The experimental Km andVmax values are in agreement with the predicted values at 50 C (Km 0.45 mg mL-1and Vmax 111.11 mol mg-1 protein min-1) and at 60 C (Km 0.36 mg mL-1and Vmax142.85 mol mg-1 protein min-1). An Arrhenius plot indicated thermal activation upto 60 C, 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 theactivity. Glucoamylase activity was inhibited by EDTA, hars/beta2.gif" BORDER=0 ALIGN="middle">-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activityand the critical role of disulfide linkages in maintaining the conformation of theenzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch(Km 2.4 mg mL-1, Vmax 50 mol mg-1 protein min-1), and it was remarkably insensitiveto glucose, suggesting its applicability in starch saccharification.