Optimization of medium components using response surface methodology for production of thermostable amylopullulanase in submerged fermentation by Clostridium thermosulfurogenes SVM17

Response surface methodology (RSM) based on central composite rotatable design (CCRD) was used to determine the optimal levels of medium components, viz., soluble starch, tapioca flour, peptone, magnesium chloride and ferrous sulphate for enhanced thermostable amylopullulanase production by Clostridium thermosulfurogenes SVM17 in submerged fermentation. The design contains a total of 54 experimental trials with first 32 organized in a fractional factorial design and experimental trials from 33-40 and 51-54 involving the replication of the central points. Within the tested range of concentrations, all medium components were found significant. The optimum levels of nutrients for maximum production of enzyme were (% w/v): potato starch, 5.2; tapioca flour, 6.3; peptone, 2.5; MgCl2·6H2O, 0.015 and FeSO4·7H2O, 6.0 ppm. After optimization of medium components, the strain SVM17 showed 96 and 409 % increased amylase and pullulanase activities, respectively when compared with the non-optimized conditions.

Purification and characterization of highly thermostable amylopullulanase from a thermophilic, anaerobic bacterium Clostridium thermosulfurogenes SVM17

A highly thermostable amylopullulanase was purified to homogeneity from the culture filtrate of the Clostridium thermosulfurogenes SVM17. On SDS-PAGE, the purified fraction having both amylase and pullulanase activities were observed as a single band. The molecular weight of the purified amylopullulanase on SDS-PAGE was 97 kDa. The optimum temperature for both amylase and pullulanase was 70 °C. The enzyme was completely stable at 70 °C for 2 h. The presence of 5% starch increased the thermal stability of the enzyme at 100 °C up to 2 h. Both amylase and pullulanase activities were optimum at pH 5.5 to 6.0 and were stable over a pH range of 4.0 to 6.5. The TLC analysis of the reaction products on starch showed that maltose was the main product along with trace amounts of glucose. The analysis of hydrolysis product of pullulan showed that maltotriose was the main product. At 5 mM concentration, Mn2+ and Ag+ strongly stimulated both amylase and pullulanase activities, where as Mg2+, Ca2+, Cu2+, Fe3+, Zn2+, Hg2+, EDTA, Cd2+ and Li2+ inhibited both amylase and pullulanase activities. When the concentration of metal ions was increased from 5 to10 mM, a further increase in amylase activity was observed in the presence of Ni2+, Mn2+ and Co2+. Where as substantial decrease was observed at 10 mM concentration of Ag+, Pb2+ and Ca2+.