Cloning, expression, and characterization of thermostable α-amylase of a thermophilic bacterium isolated from hot-spring of Sikkim and sustainable immobilization on coconut coir

Cloning and expression of the α-amylase gene (AmyK2) of thermophilic Bacillus subtilis k2cm originated from Yume Samdong hot spring, North Sikkim, India was done in Escherichia coli BL-21(DE3). The 55.0 kDa purified recombinant enzyme exhibited optimum activity at 70 °C and pH 7.0 with significant stability in temperature and pH ranges from 30 to 90 °C and 6.0–8.0, respectively. The α-amylase is Ca^+ 2 independent, and can act with reasonably high efficiency in absence of any metal. Moreover, its activity increased in the presence of Fe^+ 2 ion and was inhibited by Hg^+ 2 ion and EDTA. The recombinant enzyme showed a half-life of 53 min at 70 °C and its V_max and K_m values were 22.22 U/mg and 5.06 mg/ml, respectively. Immobilization of the purified enzyme on low-cost coconut coir with high immobilization yield (98.27% specific activity), increased half-life (71 min), and higher thermostability with successive use up to 8 cycles with high efficacy validates the techno-economic merit of use of the immobilized biocatalyst. As a whole, cloning the α-amylase gene of thermostable bacteria into the mesophilic organism and subsequent immobilization of the enzyme will unravel its secrets within the confines of the laboratory which could expedite its commercial exploitation in future.