A novel cyclodextrin glucanotransferase from an alkaliphile Microbacterium terrae KNR 9: purification and properties.
Cyclodextrin glucanotransferase (CGTase, EC. 2.1.1.19) produced using new alkaliphile Microbacterium terrae KNR 9 has been purified to homogeneity in a single step by the starch adsorption method. The specific activity of the purified CGTase was 45 U/mg compared to crude 0.9 U/mg. This resulted in a 50-fold purification of the enzyme with 33 % yield. The molecular weight of the purified enzyme was found to be 27.72 kDa as determined by SDS-PAGE. Non-denaturing gel electrophoresis and activity staining confirmed the presence of CGTase in crude and the ammonium sulfate precipitate fraction. The purified CGTase has a pI value of 4.2. The optimum pH of 6.0 and 60 °C temperature were found to be the best for CGTase activity. Purified CGTase showed 5.18 kcal/mol activation energy (Ea). The CGTase activity was increased in the presence of metal ions (5 mM): Ca+2(130 %), Mg+2(123 %), Mn+2(119 %) and Co+2(116 %). The enzyme activity was strongly inhibited in the presence of Hg+2(0.0 %), Cu+2(0.0 %) and Fe+2(3.8 %). Inhibitor N-bromosuccinimide (5 mM) showed the highest 96 % inhibition of CGTase activity. SDS and triton X-100 among different detergents and surfactants (1.0 %, w/v) tested showed 92 % inhibition. Among the organic solvents checked for their effect on enzyme activity, 5 % (v/v) toluene resulted in 48 % increased activity. Polyethylene glycol-6000 showed a 26 % increase in the CGTase activity. The kinetic parameters Kmand Vmaxwere 10 mg/ml and 146 µmol/mg min, respectively, for purified CGTase.
Department of Microbiology and Biotechnology, University School of Sciences, Gujarat University, Navrangpura, Ahmedabad, Gujarat, 380 009, India. rajputkn@yahoo.com.
Kiransinh N
KN
Rajput
Department of Microbiology, BRD School of Biosciences, Sardar Patel Maidan, Sardar Patel University, Satellite Campus, Bakrol, Vallabh Vidyanagar, Gujarat, 388 120, India.
Kamlesh C
KC
Patel
Department of Microbiology, BRD School of Biosciences, Sardar Patel Maidan, Sardar Patel University, Satellite Campus, Bakrol, Vallabh Vidyanagar, Gujarat, 388 120, India.
Ujjval B
UB
Trivedi
Alkaliphile; Cyclodextrin glucanotransferase; Microbacterium terrae; Purification; Starch adsorption