Abstract
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 K m and V max were 10 mg/ml and 146 µmol/mg min, respectively, for purified CGTase.
Highlights
Amylases are of great significance for biotechnological and industrial applications and have approximately 25 % of the world enzyme market (Reddy et al 2003)
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
We report the purification and properties of CGTase produced by Mic. terrae KNR 9
Summary
Amylases are of great significance for biotechnological and industrial applications and have approximately 25 % of the world enzyme market (Reddy et al 2003). Cyclodextrin glucanotransferase is one of the important members of the a-amylase family 13 of glycosyl hydrolases which can degrade starch. This family of enzymes exhibits diversity in reaction specificities. CGTases produce cyclodextrins (CDs) as a result of intramolecular transglycosylation (cyclization) reaction during the degradation of starch. Though CGTase production and characterization from a variety of aerobic mesophilic bacteria have been reported, there is a need of robust enzymes from other organisms considering the harsh conditions used in starch industries (Wind et al 1995). The standard protein calibration curve was prepared using pure bovine serum albumin (0–100 lg/ ml)
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