Research Article: A novel cyclodextrin glucanotransferase from an alkaliphile Microbacterium terrae KNR 9: purification and properties

Date Published: August 13, 2016

Publisher: Springer Berlin Heidelberg

Author(s): Kiransinh N. Rajput, Kamlesh C. Patel, Ujjval B. Trivedi.

http://doi.org/10.1007/s13205-016-0495-6

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 Km and Vmax were 10 mg/ml and 146 µmol/mg min, respectively, for purified CGTase.

Partial Text

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 α-amylase family 13 of glycosyl hydrolases which can degrade starch. This family of enzymes exhibits diversity in reaction specificities. Amylases generally hydrolyze glycosidic bonds in the starch molecules, but CGTases catalyze transglycosylation as a major reaction with hydrolysis being a minor activity (Van der Veen et al. 2000).

The present study describes the single step purification of cyclodextrin glucanotransferase using the starch adsorption method. The purified CGTase with 45.22 U/mg specific activity gave a single band in SDS-PAGE. To the best of our knowledge, this is the smallest molecular weight CGTase with 27.72 kDa reported so far. The purified enzyme works efficiently near neutral pH and at 60 °C temperature. The CGTase activity can be enhanced in the presence of Ca+2 and other metal ions. It remains active in several organic solvents with toluene as the best. The addition of PEG-6000 can increase the enzyme activity. These diverse properties of novel CGTase can be potentially exploited for industrial cyclodextrin production.

 

Source:

http://doi.org/10.1007/s13205-016-0495-6

 

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