Isolation of cyclodextrin glucanotransferase producing alkalophilic bacteria and optimization of culture conditions for its production

Abstract

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) producing alkalophilic bacteria were isolated using starch agar plates (pH >10.0) containing phenolphthalein and methyl orange as an indicator. Nine different soil samples were screened for CGTase producing bacteria and five isolates were obtained. An alkaliphile AND3 showed the highest CGTase production (1.99 U/ml) among the isolates obtained, so it was selected for the further study. Physical culture conditions like inoculum size, 2 % (v/v), temperature, 37 °C and Na₂CO₃ (1 %, w/v) were found optimum for CGTase production. Different carbon sources like soluble starch, refined wheat flour, rice flour and sago flour were tested for CGTase production and rice flour (6.77 U/ml) was proved as the best raw carbon source. Among the organic (peptone, yeast extract) and inorganic nitrogen sources (ammonium sulphate, ammonium nitrate, ammonium chloride, sodium nitrate) tested, ammonium sulphate showed the highest enzyme (7.4 U/ml) production. Optimization of culture conditions resulted to 3.7 fold increase in CGTase production. CGTase was partially purified by ammonium sulphate fractionation and has shown 113 U/mg specific activity, 4.85 fold purification with 60 % yield. Partially purified enzyme showed the best activity at 50 °C temperature and 6.0 pH.