Cloning, expression and characterization of ribulokinase from Anoxybacillus kestanbolensis AC26 Sari

Ribulokinases (transferase; EC 2.7.1.16) convert L-ribulose to L-ribulose 5 phosphate and D-ribulose to D-ribulose 5 phosphate. The products as a result of reactions catalyzed by this enzyme are used in pentose phosphate pathway.This study describes the cloning of a ribulokinase gene from Anoxybacillus kestanbolensis AC26 Sari, isolation, and characterization of the gene product. A 1695 nt gene coding for ribulokinase from Anoxybacillus kestanbolensis AC26 Sari sequenced and cloned into pET-28a(+) vector. Vector encoding ribulokinase gene was transformed into E.coli BL21 (DE3), expressed and purified. The purified ribulokinase showed optimal activity at 60°C in pH 9. Km value for D-ribulose was determined to be 0.94 mM. Effects of metal ions (Mg2+, Ca2+, Li, K, Zn2+), ß-mercaptoethanol and EDTA on the enzyme activity were studied. Enzyme activity was increased by Mg2+ and ß-mercaptoethanol, and inhibited by Zn2+. In addition, Ca2+, Li, K, and EDTA had no effect on the enzyme activity. Molecular mass of A. kestanbolensis AC26 Sari ribulokinase was determined to be 61.96 kDa.Key words: Anoxybacillus, Ribulokinase, Expression