Cloning, purification, and characterization of a thermophilic ribulokinase from Anoxybacillus kestanbolensis AC26Sari

Authors : Müslüm TOKGÖZ, Kadriye İNAN, Ali Osman BELDÜZ, Öznur GEDİKLİ, Sabriye ÇANAKÇI

Pages : 633-639

Doi:10.3906/biy-1401-92

View : 18 | Download : 8

Publication Date : 2014-12-01

Article Type : Research Paper

Abstract :The gene encoding ribulokinase araB from Anoxybacillus kestanbolensis AC26Sari was cloned and sequenced. The recombinant protein was expressed in Escherichia coli BL21 under the control of isopropyl-b-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as AC26RK, was purified with the MagneHis Protein Purification System. The molecular mass of the native protein, as determined by SDS-PAGE, was about 61 kDa. AC26RK was active throughout a broad pH insert ignore into journalissuearticles values(pH 5.0-10.0); and temperature insert ignore into journalissuearticles values(50-75 °C); range, and it had an optimum pH of 9.0 and optimum temperature of 60 °C. The enzyme displayed about 90%-100% of its original activities after a 30-min incubation at a pH interval of 5.0-10.0. The enzyme exhibited a high level of D-ribulose activity with apparent Km, Vmax, and Kcat values of 0.94 mM, 3.197 U/mg, and 3.31 s-1, respectively. AC26RK activity was strongly inhibited by Zn2+ but increased by Mg2+. The effects of some chemicals on the ribulokinase activity revealed that Anoxybacillus kestanbolensis AC26Sari does not need metallic cations for its activity. In this paper, we describe for the first time the cloning and characterization of a thermophilic ribulokinase from thermophilic bacteria.
Keywords : Anoxybacillus, ribulokinase, thermophilic, expression