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

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

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 (pH 5.0-10.0) and temperature (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.

Anahtar Kelimeler:

Anoxybacillus, ribulokinase, thermophilic, expression

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

Keywords:

Anoxybacillus, ribulokinase, thermophilic, expression,

PDF

___

Agarwal R, Burley SK, Swaminathan S (2012). Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase. Proteins 80: 261–268.
Ay F, Karaoglu H, Inan K, Canakci S, Belduz AO (2011). Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF1. Protein Expres Purif 80: 74–79.
Bachelard HS (1971). Allosteric activation of brain hexokinase by magnesium ions and by magnesium ion-adenosine triphosphate complex. Biochem J 125: 249–254.
Burma DP, Horecker BL (1957). Pentose fermentation by Lactobacillus plantarum. III. Ribulokinase. J Biol Chem 231: 1039–1051.
D’Souza DR, Morgan RD, Parashar V, Capalash N, Sharma P (2004). Characterization of BfιI- a thermostable, Co ++-requiring isoschizomer of BfιI from Anoxybacillus flavithermus. World J Microb Biot 20: 593–598.
Dulger S, Demirbag Z, Belduz AO (2004). Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov. Int J Syst Evol Micr 54: 1499–1503.
Englesberg E, Wilcox G (1974). Regulation: positive control. Annu Rev Genet 8: 219–242.
Goh KM, Kahar UM, Chai YY, Chong CS, Chai KP, Ranjani V, Illias R Md, Chan KG (2013). Recent discoveries and applications of Anoxybacillus. Appl Microbiol Biot 97: 1475–1488.
İnan K, Çanakçı S, Beldüz AO (2011). Isolation and characteriza- tion of xylanolytic new strains of Anoxybacillus from some hot springs in Turkey. Turk J Biol 35: 529–542.
Lee LV, Gerratana B, Cleland WW (2001). Substrate specificity and kinetic mechanism of Escherichia coli ribulokinase. Arch Bio- chem Biophys 396: 219–224.
Lee N, Bendet I (1967). Crystalline L-ribulokinase from Escherichia coli. J Biol Chem 242: 2043–2050.
Lichenstein HS, Hamilton EP, Lee N (1987). Repression and catabo- lite gene activation in the araBAD operon. J Bacteriol 169: 811–822.
Lim R, Cohen SS (1966). D-Phosphoarabinoisomerase and D-ribulokinase in Escherichia coli. J Biol Chem 241: 4304–4315.
Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning: A Laboratory Manual. 2nd ed. New York, NY, USA: Cold Spring Harbor Laboratory Press.
Schleif R (2010). AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev 34: 779–796.
Sedlak M, Ho NWY (2001). Expression of E. coli araBAD operon encoding enzymes for metabolizing L-arabinose in Saccharomyces cerevisiae. Enzyme Microb Tech 28: 16–24.
Viksİ-Nielsen A, Andersen C, Hoff T, Pedersen S (2006). Development of a new α-amylases for raw starch hydrolysis. Biocatal Biotransfor 24: 121–127.
Zhang L, Leyn SA, Gu Y, Jiang W, Rodionov DA, Yang C (2012). Ribulokinase and transcriptional regulation of arabinose metabolism in Clostridium acetobutylicum. J Bacteriol 194: 1055–1064.