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

Abstract

IntroductionAnoxybacillus is a relatively new genus compared to the well-studied genera Geobacillus or Bacillus. The genus Anoxybacillus represents aerobic or facultatively anaerobic, neutrophilic, obligately thermophilic, endospore-forming bacteria (İnan et al., 2011). Most of the reported data have revealed that the members of this genus produce interesting enzymes that are thermostable and tolerant to alkaline pH. Some of the well-studied enzymes were discovered through partnerships with industry; for example, the raw starch-degrading amylase was discovered by a Novozyme team (Viksø-Nielsen et al., 2006), and the BfιI RE was discovered by New England Biolabs (D'Souza et al., 2004;Goh et al., 2013).L-ribulokinase (RK; EC 2.7.1.16) is 1 of 3 major enzymes of the arabinose catabolic pathway. L-arabinose is 1 of the major polysaccharide components in plant cell walls and among the most abundant monosaccharides in nature. Furthermore, its utilization pathway in bacteria has been investigated extensively (Zhang et al., 2012). The arabinose regulon is 1 of many gene systems in Escherichia coli and the regulon consists of 4 operons, araBAD, araC, araE, and araFGH, which are responsible for L-arabinose catabolism, gene regulation, low-affinity transport, and high-affinity transport, respectively (Englesberg and Wilcox, 1974;Lichenstein et al., 1987). In the lowaffinity transport system, the transporter, the araE gene product, is bound to the inner membrane and utilizes the electrochemical potential to transport arabinose. The araFGH genes encode arabinose-specific components of a high-affinity transport system, ABC transporters. These are 3 proteins of the ATP-binding cassette transporter family. AraF is the periplasmic arabinose-binding protein, AraG is the ATP-binding component, and AraH is the membrane-bound component (Schleif, 2010). AraC acts directly as an inducer or an activator of gene expression.The araBAD operon encodes 3 different enzymes required for catabolism of L-arabinose, which are responsible for the conversion of L-arabinose into D-xylulose-5-phosphate. AraA, as an isomerase (L-arabinose isomerase), converts arabinose to L-ribulose; AraB, as a kinase (L-ribulokinase), phosphorylates L-ribulose; and AraD, as an epimerase (L-ribulose-5phosphate 4-epimerase), converts L-ribulose-phosphate