Biochemical characterization of a thermostable l-arabinose isomerase from a thermoacidophilic bacterium, Alicyclobacillus hesperidum URH17-3-68

The rare monosaccharide d-tagatose is a low-calorie sugar-substituting sweetener, having 92% of relative sweetness but only 1/3 of energy content of sucrose. Industrial production of d-tagatose is carried out from d-galactose by l-arabinose isomerase (l-AI). It is generally recognized that commercial l-AI for d-tagatose production requires two important enzymatic properties, thermostability and slightly acidic pH optimum. In this article, a thermostable l-AI was characterized from a novel thermoacidophilic bacterium, Alicyclobacillus hesperidum URH17-3-68, which showed promising thermostability and displayed a relatively wide pH spectrum. The araA gene encoding the Al. hesperidum l-AI was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and ion-exchange chromatography. The enzyme displayed maximal activity at 70 °C and pH 7.0, and showed more than 75% of maximal activity from pH 5.5 to 7.0. Cobalt ion was required as optimum metal cofactor for both activity simulation and thermostability improvement at high temperature. The enzyme retained 93% and 63% of initial activity after 4 and 16 h of incubation, respectively, at 75 °C in the presence of Co2+. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for substrate d-galactose were measured to be 54.7 mM, 68.0 min−1, and 1.2 mM−1 min−1, respectively. The bioconversion yield of d-tagatose by the purified enzyme after 27 h at 70 °C reached 43% and 22%, from 50 and 200 mM of d-galactose, respectively. Due to the promising thermostability and high activity at slight acidic pH, the Al. hesperidum l-AI was appropriate for use as a new source of d-tagatose producing enzyme.