Zhejiang Dongfang Pharmaceutical Co. Ltd. Taiji Group

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 com. L-AI for D-tagatose production requires two important enzymic 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 chromatog.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 temperatureThe enzyme retained 93% and 63% of initial activity after 4 and 16 h of incubation, resp., at 75 °C in the presence of Co²⁺.The Michaelis-Menten constant (K_m), turnover number (k_cat), and catalytic efficiency (k_cat/K_m) for substrate D-galactose were measured to be 54.7 mM, 68.0 min^-1, and 1.2 mM^-1 min^-1, resp.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, resp.Due to the promising thermostability and high activity at slight acidic pH, the Al. hesperidumL-AI was appropriate for use as a new source of D-tagatose producing enzyme.