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Characterization of a Thermostable l-Arabinose (d-Galactose) Isomerase from the Hyperthermophilic Eubacterium Thermotoga maritima

ABSTRACT The araA gene encoding l-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni2+ affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90°C and pH 7.5 under the assay conditions used. Its apparent Km values for l-arabinose and d-galactose were 31 and 60 mM, respectively; the apparent Vmax values (at 90°C) were 41.3 U/mg (l-arabinose) and 8.9 U/mg (d-galactose), and the catalytic efficiencies (kcat/Km) of the enzyme were 74.8 mM−1 · min−1 (l-arabinose) and 8.5 mM−1 · min−1 (d-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn2+ and/or Co2+ than in the absence of these ions. The enzyme carried out the isomerization of d-galactose to d-tagatose with a conversion yield of 56% for 6 h at 80°C.