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 V max 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.
|Number of pages||8|
|Journal||Applied and Environmental Microbiology|
|Publication status||Published - 2004 Mar|
All Science Journal Classification (ASJC) codes
- Food Science
- Applied Microbiology and Biotechnology