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

Dong Woo Lee, Hyeung Jin Jang, Eun Ah Choe, Byoung Chan Kim, Sang Jae Lee, Seong Bo Kim, Young Ho Hong, Yu Ryang Pyun

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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 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.

Original languageEnglish
Pages (from-to)1397-1404
Number of pages8
JournalApplied and Environmental Microbiology
Volume70
Issue number3
DOIs
Publication statusPublished - 2004 Mar 1

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All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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