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Applied and Environmental Microbiology, March 2004, p. 1397-1404, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1397-1404.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Dong-Woo Lee,1 Hyeung-Jin Jang,2 Eun-Ah Choe,1 Byoung-Chan Kim,3 Sang-Jae Lee,1 Seong-Bo Kim,1 Young-Ho Hong,1 and Yu-Ryang Pyun1*

Department of Biotechnology and Bioproducts Research Center, College of Engineering, Yonsei University, Seoul 120-749, Korea,1 Diabetes Section, Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224,2 Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 010033

Received 22 August 2003/ Accepted 10 December 2003

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.

* Corresponding author. Mailing address: Department of Biotechnology and Bioproducts Research Center, College of Engineering, Yonsei University, Seodaemun-Gu, Shinchon-Dong 134, Seoul 120-749, Korea. Phone: 82-2-2123-2883. Fax: 82-2-312-6821. E-mail: yrpyun{at}yonsei.ac.kr.

Applied and Environmental Microbiology, March 2004, p. 1397-1404, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1397-1404.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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