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 Ni 2؉ 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 K m 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 (k cat /K m ) of the enzyme were 74.8 mM ؊1⅐ min ؊1 (L-arabinose) and 8.5 mM ؊1 ⅐ 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 Mn 2؉ and/or Co 2؉ 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.Of the eubacteria whose genomes have been sequenced to date, Thermotoga maritima has the highest percentage of genes that are most similar to archaeal genes (20). This bacterium is also of evolutionary importance, because small-subunit rRNA phylogeny has shown that it is one of the deepest and most slowly evolving lineages of the eubacteria. Thus, it could be useful for elucidating the evolutionary relationship between thermophilic eubacteria and archaea. In addition, it metabolizes many simple and complex carbohydrates, including glucose, sucrose, starch, cellulose, and xylan (9). Thermostable enzymes from T. maritima involved in carbohydrate metabolism are very attractive for industrial applications (4,15,24).Almost 7% of the predicted coding sequences in the T. maritima genome are involved in metabolism of simple and complex sugars (20). Several genes encode proteins involved in arabinose metabolism. Among them, the araA gene encoding L-arabinose isomerase (AI) (EC 5.3.1.4), which catalyzes the conversion of L-arabinose to L-ribulose, not only is important for pentose sugar isomerization in vivo but also is very attractive for use in the bioconversion of D-galactose into D-tagatose in vitro (5, 25) (Fig. 1).The ketohexose D-tagatose has a sweetness value (92%) equivalent to that of sucrose but is poorly digested (18,32). This compound has been found to be a safe low-calorie sweetener in food products and is classified as a generally recognized as safe substance in the United States. It has also been classified as generally recognized as safe for u...

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic l‐arabinose isomerases: The effects of divalent metal ions on protein stability at elevated temperatures

“…As previously reported, it was found that the metal dependence of the AIs increased with the temperature at which they were assayed [20,21]. To further characterize this aspect, we analysed the decrease in enzyme activity of the apo and holo proteins at various GdnHCl concentrations (Fig.…”

“…Recombinant AIs were purified as described elsewhere [20,21]. The purified AIs were dialyzed against 10 mM Tris-HCl buffer [pH 7.0 except with B. halodurans (BHAI) at pH 8.0] and stored at 4°C.…”

“…Although, these AIs are highly conserved with respect to primary amino acid sequence (similarity, >70%) and secondary structure [20], in contrast to mesophilic AIs such as E. coli AI, B. halodurans AI, and Lactobacillus gayonii AI, (hyper) thermophilic AIs from T. maritima [20], T. neapolitana [22], and G. stearothermophilus [21] showed an absolute metal dependence for their thermostability as well as active conformation. In addition, they also have metal-mediated isomerization activity not only for arabinose to ribulose in vivo but also for galactose to tagatose in vitro.…”

“…For these and related reasons, we have begun to study the unfolding of bacterial L L-arabinose isomerases (AI), specifically the three homo-tetrameric metalloenzymes (M r = 225 000) from mesophilic B. halodurans, thermophilic G. stearothermophilus, and hyperthermophilic T. maritima that contain 498 residues per monomer [20,21]. Although, these AIs are highly conserved with respect to primary amino acid sequence (similarity, >70%) and secondary structure [20], in contrast to mesophilic AIs such as E. coli AI, B. halodurans AI, and Lactobacillus gayonii AI, (hyper) thermophilic AIs from T. maritima [20], T. neapolitana [22], and G. stearothermophilus [21] showed an absolute metal dependence for their thermostability as well as active conformation.…”

Facile Enzymatic Synthesis of Ketoses

“…Having overcome these problems, other conversion-related enzymes including D-xylose isomerase (XylA), [22] RhaD [23] and AphA [18] from Escherichia coli, L-arabinose and Dgalactose isomerase (AraA) from Thermotoga maritima MSB8, [24] DTE from Pseudomonas Sp, St-24 [11] were prepared as described in supporting information.…”