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

Materials.The reagents used in this study were obtained as follows: restriction enzymes, Ex-tag DNA polymerase, deoxynucleoside triphosphates, and chemicals for PCR were obtained from Takara Biomedicals; the pGEM-T Easy vector and T4 DNA ligase were obtained from Promega; the pET-22b(+) expression vector and a His-bind resin kit were obtained from Novagen; genomic-tip and a plasmid miniprep kit were obtained from Qiagen; electrophoresis reagents were obtained from Bio-Rad; Sephacryl S300 resin was obtained from Pharmacia; and all chemicals used for enzyme assays and characterization were obtained from Sigma. Oligonucleotides were synthesized by Cosmo.

Bacterial strains and culture conditions. T. maritima DSM 3109 (9) was obtained from Deutsche Sammlung von Mikroorganismen (DSM), Braunschweig, Germany. T. maritima was grown in an artificial seawater-based medium (DSM medium 343) supplemented with (per liter) 0.5 g of yeast extract, 5 g of l-arabinose, 20 g of NaCl, 0.5 g of KH₂PO₄, 0.5 g of Na₂S · 9H₂O, 2 mg of NiCl₂ · 6H₂O, 15 ml of a trace element solution (DSM medium 141), and 1.0 mg of resazurin. Cultures were grown at 80°C for 48 h in sealed serum bottles under N₂ gas. E. coli strains DH5α and BL21(DE3) were used as bacterial hosts for recombinant plasmids. The plasmid pGEM-T Easy vector was used as a cloning and sequencing vector, and pET-22b(+) was used for expression.

Cloning, expression, and purification of T. maritima AI.In a search of the microbial genome sequences in GenBank, we found a putative araA gene in T. maritima encoding AI. Genomic DNA was isolated from T. maritima and purified by using a genomic DNA extraction kit according to the manufacturer's instructions. The gene encoding AI (araA) was amplified by PCR by using genomic DNA as the template. The PCR mixture (total volume, 50 μl) contained 20 ng of genomic DNA, 10 pmol of primer F-araA-1 (5′-CATATGATAGATCTCAAGCAGTACGAG-3′ [an NdeI site is underlined]), 10 pmol of primer R-araA-2 (5′-AAGCTTTCTTTTCAAAAGCCCCCAGTA-3′ [a HindIII site is underlined]), 1× PCR buffer, each deoxynucleoside triphosphate at a concentration of 200 μM, and 2.5 U of Ex-tag DNA polymerase. After an initial denaturation for 4 min at 94°C, the DNA was amplified by 30 cycles consisting of 30 s of denaturation at 94°C, 30 s of annealing at 60°C, and 1 min of extension at 72°C, followed by a final extension step consisting of 5 min at 72°C. The PCR product was cloned into the pGEM-T Easy vector and transformed into E. coli DH5α competent cells. Transformants containing the pGEM-T Easy vector harboring the gene encoding T. maritima AI (araA) were selected on Luria-Bertani medium-ampicillin plates containing 0.01% 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). Plasmid DNA was isolated from the transformants with inserts and digested with NdeI and HindIII. The digested DNA was purified and ligated into the NdeI and HindIII sites of pET-22b, yielding pET-TMAI. The expression vector also encoded a C-terminal polyhistidine (six-His) sequence. For expression of the recombinant enzyme, E. coli BL21 cells transformed with pET-TMAI were grown in Luria-Bertani medium (0.8 liter) containing 100 μg of ampicillin per ml at 37°C to an optical density at 600 nm of 0.5 to 0.6. After induction by 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), the cells were grown for an additional 5 h and harvested by centrifugation (10,000 × g, 20 min, 4°C). Bacterial pellets were stored at −70°C.

The centrifuged cells were suspended in 40 ml of 1× His-binding buffer (500 mM NaCl, 20 mM Tris, 5 mM imidazole; pH 7.9) and disrupted by sonication. The lysate was centrifuged at 14,000 × g for 20 min to remove the cell debris, and the supernatant was heated at 80°C for 20 min. After this the suspension was centrifuged at 20,000 × g for 20 min to remove the denatured E. coli proteins, and the soluble fraction was filtered through a 0.2-μm-pore-size filter. The filtrate was loaded on a His-bind resin column (10 ml) equilibrated with the same buffer. The column was washed with 10 volumes of the same buffer, and a gradient of imidazole (from 5 mM to 1 M) was applied to elute the recombinant protein. The fractions containing enzyme activity were pooled and dialyzed against 20 mM Tris-HCl buffer (pH 7.9), and the dialyzed enzyme preparation was stored at 4°C. Protein concentrations were determined by the bicinchoninic acid method, with bovine serum albumin as the standard. Enzyme fractions were analyzed by sodium dodecyl sulfate (SDS)-12% polyacrylamide gel electrophoresis (PAGE) and visualized with Coomassie blue (14).

Enzyme activity assay.AI (d-galactose isomerase) activity was determined by measuring the accumulation of l-ribulose (d-tagatose). Unless otherwise indicated, the standard reaction mixture contained 50 mM HEPES buffer (pH 7.5 at room temperature), 1 mM CoCl₂, 10 mM MnCl₂, 0.2 ml of enzyme preparation at a suitable dilution, 0.1 M l-arabinose (d-galactose), and enough distilled water to bring the final volume to 1.25 ml. The reaction mixtures were incubated at 90°C for 20 min. The reaction was stopped by cooling on ice. The l-ribulose (d-tagatose) formed was quantified by the cysteine-sulfuric acid-carbazole method (7), and the absorbance was measured at 560 nm. One unit of isomerase activity was defined as the amount of enzyme that produced 1 μmol of product per min under the assay conditions.

Isoelectric focusing and Western blotting.Isoelectric focusing was carried out with the PROTEINII Ready Gel precast system (Bio-Rad). The range of the precast gel was pH 3.0 to 10.0. Focusing took place under constant-voltage conditions in a stepped fashion (15 min at 100 V, 15 min at 200 V, and 60 min at 450 V, all at 20°C). For Western blot analysis with l-arabinose-induced T. maritima cell extracts and polyclonal antibodies raised against purified recombinant T. maritima AI, proteins were electrophoretically transferred to a nitrocellulose membrane (Bio-Rad), blocked with 3% skim milk in phosphate-buffered saline at 4°C overnight, and incubated with polyclonal antibodies for 2 h and then with horseradish peroxidase-conjugated mouse anti-mouse immunoglobulin G antibody (Promega) for 60 min at room temperature. The membranes were washed five times with phosphate-buffered saline containing 0.05% Tween 20 and five times with distilled water and were developed with 3,3′,5,5′-tetramethylbenzidene (Promega).

Temperature and pH studies.The effect of temperature on T. maritima AI activity was measured by using the standard protocol. To determine the effect of pH on T. maritima AI, the enzyme was incubated under standard assay conditions except that the HEPES buffer was replaced by 50 mM sodium acetate buffer (pH 5 to 6), 50 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)] buffer (pH 6 to 7.5), 50 mM EPPS [N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid)] buffer (pH 7.5 to 8.5), or 50 mM sodium bicarbonate buffer (pH 9 to 10). All pHs were adjusted at room temperature, and the ΔpK_a/Δts (the latter term being the change in temperature) for each buffer were taken into account when the results were analyzed.

Effect of metal ions on enzyme activity.Metal ions were removed from the purified AI by treatment with 10 mM EDTA at 60°C for 1 h, followed by overnight dialysis against 50 mM HEPES buffer (pH 7.5) at 4°C with several changes of buffer. The effects of various metal ions were determined by adding CoCl₂ · 6H₂O, MnCl₂ · 4H₂O, MgCl₂ · 6H₂O, CaCl₂ · 2H₂O, ZnCl₂ · 6H₂O, CuCl₂ · 2H₂O, FeCl₂ · 6H₂O, or NiCl₂ · 6H₂O at concentrations of 1 and 10 mM to the dialyzed enzyme and assaying AI activity under standard conditions without 1 mM CoCl₂ and 10 mM MnCl₂. The dependence of T. maritima AI activity on metal concentration was determined by measuring AI activity under standard conditions after a 15-min preincubation at 80°C in the presence of various concentrations of metal ions.

Determination of kinetic constants of T. maritima AI.To determine kinetic parameters, assays were performed in 50 mM HEPES (pH 7.5 at room temperature) containing 1 mM CoCl₂, 5 mM MnCl₂, and 1 to 800 mM substrate (l-arabinose or d-galactose). Assay mixtures were incubated for 1 and 10 min at 90°C for l-arabinose and d-galactose, respectively, and the reactions were stopped by cooling on ice.

Analysis of isomerization product.Thin-layer chromatography of d-galactose and d-tagatose in ethyl acetate-isopropanol-water (6/3/1, vol/vol/vol) was performed by using the ascending technique and 0.2-mm silica gel-coated aluminum sheets (type 60; Merck, Darmstadt, Germany). Each plate was sprayed with 60% concentrated H₂SO₄ and then heated to visualize the spots (5).

High-performance ionic chromatography (Dionex) was carried out by using a Carbopac PA1 column (Dionex) equipped with a pulsed amperometry electrochemical detector. d-Galactose and d-tagatose were separated by isocratic elution in a 20 mM NaOH solution at a flow rate of 0.3 ml/min.

Cloning, expression, and purification of T. maritima AI.The araA gene (1.5 kb) encoding AI was amplified by PCR from T. maritima genomic DNA and cloned into the pGEM-T Easy cloning vector. For expression in E. coli, as well as to facilitate subsequent purification, the araA gene was subcloned into the pET-22b(+) expression vector, resulting in pET-TMAI. The gene for AI in pET-TMAI was successfully expressed as a C-terminal hexahistidine-tagged fusion protein in E. coli BL21(DE3) upon induction with IPTG. To obtain the SDS-PAGE pattern shown in Fig. 2A, the E. coli lysates were heated to 80°C to remove the majority of endogenous proteins, while T. maritima AI remained soluble and active. A single Ni²⁺ chelate affinity chromatography step was used to purify the AI to more than 90% purity with a yield of 73%. The apparent M_r of the fusion protein was estimated to be 57,000 by SDS-PAGE, which is consistent with the M_r (56,658.39) calculated from the presumptive amino acid sequence. The N-terminal sequence of the first 15 residues of the recombinant T. maritima AI was determined to be MIDLKQYEFWFLVGSQ by the Tufts University Analytical Core Facility. This is identical to the amino acid sequence deduced from the DNA sequence. In addition, we confirmed by Western blotting with polyclonal antiserum against purified recombinant enzyme that the recombinant AI was identical to the native AI present in an l-arabinose-induced culture of T. maritima (Fig. 2B). Thus, we successfully overexpressed and purified recombinant T. maritima AI.

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FIG. 2.

SDS-PAGE, Western blotting, and isoelectric focusing analyses of the recombinant T. maritima AI. (A) Purified recombinant T. maritima AI was analyzed by SDS-12.5% PAGE. Lane 1, molecular weight markers; lane 2, crude extract of E. coli BL21(pET-TMAI); lane 3, crude extract after heat treatment; lane 4, Ni²⁺ affinity pool (5 μg). (B) Western blotting of recombinant and native AIs from E. coli and T. maritima with mouse antiserum raised against recombinant AI. Lane 1, crude extract of uninduced E. coli BL21(pET-TMAI); lane 2, crude extract of induced E. coli BL21(pET-TMAI); lane 3, l-arabinose-induced crude extract of wild-type T. maritima. (C) Purified recombinant AI (10 μg) analyzed by isoelectric focusing (pH 3.0 to 10.0).

Biochemical characterization of T. maritima AI.As described above, the theoretical M_r of T. maritima AI was 56,658.39, and SDS-PAGE confirmed that the apparent M_r was 57,000. However, gel filtration chromatography on Sephacryl S300 yielded an estimated M_r of 230,000 (Fig. 3), suggesting a homotetrameric structure, compared with the homohexameric structure of E. coli AI (molecular mass, 362 kDa) (22, 30). The experimentally determined isoelectric point of the recombinant T. maritima AI was pH 5.7 (Fig. 2C).

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FIG. 3.

Time course of tagatose production during T. maritima AI-catalyzed isomerization of d-galactose. The reaction mixtures (1 ml) contained 50 mM HEPES buffer (pH 7.5), 10 mM d-galactose, 1 mM Co²⁺, 5 mM Mn²⁺, and 5 mg of T. maritima AI. Aliquots were withdrawn after various periods of incubation at 70°C (□), 80°C (•), and 90°C (○).

The temperature dependence of the recombinant enzyme was determined in the presence and absence of divalent metal ions (1 mM Co²⁺ and 5 mM Mn²⁺) after 20 min of incubation at various temperatures (Table 1). In the presence of metal ions, the apparent optimum temperature for the recombinant enzyme was 90°C, whereas in the absence of metal ions the apparent optimum temperature was 85°C. The slight decrease in the apparent optimum temperature may reflect an effect of metal ions on enzyme conformation. This phenomenon is discussed further below. The apparent optimum pH at 90°C in the presence of 1 mM Co²⁺ and 5 mM Mn²⁺ was 7.5 (Table 1).

AI is known to convert d-galactose into d-tagatose and to convert l-arabinose into l-ribulose (5, 10). However, it has been noted that d-galactose, d-fucose, and l-arabinose are extremely poor substrates for AIs from mesophilic microorganisms (3, 11, 19, 22). In particular, the AIs from E. coli and L. gayonii have negligible activity for converting d-galactose into d-tagatose (19, 22). Figure 3 shows the time course of d-tagatose production from d-galactose by the purified T. maritima AI at a range of temperatures. Aliquots of the reaction mixture were withdrawn periodically and analyzed by thin-layer chromatography and high-performance ionic chromatography. The ratio of conversion of d-galactose to tagatose at 80°C was higher than that at 70°C. This is consistent with the previous results for T. neapolitana AI that showed that the ratio of conversion of d-galactose to d-tagatose depends on the reaction temperature (12). Maximum yields of 56 and 50% conversion of d-galactose to d-tagatose could be obtained after 6 h of incubation at 80 and 70°C, respectively. However, at 90°C the enzyme became inactivated before the equilibrium was reached. l-Arabinose, a major substrate for AI, was converted to l-ribulose with a 70% yield in 6 h at 80°C under the same conditions (data not shown). Even though we could not conclude that the affinity of T. maritima AI for d-galactose is unusually high, it seems clear that T. maritima AI catalyzes the conversion of d-galactose into d-tagatose very efficiently compared to mesophilic AIs (12) and that, at equilibrium, the ratio of d-galactose to d-tagatose is dependent on the reaction temperature.

Comparison of the protein sequences.The amino acid sequence of T. maritima AI was compared with those of other mesophilic AIs. Table 2 shows the percentages of identity and similarity for reported AI sequences. The T. maritima AI exhibits high levels of sequence similarity to thermolabile AIs from E. coli (72%), Salmonella enterica serovar Typhimurium (72%), Bacillus halodurans (73%), and Bacillus subtilis (71%), as well as thermostable AIs from T. neapolitina (98%) and Geobacillus stearothermophilus (76%). Interestingly, the AIs have very low sequence homology (<10%) with other sugar isomerases (e.g., XI, fucose isomerase, etc.) (data not shown).

As shown in Fig. 4, an amino acid sequence alignment of several AIs with the enzyme from T. maritima revealed that these enzymes contain several highly conserved domains. Secondary structure analysis demonstrated that the secondary structure elements are also highly conserved in these microbial AIs (Fig. 4). It is difficult to identify differences in secondary structure between T. maritima AI and other microbial AIs. However, AIs from mesophiles and thermophiles differ greatly in stability (Table 1), but as no AI crystal structure is available, it is not yet possible to account for these differences. Nonetheless, we were able to pinpoint a few characteristic features by comparing the amino acid compositions of T. maritima AI and other microbial AIs (Table 3). The pI of T. maritima AI is higher than that of mesophilic AIs due to a higher content of basic amino acids and a lower content of acidic amino acids. Another characteristic of T. maritima AI is that its hydrophobic amino acid content is higher than that of mesophilic AIs. When hydrophobicity was estimated from the grand average of hydropathicity index (http://www.expasy.org/tools/protparam.html ) (13), the value for T. maritima AI was greater than that for mesophilic AIs, indicating that T. maritima AI is much more hydrophobic than other mesophilic AIs. This result is consistent with the fact that hydrophobic interactions are important for protein thermostability. Moreover, the content of charged amino acids in T. maritima and T. neapolitana AIs is higher than that in mesophilic enzymes. This suggests that the higher content of charged amino acids in thermostable AIs may be an adaptation to enhance the quaternary structure of the enzymes by generating a network of intersubunit ion pairs or salt bridges.

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FIG. 4.

Alignment of the amino acid sequences of T. maritima and other microbial AIs. The residues of T. maritima AI involved in α-helices or β-strands are indicated by H and E, respectively. Secondary structure prediction was carried out by using the PROFsec (B. Rost; http://maple.bioc.columbia.edu/predictprotein ) and Gor4 (8) programs. TMAI, T. maritima AI (National Center for Biotechnology Information protein database accession no. AE001709 ); TNAI, T. neapolitana AI (AY028379); GSAI, G. stearothermophilus AI (AF160811); ECAI, E. coli AI (M15263); BSAI, B. subtilis AI (1769994); STAI, S. enterica serovar Typhimurium AI (M11047) (16); MSAI, Mycobacterium smegmatis AI (7242883).

Effect of metal ions on enzyme activity and stability.To investigate the effect of divalent metal ions on AI activity, the purified enzyme was dialyzed against 5,000 volumes of 50 mM HEPES buffer (pH 7.5) containing 10 mM EDTA. No activity was measurable in the absence of divalent metal ions. However, activity was recovered when Mn²⁺ or Co²⁺ was added. Thus, like all other l- and d-AIs previously reported (3, 12, 19, 22, 31), T. maritima AI has a requirement for Mn²⁺ or Co²⁺. As shown in Fig. 5A, about 1 mM Co²⁺ restored activity, whereas 5 mM Mn²⁺ was needed for full activity. Compared to Mn²⁺ and Co²⁺, other metal ions, such as Mg²⁺, Ca²⁺, Fe²⁺, and Ni²⁺, were poor activators (data not shown). It is evident that Mn²⁺ and Co²⁺ are effective activators. Although the mesophilic AI from L. gayonii was slightly activated by Co²⁺, Mn²⁺ is the only activating metal ion for E. coli AI. We performed kinetic experiments on the effect of Mn²⁺ and Co²⁺ on T. maritima AI. Double-reciprocal plots of velocity versus substrate concentration and velocity versus metal ion concentration gave a family of lines in which the slope and intercept on the vertical axis varied with the Co²⁺ concentration as well as the Mn²⁺ concentration (Fig. 5B and C). The results of these kinetic studies reflect the fact that not only Mn²⁺ but also Co²⁺ is able to activate T. maritima AI. However, it was found that there is a decrease in velocity at high metal concentrations, as observed for substrate inhibition. This was manifested by an upward curvature at low values of 1/[Co²⁺], where an upward curvature is observed at cobalt concentrations above 5 mM (Fig. 5C). This phenomenon is known as substrate inhibition and is usually interpreted in terms of the existence of two types of substrate-binding sites in the enzyme. At high metal concentrations, a low-affinity type of site becomes occupied, and this is presumed to inhibit the catalytic reaction taking place at the high-affinity type of site. For a fuller investigation of this phenomenon, the three-dimensional structure of the enzyme complex with divalent metal ions should be investigated.

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FIG. 5.

Effect of concentration of divalent metal ions on the activity of purified T. maritima AI. (Top) EDTA-pretreated T. maritima AI (0.5 mg/ml) activity was measured at various Mn²⁺ (○) and Co²⁺ (•) concentrations in 50 mM HEPES buffer (pH 7.5) containing 40 mM l-arabinose. (Middle and bottom) The purified enzyme (0.5 mg/ml) was treated with 10 mM EDTA and then dialyzed against 50 mM HEPES buffer (pH 7.5) overnight at 4°C. The EDTA-treated and dialyzed enzyme was incubated at 90°C for 1 min in the presence of 0.1 mM Mn²⁺ (▴), 1 mM Mn²⁺ (•), or 10 mM Mn²⁺ (▪) (middle) or in the presence of 2.5 mM Co²⁺ (•), 5 mM Co²⁺ (○), 10 mM Co²⁺ (▴), 20 mM Co²⁺ (▵), and 40 mM Co²⁺ (▪) (bottom).

Interestingly, T. maritima AI also required Mn²⁺ or Co²⁺ for thermostability (Fig. 6). The apoenzyme proved to be very unstable at 90°C in the absence of metal ions. Addition of 5 mM Mn²⁺ or 1 mM Co²⁺ significantly increased the thermostability. Indeed, incubation in the presence of Mn²⁺ or Co²⁺ at 80°C for 240 min resulted in little loss of activity.

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FIG. 6.

Time course of irreversible thermal inactivation of purified T. maritima AI. After various periods of incubation at 80°C (top) and 90°C (bottom) in the absence of divalent metal ions (○) or in the presence of 1 mM Co²⁺ (•) or 5 mM Mn²⁺ (□), aliquots were withdrawn, and their residual activities were measured under the standard conditions.

It has been noted that all of the simple sugar isomerases, such as XI and fucose isomerase, are metalloproteins, and it has been suggested that the metal plays the same role in these enzymes that phosphate plays in the phosphosugar isomerases (1). Nuclear magnetic resonance experiments with l-arabinose showed that AI isomerizes l-arabinose to l-ribulose by exchanging the proton at carbon 2 of the substrate (1). The presence of Mn²⁺ in E. coli B/r produces an enzyme with greater intrinsic activity and heat stability (23), and divalent metal ions have also been found to be required for the thermal stability of other thermostable enzymes. For example, the XIs of T. maritima, T. neapolitana, and Thermoanaerobacterium strain JW/SL-YS 489 require metal ions, such as Mn²⁺ and Co²⁺, for heat stability, as well as catalytic activity (17, 29). Thermostable enzymes, such as aminoacylases and carboxypeptidases, are known to contain Zn²⁺ ions that maintain or stabilize the active conformation (26, 28). Ca²⁺ also enhanced the thermostability of an extracellular amylase from Thermococcus profundus DT5432 (6).

In order to exploit quantitative data for the fraction of folded and unfolded protein as a function of temperature, the change in ellipticity at 225 nm was monitored. The effects of Mn²⁺ and Co²⁺ on the thermal transition of T. maritima AI were determined, as shown in Fig. 7. In the absence of metal ions, the apoenzyme began to denature at 80°C, and about 50% of the total enzyme was denatured at 88°C. On the other hand, in the presence of Mn²⁺ and Co²⁺, over 50% of the enzyme remained in the native state at 94°C. Divalent metal ions appear, therefore, to increase the thermostability of the enzyme by at least 5 to 6°C. Hence, we propose that divalent metal ions, such as Mn²⁺ and Co²⁺, are key stabilizing factors for the tetrameric structure of T. maritima AI.

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FIG. 7.

Thermal transitions of T. maritima AI in the presence of 5 mM Mn²⁺ (□) or 1 mM Co²⁺ (•) or in the absence of divalent metal ions (○). Each purified enzyme sample (0.5 mg/ml) in 20 mM HEPES buffer (pH 7.5) was scanned at temperatures from 30 to 110°C. The temperature was increased at a constant rate of 1°C/min. Protein unfolding was monitored by determining the temperature-induced changes in α-helical ellipticity at 225 nm. The fraction of unfolded protein was calculated as described previously (21).

Determination of kinetic parameters of T. maritima AI.The kinetic parameters were determined for l-arabinose and d-galactose as substrates from Lineweaver-Burk plots. The K_m for l-arabinose was 31 mM, and the V_max was 41.3 U/mg, whereas for d-galactose the K_m and V_max were 60 mM and 8.9 U/mg, respectively. The catalytic efficiency (k_cat/K_m) for l-arabinose (74.8 mM⁻¹ · min⁻¹) was approximately nine times higher than that for d-galactose (8.5 mM⁻¹ · min⁻¹). Although a direct comparison is difficult because of the different reaction temperatures, T. maritima AI appears to have a lower K_m and a higher efficiency than T. neapolitana AI (12). However, with d-galactose and l-arabinose, the V_max of T. maritima AI was lower than that of T. neapolitana AI (the V_max values were 41.3 and 8.9 U/mg at 90°C, compared to 119 and 14.3 U/mg for the T. neapolitana AI at 85°C).

The biochemical properties of T. maritima AI are compared with those of mesophilic AIs in Table 1. The differences between these microbial AIs are very interesting in view of the similarities of the primary and secondary structures of the molecules. Since no three-dimensional AI structure is available, these differences cannot be explained yet. For industrial applications, the catalytic efficiency for d-galactose needs to be increased for the enzymes to be useful in the commercial production of tagatose as a low-calorie bulk sweetener. The productivity of the enzymes can be enhanced by addition of metal ions, such as Co²⁺, Mn²⁺, or Mg²⁺, etc., and the nature of the selected ion and its optimum concentration are different for different enzymes. However, certain ions, such as Co²⁺, cannot be used in nutritional applications. To satisfy these requirements, it would be desirable to modify the enzymes by directed evolution. In this connection an X-ray crystallographic study of AI would be desirable to facilitate rational structure-based mutagenesis, as has been done for similar XIs. Since AIs from mesophiles and (hyper)thermophiles differ only in a small number of amino acids, the possible use of systematic or random mutagenesis to correlate increases in stability with specific amino acid substitutions seems most promising. T. maritima AI may be a good model for analysis of metal-mediated thermostabilization and for industrial application in the production of d-tagatose as a novel sweetener.