Dihydroxybenzoate decarboxylase

COO H

OH OH

COO H

OH OH

Scheme 4

A novel decarboxylase, 2,6-dihydroxybenzoate decarboxylase, was found in Agrobacterium tumefaciens IAM 12048 at first.36 Thereafter, the same activity was found in Rhizobium species by two groups independently.37 38 Furthermore, Pandoraea sp. 12B-2, the most powerful producer of 2,6-dihydroxybenzoate decarboxylase, was isolated.39 These enzymes have been purified and characterized.

2,6-Dihydroxybenzoate decarboxylase activity of these bacteria was induced specifically by 2,6-dihydroxybenzoate. The enzyme activity in a cell-free extract of A. tumefaciens IAM 12048 was stable during storage at 4°C for 7 days in potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol. Different from 4-hydroxybenzoate decarboxylase and 3,4-dihydroxybenzoate decar-boxylase, 2,6-dihydroxybenzoate decarboxylase was much less labile and barely exhibited the sensitivity to oxygen. Therefore, the enzyme purification was performed with relatively high yield. The purified enzyme of A. tumefaciens IAM 12048 was a homotetramer of identical 38 kDa subunits. The enzyme showed no characteristic absorption maximum other than 280 nm, indicating the absence of a prosthetic group such as pyridoxal 5'-phosphate and thiamine pyrophosphate. The enzyme activity was not diminished after the incubation at 50° C for 30 min. Treatment at 60 and 70°C caused 18 and 100% losses of initial activity, respectively. The enzyme was stable on incubation at 30°C for 20 min in the pH range of 5.0-11.0. When the decarboxylation reaction was carried out for 20 min at various temperatures, the activity was maximal at 60°C. The highest activity was observed at pH 8.5 (10 mM Tris-HCl buffer) and the activities under pH 5.0-10.0 were over 80% of that at pH 8.5. 2,6-Dihydroxybenzoate decarboxylases of A. tumefaciens IAM 12048 and Rhizobium species had similar properties.

The purified enzyme of A. tumefaciens IAM 12048 catalyzed the regiose-lective carboxylation of 1,3-dihydroxybenzene into 2,6-dihydroxybenzoate in the presence of 15 mM 1,3-dihydroxybenzene and 3 M KHCO3, with a molar conversion yield of 30%. The Rhizobium enzymes were also confirmed to catalyze the reverse carboxylation. The regioselective carboxylation of 1,3-dihydroxybenzene was optimized using the whole cells of Pandoraea sp. 12B-2. The carboxyla-tion reaction was carried out at 30°C in a tightly sealed reaction vessel to avoid the leakage of CO2 gas. The standard reaction mixture contained 12.5 mM 1,3-dihydroxybenzene, 3 M KHCO3, 100 mM potassium phosphate buffer (pH 7.0), and whole cells (22.0 mg dry weight per 1 ml of reaction mixture).

The effect of the KHCO3 concentration on the production of 2,6-dihydroxybenzene was investigated.39 In the presence of 0.1 M KHCO3, 0.5 mM 2,6-dihydroxybenzoate was formed from 12.5 mM 1,3-dihydroxybenzene. The addition of 1-3 M KHCO3 in the reaction mixture significantly promoted the formation of 2,6-dihydroxybenzoate (Fig. 5a). With 3 M KHCO3, the molar conversion ratio of 1,3-dihydroxybenzene reached its highest value (48%). The effect of temperature on 2,6-dihydroxybenzoate formation was examined (Fig. 5b). When the reaction was carried out for 6 h at 40 or 50°C, the initial velocity of 2,6-dihydroxybenzoate formation was accelerated. However, the prolonged incubation over 40°C resulted in the degradation of 2,6-dihydroxybenzoate. To enhance the productivity of 2,6-dihydroxybenzoate, the addition of various kinds of organic solvent was tested. Among them, the addition of 10-20% (v/v) acetone accelerated the initial velocity of 2,6-dihydroxybenzoate formation (Fig. 5c); however, when the equilibrium was achieved, the final molar conversion yield of 1,3-hydroxybenzene reached the same level as in the case without adding acetone. The addition of acetone decreased the viscosity of the whole cells of Pandoraea sp. 12B-2 in the reaction mixture.

Figure 5: Effect of KHCO3 concentraion (a), temperature (b), and acetone (c) on the carboxylation of 1,3-dihydroxybenzene. (a) The following concentrations of KHCO3 were added in the reaction mixture: open triangles, 0.1 M; closed diamonds, 1 M; open squares, 2 M; closed circles, 3 M. (b) Reactions were carried out using 100 mM 1,3-dihydroxybenzene at 20° C open triangles; 30° C closed circles; 35°C closed diamonds; 40°C open squares. (c) Reactions were carried out using 200 mM 1,3-dihydroxybenzene. Acetone was added at the following concentrations: open squares, 0% (v/v); closed circles 10% (v/v); closed diamonds 20% (v/v).

Figure 5: Effect of KHCO3 concentraion (a), temperature (b), and acetone (c) on the carboxylation of 1,3-dihydroxybenzene. (a) The following concentrations of KHCO3 were added in the reaction mixture: open triangles, 0.1 M; closed diamonds, 1 M; open squares, 2 M; closed circles, 3 M. (b) Reactions were carried out using 100 mM 1,3-dihydroxybenzene at 20° C open triangles; 30° C closed circles; 35°C closed diamonds; 40°C open squares. (c) Reactions were carried out using 200 mM 1,3-dihydroxybenzene. Acetone was added at the following concentrations: open squares, 0% (v/v); closed circles 10% (v/v); closed diamonds 20% (v/v).

When the whole-cell reaction was carried out in the presence of 0.1-3 M 1,3-dihydroxybenzene and 3 M KHCO3, the molar conversion ratio of 1,3-dihydroxybenzene always reached 45-50% (Fig. 6). The high concentration of 1,3-dihydroxybenzene was not inhibitory on 2,6-dihydroxybenzoate decar-boxylase. When 3 M 1,3-dihydroxybenzene was incubated with the whole cells of Pandoraea sp. 12B-2 (43.9 mg as dry cell weight) in the presence of 3 M KHCO3, 220 mg ml-1 of 2,6-dihydroxybenzoate (1.42 M) accumulated after 120 h incubation, with a conversion ratio of 48%. During the carboxylation of 1,3-dihydroxybenzene, no other product except for 2,6-dihydroxybenzoate was formed.

Figure 6: 2,6-Dihydroxybenzoate production using whole cells of Pandoraea sp. 12B-2. Reactions were carried out in the reaction mixture containing the following concentrations of 1,3-dihydroxybenzne: closed circles, 3 M; open squares, 2 M; closed diamonds, 1 M; open triangles, 0.1 M.

Figure 6: 2,6-Dihydroxybenzoate production using whole cells of Pandoraea sp. 12B-2. Reactions were carried out in the reaction mixture containing the following concentrations of 1,3-dihydroxybenzne: closed circles, 3 M; open squares, 2 M; closed diamonds, 1 M; open triangles, 0.1 M.

The product was isolated and identified by NMR and 13C NMR analyses comparing with the authentic 2,6-dihydroxybenzoic acid as a reference.

Carboxylation of 20-300 mM 1,2-dihydroxybenzene was carried out using 36.9 mg (as dry cell weight) of whole cells in the presence of 3 M KHCO3 in 1 ml of the reaction mixture. The molar conversion yields were almost the same using 20, 100, and 200 mM 1,2-dihydroxybenzene (approximately 25%) as shown in Fig. 7.

Figure 7: Time course of carboxylation of 1,2-dihydroxybenzene using whole cells of Pandoraea sp. 12B-2. Reactions were carried out in the reaction mixture containing the following concentration of 1,2-dihydroxybenzene: closed circles, 300 mM; open squares, 200 mM; closed diamonds, 100 mM; open triangles,

Figure 7: Time course of carboxylation of 1,2-dihydroxybenzene using whole cells of Pandoraea sp. 12B-2. Reactions were carried out in the reaction mixture containing the following concentration of 1,2-dihydroxybenzene: closed circles, 300 mM; open squares, 200 mM; closed diamonds, 100 mM; open triangles,

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