We found a bacterium, Pandoraea sp. 12B-2, of which whole cells catalyzed not only the decarboxylation of 2,6-dihydroxybenzoate but also the regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate. The whole cells of Pandoraea sp. 12B-2 also catalyzed the regioselective carboxylation of phenol and 1,2-dihydroxybenzene to 4-hydroxybenzoate and 2,3-dihydroxybenzoate, respectively. The molar conversion ratio of the carboxylation reaction depended on the concentration of KHCO(3) in the reaction mixture. Only 5 or 48 % of 1,3-dihydroxybenzene added was converted into 2,6-dihydroxybenzoate in the presence of 0.1 M or 3 M KHCO(3), respectively. The addition of acetone to the reaction mixture increased the initial rate of the carboxylation reaction, but the final molar conversion yield reached almost the same value. When the efficient production of 2,6-dihydroxybenzoate was optimized using the whole cells of Pandoraea sp. 12B-2, the productivity of 2,6-dihydroxybenzoate topped out at 1.43 M, which was the highest value so far reported. No formation of any other products was observed after the carboxylation reaction.
3,4-Dihydroxybenzoate decarboxylase in Enterobacter cloacae P241 was induced by adding 3,4-dihydroxybenzoic acid, 3-hydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid or 4-acetamidobenzoic acid to the culture medium. After stabilizing the enzyme activity by adding 5 mM dithiothreitol and 20 mM Na(2)S(2)O(3) to a cell-free extract, catechol at 50 mM was carboxylated in the presence of 3 M KHCO(3) to 3,4-dihydroxybenzoic acid with a molar conversion ratio of 28% after 14 h at 30 degrees C.
We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its Km value for 4-hydroxybenzoate was 596 microM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the Km value was 6.80 mM. In the presence of 3 M KHCO3 and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The Km value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96-97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.
A nonoxidative decarboxylase, 2,6-dihydroxybenzoate decarboxylase, was found in Agrobacterium tumefaciens IAM12048. The enzyme activity was induced specifically by 2,6-dihydroxybenzoate. The purified enzyme was a homotetramer of identical 38 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 2,6-dihydroxybenzoate and 2,3-dihydroxybenzoate without requiring any cofactors. In the presence of KHCO(3), the enzyme also catalyzed the regioselective carboxylation of 1,3-dihydroxybenzene into 2,6-dihydroxybenzoate at a molar conversion ratio of 30%.
The reversible photoreaction on seed germination of two varieties of lettuce differs remarkably not only with the variety but also with the germination temperature and the physiological conditions of seeds caused by after-ripening.Grand Rapids showed reversible photoreaction at 25 C with 0 to 6 months and at 30 C with 5 to 6 months after-ripening. MSU 16 did not show any reversible photoreaction at 30 C with 0 to 6 months or at 25 C with 0 to 3 months after-ripening although they were completely reversible with 4 to 6 months after-ripening.These two varieties of lettuce seeds, however, showed reversible photoreaction at 20 C when they were sown immediately after harvest or after I and 2 months after-ripening. The photoreversion decreased after the 4-to 6-month stage.It is wellknown that the typical phytochrome system is involved in the germination of photosensitive lettuce seeds (1). There is, however, considerable variation between different seed stocks, which depends largely on the history of the parent plant (7) and of the seeds after harvest (4, 12).An analysis of the relationships between the temperature and the after-ripening of seeds in phytochrome-mediated germination was not made. We studied the functioning of the phytochrome system in lettuce seed germination at different temperatures, using the seeds of two varieties in 0 to 6 months after-ripening. moistened with 1 ml distilled H20 in a 4-cm diameter Petri dish. The dishes then were wrapped with a light-proof paper, which was removed when irradiation was given. These dishes were placed in temperature-controlled chambers (卤 1 C). R5 (660 nm) and FR (730 nm) light were obtained by biological spectrograph (9) and light intensity at the level of seeds was adjusted to 3,000 ergs cm-2 s-1 for both R and FR. MATERIALS AND METHODSThe per cent germination was determined 48 h after sowing and is expressed as an average of four dishes 卤 the standard error. RESULTSSeeds of both varieties of lettuce showed phytochrome control of germination at 20 C when they were sown immediately after harvest or with 1 and 2 months after-ripening (Table I). Their reversibility, however, began to decrease gradually with 2 to 3 months after-ripening. Neither Grand Rapids nor MSU 16 seeds showed any reversibility with after-ripening for 4 to 6 months. This is because both varieties germinated 88 to 76 % in darkness or under 5 min FR irradiation.Grand Rapids seeds were completely reversible at 25 C with 0 to 6 months of after-ripening and at 30 C with 5 to 6 months of after-ripening (Table I). MSU 16 was completely reversible at 25 C with 4 to 6 months after-ripening.Grand Rapids did not show any reversibility at 30 C with 0 to 4 months after-ripening and MSU 16 did not show it, either, at 25 C with 1 to 3 months after-ripening. This is because both varieties were dormant and were not induced to germinate by 5-min exposure to R light at each temperature. DISCUSSIONGrand Rapids seeds lose their thermal dormancy when afterripened for 5 to 6 months. Early during thermal dormanc...
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