RESUMEN
Genes encoding 3-hydroxybutyrate oligomer hydrolase (PhaZc) and 3-hydroxybutyrate dehydrogenase (Hbd) were isolated from Paracoccus denitrificans. PhaZc and Hbd were overproduced as His-tagged proteins in Escherichia coli and purified by affinity and gel filtration chromatography. Purified His-tagged proteins had molecular masses of 31 kDa and 120 kDa (a tetramer of 29-kDa subunits). The His-tagged PhaZc hydrolyzed not only 3-hydroxybutyrate oligomers but also 3-hydroxyvalerate oligomers. The His-tagged Hbd catalyzed the dehydrogenation of 3-hydroxyvalerate as well as 3-hydroxybutyrate. When both enzymes were included in the same enzymatic reaction system with 3-hydroxyvalerate dimer, sequential reactions occurred, suggesting that PhaZc and Hbd play an important role in the intracellular degradation of poly(3-hydroxyvalerate). When the phaZc gene was disrupted in P. denitrificans by insertional inactivation, the mutant strain lost PhaZc activity. When the phaZc-disrupted P. denitrificans was complemented with phaZc, PhaZc activity was restored. These results suggest that P. denitrificans carries a single phaZc gene. Disruption of the phaZc gene in P. denitrificans affected the degradation rate of PHA.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Hidroxibutirato Deshidrogenasa/metabolismo , Hidroxibutiratos/metabolismo , Paracoccus denitrificans/enzimología , Paracoccus denitrificans/metabolismo , Poliésteres/metabolismo , Valeratos/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Clonación Molecular , Elementos Transponibles de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Expresión Génica , Prueba de Complementación Genética , Hidroxibutirato Deshidrogenasa/química , Hidroxibutirato Deshidrogenasa/genética , Hidroxibutirato Deshidrogenasa/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Insercional , Multimerización de Proteína , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADNRESUMEN
D-(-)-3-Hydroxybutyrate (3HB) oligomer hydrolase was purified from Paracoccus denitrificans. The enzyme was a monomeric protein with an approximate molecular mass of 31 kDa. The isoelectric point of the enzyme was 5.2. Optimum temperature and pH were 35-40 degrees C and 8.0, respectively. The enzyme activity was not affected by sulfhydryl reagents but strongly inhibited by serine proteinase inhibitors. Both 3HB trimer and 3HB dimer were hydrolyzed by the enzyme, indicating that the enzyme is not 3HB dimer hydrolase but 3HB oligomer hydrolase. para-Nitrophenyl esters of short-chain fatty acids were also hydrolyzed by the enzyme. 3HB dimer was hydrolyzed somewhat faster than 3HB trimer. The level of the enzyme activity was almost constant, irrespective of carbon sources for the bacterial growth and of the cultivation conditions.