Local sequence dependence of polyhydroxyalkanoic acid degradation in Hydrogenophaga pseudoflava

The first order intracellular degradation of various polyhydroxyalkanoic ac id (PHA) inclusions in Hydrogenophaga pseudoflava cells was investigated by analyzing the compositional and microstructural changes of the PHA. using gas chromatography, C-13 NMR spectroscopy, and differential scanning calori metry. Two types of PEA, copolymers and blend-type polymers, were separatel y accumulated in cells for comparison. The constituent monomers were S-hydr oxybutyric acid (3HB), 4-hydroxybutyric acid (4HB), and 3-hydroxyvaleric ac id (3HV), It was found that the 3HB-4HB copolymer was degraded only when th e polymer contained a minimal level of 3HB units. With the cells containing a 3HB/4HB blend-type polymer, only poly(3HB) was degraded, whereas poly(4H B) was not degraded, indicating the totally inactive nature of the intracel lular depolymerase against poly(4HB). On the basis of the magnitude of the first order degradation rate constants, the relative substrate specificity of the depolymerase toward the constituting monomer units was determined to decrease in the order 3HB > 3KV > 4HB, C-13 NMR resonances of the tetrad, triad, and dyad sequences were analyzed for the samples isolated before and after degradation experiments. The results showed that the intracellular d egradation depended on the local monomer sequence of the copolymers, The re lative substrate specificity of the depolymerase determined from the NMR lo cal sequence analysis agreed well with that obtained from the kinetics anal ysis. It is suggested that, without isolation and purification of the intra cellular PHA depolymerase and "native" PHA substrates, the relative specifi city of the enzyme as well as the microstructural heterogeneity of the PHA could be determined by measuring in situ the first order degradation rate c onstants of the PHA in cells.