H-1-NMR IMAGING STUDY OF ENZYMATIC DEGRADATION IN POLY(3-HYDROXYBUTYRATE) AND POLY(3-HYDROXYBUTYRATE-CO-3-HYDROXYVALERATE) - EVIDENCE FOR PREFERENTIAL DEGRADATION OF THE AMORPHOUS PHASE BY PHB DEPOLYMERASE-B FROM PSEUDOMONAS-LEMOIGNEI

H-1 NMR imaging has been used to monitor the enzymatic degradation of poly(3-hydroxybutyrate), PHB, and poly(3-hydroxybutyrate-co-23%-3-hydr oxyvalerate), PHB/V, by PHB depolymerase B from Pseudomonas lemoignei. The samples examined were thin films of approximately 200 mu m thickn ess produced by melt casting. Since only the amorphous regions of the semicrystalline films contribute to the H-1 NMR image intensity, this technique provides unique information regarding the degradation proces s in the amorphous regions of the films when combined with total weigh t loss measurements. It was found that although the total weight loss rate of both the PHB and PHB/V films was constant, as previously repor ted, the initial amorphous material consumption rate was exponential. During the initial stages of the degradation process, up to 40-60 h, p referential consumption of amorphous material by depolymerase B was fo und to take place. At later stages the preference for amorphous materi al diminished, and both crystalline and amorphous phases were degraded indiscriminately. This initial consumption of amorphous material supp orts evidence that this stage is necessary to provide access to lamell ar crystalline regions. The initial amorphous polymer consumption was verified by optical microscopy of the PHB film surface, which revealed the well-known circular erosion pattern associated with this type of enzymatic activity. Values of 0.020 and 0.049 h(-1) for the rate const ant of amorphous PHB and PHB/V consumption by depolymerase. B were cal culated from the H-1 imaging data during the early stages of degradati on. The factors responsible for the observed behavior of the depolymer ase B enzyme and the implications for the mechanism of enzymatic degra dation in PHAs are discussed.