Visualization of enzymatic degradation of poly[(R)-3-hydroxybutyrate] single crystals by an extracellular PHB depolymerase

Single crystals of bacterial poly[(R)-3-hydroxybutyrate] (P(3HB)) with different molecular weights (M(n)=2500 and M(n)=48000) were prepared in a mixture of chloroform and ethanol. The enzymatic degradation of P(3HB) single crystals with extracellular PHB depolymerase purified from Pseudomonas stutzeri YM1006 was investigated by transmission electron microscopy, atomic force microscopy, and gel-permeation chromatography. Adsorption of PHB depolymerase on P(3HB) single crystals was examined using an immune-gold labeling technique, which demonstrated a homogeneous distribution of enzymes on the surface of crystals. Unselective adsorption of PHB depolymerase suggests that the enzyme adheres to hydrophobic surfaces of single crystals and contributes to increase in the mobility of P(3HB) chains as a whole. Enzymatic degradation of single crystals progressed from the edge of crystals, or along their long axis as making a small fragment. Both lamellar thicknesses of single crystals and molecular weights of P(3HB) chains remained unchanged during the enzymatic hydrolysis. It has been concluded that the attack by the active site of PHB depolymerase takes place preferentially at the disordered chain-packing regions of crystal edge rather than the chain-folding surfaces of single crystals, in spite of the unselective adsorption of enzyme and that untied or exposed P(3HB) chains are degraded predominantly by exo-type behavior of enzyme.