Enzymatic Degradation and Adsorption on Poly[(R)-3-hydroxybutyrate] Single Crystals with Two Types of Extracellular PHB Depolymerases from Comamonas acidovorans YM1609 and Alcaligenes faecalis T1

Abstract

Poly[(R)-3-hydroxybutyrate] (P(3HB)) single crystals, which had different morphologies with and without chain-folding surfaces, were grown from dilute solutions of chloroform and ethanol. Two types of extracellular PHB depolymerases purified from Alcaligenes faecalis T1 and Comamonas acidovorans YM1609, defined as types I and II according to the position of the lipase box in the catalytic domain, were used in the enzymatic degradation and adsorption experiments. The enzymatic degradation of P(3HB) single crystals was investigated by means of transmission electron microscopy, atomic force microscopy, and gel-permeation chromatography. Adsorption of PHB depolymerase on P(3HB) single crystals was examined using an immuno-gold labeling technique. Enzymatic degradation of single crystals progressed from the edges and ends of crystals to yield the narrow cracks and the small crystal fragments along their long axis, independent of both surface morphologies of single crystals and types of extracellular PHB depolymerases. Lamellar thicknesses of single crystals and molecular weights of P(3HB) chains remained unchanged during the enzymatic hydrolysis. Adsorption of extracellular PHB depolymerase demonstrated a homogeneous distribution of enzyme molecules on the chain-folding surface of crystals. The above results suggest that the adsorption of enzyme increases with the mobility of P(3HB) chains of single crystals as a whole and that the endo − exo type attack by the active-site of PHB depolymerase takes place preferentially at the disordered chain-packing regions of crystal edges and ends rather than the chain-folding surfaces of single crystals, in spite of the difference in the structure of enzymes.

Crystal structure and biodegradation of aliphatic polyester crystals

“…Furthermore, in case of P(3HB-co-8%-3HH) and P(3HB-co-10%-4HB), their single crystals showed a lath-shaped morphology 47) . Each monolamellar part of lath-shaped copolymer single crystals yields a sharp electron diffraction pattern, and this diffraction pattern is consistent with a p 2gg symmetry, which coincides with that of P(3HB) single crystals 11,12,33) . In our study there is no essential difference in the d-spacings of electron diffraction diagrams between P(3HB) copolymer and homopolymer single crystals grown from a mixture of chloroform and ethanol.…”

“…15 A. This homogeneous distribution was also observed in case of the other PHB depolymerases from Pseudomonas stutzeri YM1006 11) and Comamonas acidovorans YM1609 12) . Furthermore, the glutathion S-transferase fusion protein with substrate-binding domain of the depolymerase adsorbed on P(3HB) single crystals tightly and homogeneously, suggesting that the substratebinding domain contributes to the adsorption of enzyme on solid P(3HB) without site specificity 101,107,108) .…”

Visualization of the Recrystallization of Solution-Grown Poly[(R)-3-hydroxybutyrate] Lamellar Crystals

“…[14,15] SGCs were prepared from an octanol-diluted solution, collected by centrifugation, washed several times with methanol, and then re-suspended in methanol. The samples were prepared by putting drops of crystal suspensions onto cleaned silicon wafers (3 6 3 mm 2 ).…”

Degradation of Natural and Artificial Poly[(R)-3-hydroxyalkanoate]s: From Biodegradation to Hydrolysis

“…Enzymatic degradation of PHB single crystals with PHB depolymerases from Pseudomonas lemoignei (Nobes et al 1996) and R. pickettii T1 (Iwata et al 1997b;Murase et al 2001a) was observed with the transmission electron microscope and the atomic force microscope. These studies demonstrated that enzymatic hydrolysis progresses from the short ends of the lath-like-shaped PHB lamellar single crystals to form cracks along the long axis.…”