Adsorption effects of poly(hydroxybutyric acid) depolymerase on chain-folding surface of polyester single crystals revealed by mutant enzyme and frictional force microscopy

Abstract

Adsorption effects of poly(hydroxybutyric acid) (PHB) depolymerase from Ralstonia pickettii T1 on various polymer single crystals were studied using a catalytically inactive mutant of PHB depolymerase by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), and frictional force microscopy (FFM). Six types of polymer single crystals, poly[( R)-3-hydroxybutyric acid] (P(3HB)), poly[( R)-3-hydroxybutyric acid- co-6 mol% ( R)-3-hydroxyvaleric acid] (P(3HB- co-6 mol% 3HV)), poly[( R)-3-hydroxybutyric acid- co-8 mol% ( R)-3-hydroxyhexanoic acid] (P(3HB- co-8 mol% 3HH)), poly( l-lactic acid) (PLLA), poly( d-lactic acid) (PDLA), and polyethylene (PE), were prepared to examine the influence of an ester bond and stereoregularity of a polymer on the enzymatic adsorption. The numbers of PHB depolymerase enzymes adsorbed on P(3HB) and P(3HB- co-6 mol% 3HV) single crystals were determined as 171 and 183 enzymes/μm 2 by AFM, respectively. AFM observation revealed that the concentration of PHB depolymerase enzymes adsorbed onto PLLA and PDLA single crystals is much higher compared to those on a P(3HB) single crystal, whereas the concentration of enzyme adsorbed onto PE and P(3HB- co-8 mol% 3HH) single crystals is much less. In addition, the single crystals of each polymer were characterized by TEM and FFM before and after enzymatic treatment by mutant for 1 h at 37 °C. The surface properties of P(3HB), P(3HB- co-6 mol% 3HV), and P(3HB- co-8 mol% 3HH) single crystals were changed by the enzymatic adsorption, whereas the internal structures were not affected. On the basis of these results, the properties of the binding domain of PHB depolymerase to polymer chain-folding surfaces have been discussed.