Nonhydrolytic Fragmentation of a Poly[(R)-3-hydroxybutyrate] Single Crystal Revealed by Use of a Mutant of Polyhydroxybutyrate Depolymerase

Abstract

This paper reports the initial process of the enzymatic degradation of solution-grown lamellar single crystals of bacterial poly[(R)-3-hydroxybutyrate] (P(3HB)) with an extracellular polyhydroxybutyrate (PHB) depolymerase purified from Alcaligenes faecalis T1. We used a hydrolytic-activity-disrupted mutant of the PHB depolymerase in order to avoid the influence of hydrolytic reaction in the system. The effect of addition of the mutant enzyme upon the P(3HB) single crystals was investigated by turbidimetric assay, high-performance liquid chromatography (HPLC), and atomic force microscopy (AFM). Suspension turbidity of the P(3HB) single crystals increased after addition of the mutant enzyme having no hydrolytic activity. No soluble product from the P(3HB) single crystals with the mutant enzyme was detected by HPLC. AFM observation of the P(3HB) single crystals adsorbed on highly ordered pyrolytic graphite revealed that the mutant enzyme yielded a lot of lengthwise crystal fragments from the P(3HB) single crystals. On the basis of these results, we concluded that the mutant enzyme disturbs the molecular packing of the P(3HB) polymer chain around the loose chain packing region in the single crystal, resulting in the fragmentation. Therefore, it is suggested that the enzymatic degradation of P(3HB) single crystals with a wild-type PHB depolymerase progresses via three steps: (1) adsorption of the enzyme onto the surface of the single crystal; (2) disturbance of the molecular packing of P(3HB) polymer chain in the single crystal by the adsorbed enzyme; and (3) hydrolysis of the disturbed polymer chain by the adsorbed enzyme.

Effects of Mutations in the Substrate-Binding Domain of Poly[( R )-3-Hydroxybutyrate] (PHB) Depolymerase from Ralstonia pickettii T1 on PHB Degradation

“…(16,17). Furthermore, AFM analysis with PHB single crystals and a mutant of dPHB depolymerase with disrupted hydrolytic activity has demonstrated that the SBD of dPHB depolymerase disturbs the molecular packing of PHB polymer chains, resulting in the fragmentation of PHB single crystals (23). Similarly, in the field of enzymatic degradation of cellulose by cellulase or of chitin by chitinase, several researchers reported that the binding domains of these enzymes enhance the specific physical disruption of their substrates (4,35).…”

The Crystal Structure of Polyhydroxybutyrate Depolymerase from Penicillium funiculosum Provides Insights into the Recognition and Degradation of Biopolyesters

“…In the case of a multi-domain depolymerase, the enzyme can reside on the surface of PHB via the substrate-binding domain. 10,12,[15][16][17] The single domain depolymerase from P. funiculosum lacks such a functionally distinct domain, but retains the ability to reversibly bind to polymers. 19 Therefore it must have a region on its molecular surface for binding to the hydrophobic surface of a polymer.…”

“…19 The issue of whether the adsorption site contributes to the destabilization of ordered regions of PHB chains is an interesting topic. Studies of the degradation of PHB by multi-domain depolymerases 10,12,[15][16][17] indicate that the substrate-binding domain may play a critical role in introducing mechanical damage to ordered regions of the polymer upon binding to that region, as well as providing the catalytic domain a toehold to facilitate the hydrolysis. The introduction of mechanical damage may be the result of strong hydrophobic interactions between the substrate-binding domain and PHB chains, which destabilize the hydrophobic intra-chain and inter-chain interactions of the PHB chains.…”

“…8,9 It has recently been proposed that the substrate-binding domain has an additional and more active function of disrupting the structure of the polymer. 10,11,12 The role of the linker domain is not clearly understood, but it may function as a spacer that introduces a flexible region between the catalytic and substrate-binding domains to increase the hydrolytic efficiency of the catalytic domain. 13 A recent study of a homologous protein to the linker domain, the fibronectin type III homology domain of cellobiohydrolase ChbA of Clostridium thermocellum, indicates that the linker domain may also exhibit a disruptive function against crystalline substrates, analogous to the substrate-binding domain.…”

“…Since these three functional domains are essential for the enzymatic degradation of water-insoluble polymers, 13 it has been proposed that the degradation of the crystalline region of the polymer should proceed in three steps: adsorption of the enzyme to the polymer, non-hydrolytic disruption of the structure of the polymer, and hydrolysis. 10,12,[15][16][17] An extracellular PHB depolymerase from Penicillium funiculosum has been purified and characterized. 18,19 It has a molecular mass of about 33,000, and is the smallest of the known extracellular depolymerases, typically ranging from 40,000 to 57,000.…”