bIn this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaC Ac ). In in vitro assays, among the three PhaCs tested, PhaC Ac was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaC Ac ), whereas the prepolymerization PhaC Re (derived from Ralstonia eutropha) and PhaC Da (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaC Ac showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C 6 ). PhaP Ac also activated PhaC Ac when it was added during polymerization (polymer-elongating PhaC Ac ), while this effect was not observed for PhaC Re . In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaP Ac expression on PHA synthesis by PhaC Ac or PhaC Re was examined. As PhaP Ac expression increased, PHA production was increased by up to 2.3-fold in the PhaC Ac -expressing strain, whereas it was slightly increased in the PhaC Re -expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaC Ac both in vitro and in vivo but do not activate PhaC Re . This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaC Ac .

ε-Poly-l-lysine (ε-PL) is one of the few homopoly(amino-acid)s occurring in nature. ε-PL, which possesses multiple amino groups, is highly soluble in water, where it forms the antimicrobial polycationic chain (PL). Although the high water-solubility is advantageous for the use of ε-PL as a food preservative, it has limited the applicability of ε-PL as a biopolymer plastic. Here, we report on the preparation and availability of a water-insoluble complex formed with PL and an anionic surfactant, bis(2-ethylhexyl) sulfosuccinate (BEHS, is also commercialized as AOT) anion. The PL/BEHS-complex, which is soluble in organic solvents, was successfully used as a coating material for a cellulose acetate membrane to create a water-resistant antimicrobial membrane. In addition, the thermoplastic PL/BEHS-complex was able to be uniformly mixed with polypropylene by heating, resulting in materials exhibiting antimicrobial activities.

We have developed a moldable and self-healable material from lignosulfonate by taking advantage of the ionic interactions between lignosulfonate and various cationic polyelectrolytes. Three lignosulfonate complexes with different cationic polyelectrolytes were prepared. The complex of sodium lignosulfonate (L-SO3Na) and poly­(diallyldimethylammonium chloride) (PDADMACl) was deemed a promising moldable material based on its mechanical toughness in tensile tests (>3 MJ/m3). The mechanical properties of the L-SO3Na/PDADMACl complex were further characterized with regard to the effects of composition, the molecular weight of PDADMACl, and relative humidity. Our findings were as follows: (i) the addition of adequate amounts of lignosulfonate to PDADMACl improved the toughness of the complex (from 1.8 to 3.6 MJ/m3 at maximum), (ii) using higher-molecular-weight (>200,000) PDADMACl enhanced the mechanical properties of the complex, and (iii) increasing the relative humidity softened the complex through water absorption. The self-healing properties of the L-SO3Na/PDADMACl complex were also evaluated. The healing efficiency, in terms of toughness recovery, was only 14% at 50% relative humidity but increased to almost 100%, i.e., the sample did not break at the healed position, at 60% relative humidity. This study proposes the use of lignosulfonate as a component of humidity-responsive and completely self-healable materials.

The polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) shows a lag time for the start of its polymerization reaction, which complicates kinetic analysis of PhaC(Re). In this study, we found that the lag can be virtually eliminated by addition of 50 mg/L TritonX-100 detergent into the reaction mixture, as well as addition of 2.5 g/L Hecameg detergent as previously reported by Gerngross and Martin (Proc Natl Sci USA 92: 6279-6283, 1995). TritonX-100 is an effective lag eliminator working at much lower concentration than Hecameg. Kinetic analysis of PhaC(Re) was conducted in the presence of TritonX-100, and PhaC(Re) obeyed Michaelis-Menten kinetics for (R)-3-hydroxybutyryl-CoA substrate. In inhibitory assays using various compounds such as adenosine derivatives and CoA derivatives, CoA free acid showed competitive inhibition but other compounds including 3'-dephospho CoA had no inhibitory effect. Furthermore, PhaC(Re) showed a considerably reduced reaction rate for 3'-dephospho (R)-3-hydroxybutyryl CoA substrate and did not follow typical Michaelis-Menten kinetics. These results suggest that the 3'-phosphate group of CoA plays a critical role in substrate recognition by PhaC(Re).

cThe type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter ؊1 ), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter ؊1 ) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.