5-fold reduction of PhaC activity could be demonstrated for PhaI- granules of P. putida GPo1001 [23]. These results indicate that PhaI has more impact on PhaC activity than PhaF. Yet, the highest impact is observed when both phasins are absent. The influence of PhaF and PhaI on the specific activity

of PhaZ could not be investigated due to lack of accuracy in determining the amount of granule-BMS-907351 supplier associated PhaZ. Discussion Two activity assays were developed which allow rapid measurements of PHA polymerases and PHA depolymerases in crude extracts from cells harvested at different growth stages (Figures 1 and 2). Using these assays with whole cell lysates, we demonstrated a 5-fold decrease in the activity of PhaC and a 1.5-fold increase in the activity of PhaZ during exponential to stationary phase growth of P. putida U on octanoate (Figure 3). These results were consistent Selleck GF120918 with the in vitro activity studies using isolated PHA granules harvested at different growth stages [23]. The results obtained here also confirm previous data in which parallel PHA accumulation and degradation was demonstrated [19, 27]. Regarding the decrease of PhaC activity with the growth of bacteria, previously we have shown that the PhaC activity is influenced by the physiological stage of the cells: the activity of PhaC is stimulated by the high ratio of [3-hydroxyacyl-CoA]/[CoA] [19]. It

is likely that at the beginning of the growth phase (high growth rate), CoA and NAD+ are consumed, and acetyl-CoA and NADH are produced via Fenbendazole β-oxidation for growth, leading to high ratios of [acetyl-CoA]/[CoA] and [NADH]/[NAD], which further resulting in high ratio Fludarabine of [3-hydroxyacyl-CoA]/[CoA] [19], thus, higher activity of PhaC. In contrast, when cells enter the stationary growth phase, β-oxidation is not highly active anymore, the ratios of [acetyl-CoA]/[CoA] and [NADH]/[NAD] are likely to decrease, leading to lower ratio of [3-hydroxyacyl-CoA]/[CoA] [19], thus lower activity of PhaC. Therefore, even through PhaC content

was increased with the growth of bacteria (Figure 4), the activity of PhaC was decreased (Figure 3). In addition to the effect of physiological reagents on PhaC activity, in this study, we further investigated the influence of phasins and found that availability of both PhaI and PhaF have significant impact the activity of PhaC (Table 1). Although the PHA granules became larger as the culture aged [28, 29], this was not associated with an increase of the amount of phasins (Figure 5). The availability of phasins could be one of the reasons for the observed changes in enzyme activities of PhaC. At the initial accumulation stage, young PHA granules may be fully covered with phospholipids and proteins. Interactions between the enzymes and granule-bound phasins may be important for optimal polymerase activity because in the absence of phasins the specific PHA polymerase activity was reduced (Table 1).