Substrate Report for: unsaturated-poly-3-hydroxynonanoate

Although poly-3-hydroxyalkanoates (PHAs) and particularily medium-chain-length (mcl)-PHAs are likely to find industrial applications in a latex form, very few studies have examined their behavior in aqueous suspension and none have examined the dense suspensions required commercially. For this reason, the stability of mcl-PHA latexes containing saturated aliphatic (65 mol% 3-hydroxynonanoate, PHN), and for the first time, with vinyl (PHNU) or carboxylated side chains was examined. At 4 g L -1 with no stabilizing agent, PHNU nanoparticles (199.4 +/- 3.6 nm) were significantly smaller than those of PHN (211.5 +/- 6.4 nm) while carboxylated PHN nanoparticles (76.1 +/- 6.4 nm) were substantially smaller than those of either PHN or PHNU with particles stable for more than 110 days. Increasing the PHN concentration to 10 g L -1 also resulted in stable latexes but with larger particles (410.8 +/- 5.2 nm). Adjusting the pH of the suspending medium (water) before addition of the polymer (dissolved in acetone) resulted in much smaller PHN particles at pH = 11.3 (134 +/- 2 nm) than at pH = 4.3 (312 +/- 8 nm) at a 4 g L -1 final polymer concentration. Zeta potentials of PHN suspensions decreased with pH, likely due to the carboxyl end groups. Above a pH of 4.0, adjusting the pH after particle formation had little effect. NaCl addition could be used to agglomerate and ultimately precipitate the particles. Stabilizers such as surfactants will likely be required to produce denser mcl-PHA latexes with suitable particle size for certain applications such as coatings and toner production.

A bacterial strain M4-7 capable of degrading various polyesters, such as poly(epsilon-caprolactone), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyoctanoate), and poly(3-hydroxy-5-phenylvalerate), was isolated from a marine environment and identified as Pseudomonas alcaligenes. The relative molecular mass of a purified extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase (PhaZ(PalM4-7)) from P. alcaligenes M4-7 was 28.0 kDa, as determined by SDS-PAGE. The PhaZ(PalM4-7) was most active in 50 mM glycine-NaOH buffer (pH 9.0) at 35 degrees C. It was insensitive to dithiothreitol, sodium azide, and iodoacetamide, but susceptible to p-hydroxymercuribenzoic acid, N-bromosuccinimide, acetic anhydride, EDTA, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, Tween 80, and Triton X-100. In this study, the genes encoding MCL-PHA depolymerase were cloned, sequenced, and characterized from a soil bacterium, P. alcaligenes LB19 (Kim et al., 2002, Biomacromolecules 3, 291-296) as well as P. alcaligenes M4-7. The structural gene (phaZ(PalLB19)) of MCL-PHA depolymerase of P. alcaligenes LB19 consisted of an 837 bp open reading frame (ORF) encoding a protein of 278 amino acids with a deduced M((r)) of 30,188 Da. However, the MCL-PHA depolymerase gene (phaZ(PalM4-7)) of P. alcaligenes M4-7 was composed of an 834 bp ORF encoding a protein of 277 amino acids with a deduced Mr of 30,323 Da. Amino acid sequence analyses showed that, in the two different polypeptides, a substrate-binding domain and a catalytic domain are located in the N-terminus and in the C-terminus, respectively. The PhaZ(PalLB19) and the PhaZ(PalM4-7) commonly share the lipase box, GISSG, in their catalytic domains, and utilize 111Asn and 110Ser residues, respectively, as oxyanions that play an important role in transition-state stabilization of hydrolytic reactions.