US EPA

1149744 

Journal Article 

In vivo C-13-NMR spectroscopic study of polyhydroxyalkanoic acid degradation kinetics in bacteria 

Oh, JS; Choi, MH; Yoon, SC 

2005 

Yes 

Journal of Microbiology and Biotechnology
ISSN: 1017-7825
EISSN: 1738-8872 

15 

6 

1330-1336 

WOS:000234333800024 

Polyhydroxyalkanoic acid (PHA) inclusion bodies were analyzed in situ by C-13-nuclear magnetic resonance (C-13-NMR) spectroscopy. The PHA inclusion bodies studied were composed of poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-4-hydroxybutyrate), which was accumulated in Hydrogenophaga pseudoflava, and medium-chain-length PHA (MCL-PHA), which was accumulated in Pseudomonas fluorescens BM07 from octanoic acid or 11-phenoxyundecanoic acid (11-POU). The quanti fication of the C-13-NMR signals was conducted against a standard compound, sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). The chemical shift values for the in vivo NMR spectral peaks agreed well with those for the corresponding purified PHA polymers. The intracellular degradation of the PHA inclusions by intracellular PHA depolymerase(s) was monitored by in vivo NMR spectroscopy and analyzed in terms of first-order reaction kinetics. The H. pseudoflava cells were washed for the degradation experiment, transferred to a degradation medium without a carbon source, but containing 1.0 g/l ammonium sulfate, and cultivated at 35 degrees C for 72 h. The in vivo NMR spectra were obtained at 70 degrees C for the short-chain-length PHA cells whereas the spectra for the aliphatic and aromatic MCL-PHA cells were obtained at 50 degrees C and 80 degrees C, respectively. For the H. pseudoflava cells, the in vivo NMR kinetics analysis of the PHA degradation resulted in a first-order degradation rate constant of 0.075/h (r(2)=0.94) for the initial 24 h of degradation, which was close to the 0.050/h determined when using a gas chromatographic analysis of chloroform extracts of sulfuric acid/methanol reaction mixtures of dried whole cells. Accordingly, it is suggested that in vivo C-13-NMR spectroscopy is an important tool for studying intracellular PHA degradation in terms of kinetics. 

PHA degradation kinetics; in vivo C-13-NMR; bacteria; PHA