Molecular characterization of Pseudomonas sp. LDC-5 involved in accumulation of poly 3-hydroxybutyrate and medium-chain-length poly 3-hydroxyalkanoates

Abstract

Polyhydroxyalkanoates (PHAs) are biological polyesters, of which, Short-Chain-Length-Medium-Chain-Length (SCL-MCL) PHA copolymers are important because of their wide range of applications. The present study focused on molecular characterization of Pseudomonas sp. LDC-5 that is identified as SCL-MCL producer. Phase contrast, fluorescent and electron microscopic observation confirmed the presence of PHA granules in Pseudomonas sp. LDC-5. PCR analysis indicated the presence of expected amplicon for SCL phaC gene ( approximately 500 bp), MCL phaC1 with phaZ ( approximately 1.3), and phaC2 with phaZ ( approximately 1.5 kb). Sequence analysis of the PHA synthase gene of Pseudomonas sp. LDC-5 revealed significant differences in phaC1 and phaC2 which were further confirmed by recombinant studies. Recombinant Escherichia coli harboring the partial phaC1 gene was able to accumulate PHA, whereas E. coli with phaC2 did not accumulate PHA as verified by fold analysis, immunoblotting, Gas Chromatography (GC), Differential scanning calorimetry (DSC), and FTIR studies. The predicted theoretical three-dimensional structure revealed that PhaC1 is consistent with alpha/beta hydrolase fold. Monomer composition showed the presence of monomer ranging from C4 to C12: 1 when glucose and sodium octanoate fed as the carbon source. DSC revealed melting temperature peak at 153.12 degrees C and glass transition (T(g)) peaks at -0.37 degrees C. Thermogravimetric analysis revealed that the polymer was stable up to 276 degrees C. Fourier Transform Infrared Spectroscopy (FT-IR) spectral analysis showed the PHA specific wave number at 1,739.67 and 1,161.07 cm(-1). The potential of Pseudomonas sp. LDC-5 and its properties are discussed.