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

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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 (∼500 bp), MCL phaC1 with phaZ (∼1.3), and phaC2 with phaZ (∼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 α/β 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°C and glass transition (Tg) peaks at −0.37°C. Thermogravimetric analysis revealed that the polymer was stable up to 276°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.

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Abbreviations

3-D:

Three-dimensional

BCIP:

5-bromo-4-chloro-3-indolylphosphate

BLAST:

Basic local alignment search tool

EMBL:

European Molecular Biology Laboratory

EXPASY:

Expert Protein Analysis System

FASTA:

Fast Alignment Search Tool A

FT-IR:

Fourier transform infrared spectroscopy

GC:

Gas chromatography

MCL:

Medium chain length

NBT:

Nitro blue tetrazolium

PVDF:

Polyvinylidene difluoride

RMSD:

Root mean square deviation

SCL:

Short chain length

TEM:

Transmission electron microscopy

Tg :

Glass transition temperature (°C)

Tm :

Melting temperature (°C)

TrEMBL:

Translated EMBL

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Acknowledgments

The authors are grateful to Dr. Kumar Sudesh (Universiti Sains Malaysia, School of Biological Sciences) for his generous help in Gas Chromatography (GC) analysis. The authors thank Dr. A. H. Bishop, Greenwich University, London for TEM microscopy, Dr. S. Muthusubramaniam and Dr. Sivakolunthu, Madurai Kamaraj University, School of Chemistry, Madurai and Mrs. Geetha sivasubramaniam for their help in spectral studies. The authors express their gratitude to the Department of Science and Technology (DST-WOS-ANo: No.SR/WOS-A/LS-137/2004), New Delhi for its financial support to complete this work.

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  1. PG and Research Department of Zoology and Biotechnology, Lady Doak College, Madurai, 625 002, Tamilnadu, India

    Kabilan Sujatha, Ayyasamy Mahalakshmi & Rajaiah Shenbagarathai

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  1. Kabilan Sujatha
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  2. Ayyasamy Mahalakshmi
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Sujatha, K., Mahalakshmi, A. & Shenbagarathai, R. Molecular characterization of Pseudomonas sp. LDC-5 involved in accumulation of poly 3-hydroxybutyrate and medium-chain-length poly 3-hydroxyalkanoates. Arch Microbiol 188, 451–462 (2007). https://doi.org/10.1007/s00203-007-0265-2

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