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Cloning, Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome

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Abstract

The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6–7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.

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Acknowledgments

Authors thank Department of Science and Technology, India for providing Innovation in Science Pursuit for Inspired Research (INSPIRE) fellowship to AP. The Centre for Advanced studies in Functional Genomics, Centre for Excellence in Genomic Sciences, and the Networking Resource Centre in Biological Sciences at Madurai Kamaraj University are highly acknowledged for the support facilities. The School of Chemistry, MKU is gratefully acknowledged for providing the circular dichroism facility.

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Authors and Affiliations

  1. Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, India

    Arivaradarajan Preeti, Devaraj Hemalatha, Jeyaprakash Rajendhran & Paramasamy Gunasekaran

  2. Division of Microbial Diseases, UCL Eastman Dental Institute, London, UK

    Peter Mullany

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  1. Arivaradarajan Preeti
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  2. Devaraj Hemalatha
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  3. Jeyaprakash Rajendhran
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  4. Peter Mullany
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  5. Paramasamy Gunasekaran
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Correspondence to Paramasamy Gunasekaran.

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Preeti, A., Hemalatha, D., Rajendhran, J. et al. Cloning, Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome. Indian J Microbiol 54, 284–292 (2014). https://doi.org/10.1007/s12088-014-0455-y

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  • DOI: https://doi.org/10.1007/s12088-014-0455-y

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