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Characterization and evolution of a metagenome-derived lipase towards enhanced enzyme activity and thermostability

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Abstract

In the present investigation, we used directed evolution approach to engineer a lipase from metagenomic origin. A variant S311C, was generated, characterized in detail and compared with wild type. Wild type and variant lipases were overexpressed and purified to homogeneity. The temperature optima of the purified lipases (Variant and wild type) were almost same, and found to be 45 and 50 °C, respectively. The variant protein was highly thermostable (54 times) as compared with the wild type at 60 °C. The variant displayed very high kinetic efficiency over the wild type protein. Analysis of the homology models of wild type and variant lipase showed that the substitution is on the surface of the protein. This substitution, along with hydrophobic residues in near vicinity may be involved in formation of strong hydrophobic channel leading to active site. This study identifies the role of hydrophobic interactions in protein stability along with enhancement of enzyme activity.

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Acknowledgments

Author acknowledges the financial assistance to Dr. Jagdeep Kaur from the Department of Science and Technology, New Delhi, India.

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Authors and Affiliations

  1. Department of Biotechnology, Sector 14, Panjab University, Chandigarh, 160014, India

    Rakesh Kumar, Monika Sharma & Jagdeep Kaur

  2. National Centre for Human Genome Studies and Research, Panjab University, Chandigarh, 160014, India

    Ranvir Singh

Authors
  1. Rakesh Kumar
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  2. Monika Sharma
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  3. Ranvir Singh
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  4. Jagdeep Kaur
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Corresponding author

Correspondence to Jagdeep Kaur.

Additional information

Ranvir Singh and Jagdeep Kaur contributed equally to this study.

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Supplementary material 1 (DOC 76 kb)

11010_2012_1483_MOESM2_ESM.doc

SD Fig-1: Showing multiple sequence alignment of Wild type (pJKR1), Uncultured bacterium (JKP01), BSL, BSP and SF1 having accession no- AEL99899, ACJ07039, AAC12257, AAF40217 and AAY82869 respectively. Active site residues have been shown in blue colours (Ser is in conserved pentapeptide (AHSQG)). Signal peptide is shown is yellow colour. (DOC 32 kb)

11010_2012_1483_MOESM3_ESM.doc

SD Fig-2: Hydropathy plots of the proteins LipR1 (5A) and LipR2 (5B). Blue lines denote Hydropathy. Red lines denote Amphipathicity. The circles are showing region of change in hydropathy due to change in amino acid S311C. (DOC 230 kb)

SD Fig-3 Model quality of the Wild type (LipR1) and Variant (LipR2) using PROSA server. (DOC 171 kb)

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Kumar, R., Sharma, M., Singh, R. et al. Characterization and evolution of a metagenome-derived lipase towards enhanced enzyme activity and thermostability. Mol Cell Biochem 373, 149–159 (2013). https://doi.org/10.1007/s11010-012-1483-8

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  • DOI: https://doi.org/10.1007/s11010-012-1483-8

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