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Stability and Activity of Porcine Lipase Against Temperature and Chemical Denaturants

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Abstract

Lipases are the class of hydrolases with wide industrial applications. The present study analyses the stability of porcine pancreatic lipase (PPL) against urea, guanidine hydrochloride (Gdn), sodium dodecyl sulphate (SDS), and temperature using different spectroscopic techniques. Interestingly, this two-domain protein shows a two-state unfolding transition against urea and Gdn. The free energy of unfolding of PPL calculated from global analysis of the unfolding transitions obtained from different spectroscopic techniques is ~2.2 kcal/mol. In the presence of SDS, PPL shows a cooperative loss of secondary and tertiary structures above 0.2 mM of SDS. At above 2 mM of SDS, PPL forms irreversible, non-native, thermally stable structure. PPL loses its activity even at lower concentrations of urea (3 M), Gdn (0.5 M), and SDS (0.8 mM). Thermal denaturation of PPL shows an irreversible unfolding, and the protein lost its activity even by increasing the temperature to 45 °C. Though PPL in higher concentrations of SDS (>5 mM) shows stable conformation against temperature, its activity is completely lost. The results suggest that the structure and activity of PPL are more sensitive against chemical denaturants and temperature, and forms irreversible, non-native (in SDS) or completely unfolded (in urea, Gdn, and at higher temperature) conformations in different denaturing conditions.

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Abbreviations

ANS:

1-Anilinonaphthalene-8-sulfonic acid

CD:

Circular dichroism

Gdn:

Guanidine hydrochloride

NPhP:

p-Nitrophenyl palmitate

SDS:

Sodium dodecyl sulphate

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Acknowledgments

The work was supported by the Department of Biotechnology, India (Grant BT/PR15029/GBD/27/288/2010).

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Authors and Affiliations

  1. Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, India

    P. Krishna Chaitanya & N. Prakash Prabhu

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  1. P. Krishna Chaitanya
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  2. N. Prakash Prabhu
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Correspondence to N. Prakash Prabhu.

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Chaitanya, P.K., Prabhu, N.P. Stability and Activity of Porcine Lipase Against Temperature and Chemical Denaturants. Appl Biochem Biotechnol 174, 2711–2724 (2014). https://doi.org/10.1007/s12010-014-1220-8

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  • DOI: https://doi.org/10.1007/s12010-014-1220-8

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