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Biochemical Characterization and Molecular Modeling of Pancreatic Lipase from a Cartilaginous Fish, the Common Stingray (Dasyatis pastinaca)

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Abstract

In order to identify fish enzymes displaying novel biochemical properties, we have chosen the common stingray (Dasyatis pastinaca), one of the most primitive living jawed aquatic vertebrates as a starting biological material to purify a lipase. A stingray pancreatic lipase (SPL) was purified from delipidated pancreatic powder. The SPL molecular weight was around 55 kDa which is slightly higher than that of known classical pancreatic lipases (50 kDa). This increase in the molecular weight was due to glycosylation. Like classic pancreatic lipases, SPL was found to be much more active on short-chain triacylglycerols than on long-chain ones. Natural detergents act as inhibitors of the SPL activity. This inhibition can be reversed by the addition of stingray colipase. Starting from total pancreatic messenger RNAs (mRNAs), partial stingray pancreatic lipase complementary DNA (cDNA) was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR) and cloned into the PGEM-T vector. Partial amino acid sequence of the SPL was homologous to that of Japanese eel, porcine, and human pancreatic lipases. A 3D structure model of the sequenced part of SPL was built using the 3D structure of porcine pancreatic lipase as template, since both lipases shared an amino acid sequence identity of 60 %.

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Abbreviations

AG:

Arabic gum

BSA:

Bovine serum albumin

DTNB:

5,5′-Dithiobis-(2-nitrobenzoïc acid)

HPL:

Human pancreatic lipase

SPL:

Stingray pancreatic lipase

PPL:

Porcine pancreatic lipase

APL:

Anguilla pancreatic lipase

NaDC:

Sodium deoxycholate

NaTDC:

Sodium taurodeoxycholate

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TC3 :

Tripropionin

TC4 :

Tributyrin

TC8 :

Trioctanoin

TPL:

Turkey pancreatic lipase

TX-100:

Triton X-100

RT-PCR:

Reverse transcriptase-polymerase chain reaction

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Acknowledgments

This work received financial support from the Tunisian Ministry of Higher Education and Scientific Research granted to the « Laboratoire de Biochimie et de Génie Enzymatique des Lipases ». Special thanks to Pr. H. Mejdoub (FSS, Tunisia) for the sequencing of the NH2-terminal of SPL. We do thank Dr. Angela Perrin from Hoffman-La Roche for providing us with orlistat.

Compliance with Ethical Standards

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We also confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.

The manuscript does contain experiments using animals. The experimental protocols and procedures used in the present work were approved by the Ethics Committee of the University of Sfax (Sfax, Tunisia) for the care and use of laboratory animals.

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Authors and Affiliations

  1. Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS, Route Soukra, BPW 1173-3038, Sfax, Tunisia

    Emna Bouchaâla, Madiha BouAli, Yassine Ben Ali, Nabil Miled, Youssef Gargouri &  Ahmed Fendri

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  1. Emna Bouchaâla
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  2. Madiha BouAli
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  3. Yassine Ben Ali
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  4. Nabil Miled
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  5. Youssef Gargouri
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  6. Ahmed Fendri
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Correspondence to Ahmed Fendri.

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Bouchaâla, E., BouAli, M., Ali, Y.B. et al. Biochemical Characterization and Molecular Modeling of Pancreatic Lipase from a Cartilaginous Fish, the Common Stingray (Dasyatis pastinaca). Appl Biochem Biotechnol 176, 151–169 (2015). https://doi.org/10.1007/s12010-015-1564-8

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