Chapter title |
Heterologous Expression and Functional Characterization of Sparidae Fish Digestive Phospholipase A2
|
---|---|
Chapter number | 9 |
Book title |
Lipases and Phospholipases
|
Published in |
Methods in molecular biology, August 2018
|
DOI | 10.1007/978-1-4939-8672-9_9 |
Pubmed ID | |
Book ISBNs |
978-1-4939-8671-2, 978-1-4939-8672-9
|
Authors |
Nabil Smichi, Neila Achouri, Alexandre Noiriel, Vincent Arondel, Abdelkarim Abousalham, Youssef Gargouri, Nabil Miled, Ahmed Fendri, Smichi, Nabil, Achouri, Neila, Noiriel, Alexandre, Arondel, Vincent, Abousalham, Abdelkarim, Gargouri, Youssef, Miled, Nabil, Fendri, Ahmed |
Abstract |
In this study, we have produced for the first time a fish phospholipase (PLA2) in heterologous system (E. coli). The Diplodus annularis PLA2 (DaPLA2) was then refolded from inclusion bodies and purified by Ni-affinity chromatography. We used the pH-stat method (with emulsified phosphatidylcholine as substrate) and the monomolecular film technique (using various glycerophospholipids substrates spread in the form of monomolecular films at the air-water interface) to access the biochemical and kinetic properties of the recombinant DaPLA2. The DaPLA2 was found to be active and stable at higher temperatures (37-50 °C) than expected. Interestingly, DaPLA2 hydrolyzes efficiently both purified phosphatidylglycerol and phosphatidylethanolamine at 20 mN/m. These analytical results corroborate with the fact that the catalytic activity of DaPLA2, measured with the pH-stat using egg yolk as substrate, is mainly due to the hydrolysis of the PE fraction present in egg yolk, whereas the phosphatidylglycerol is a hallmark substrate for the most secreted PLA2-IB. |
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