Accéder au contenu
Merck

Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region.

Biochimica et biophysica acta (2014-12-03)
Jannet Kamoun, Mathieu Schué, Wala Messaoud, Justine Baignol, Vanessa Point, Eduardo Mateos-Diaz, Pascal Mansuelle, Youssef Gargouri, Goetz Parsiegla, Jean-François Cavalier, Frédéric Carrière, Ahmed Aloulou
RÉSUMÉ

Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.

MATÉRIAUX
Référence du produit
Marque
Description du produit

Sigma-Aldrich
Acétonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Acide trifluoroacétique, ReagentPlus®, 99%
Sigma-Aldrich
Acide trifluoroacétique, suitable for HPLC, ≥99.0%
Sigma-Aldrich
Acétonitrile, HPLC Plus, ≥99.9%
Sigma-Aldrich
Acide phosphorique, ACS reagent, ≥85 wt. % in H2O
Sigma-Aldrich
sulfate de magnésium, anhydrous, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Iode, ACS reagent, ≥99.8%, solid
Sigma-Aldrich
Acétonitrile, anhydrous, 99.8%
Sigma-Aldrich
Acétonitrile, ACS reagent, ≥99.5%
Sigma-Aldrich
Acide phosphorique, 85 wt. % in H2O, 99.99% trace metals basis
Sigma-Aldrich
Acétonitrile, for HPLC, for UV, ≥99.9% (GC)
Sigma-Aldrich
Acide trifluoroacétique, puriss. p.a., suitable for HPLC, ≥99.0% (GC)
Sigma-Aldrich
Chlorure de calcium solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Acide phosphorique, ACS reagent, ≥85 wt. % in H2O
Sigma-Aldrich
Acétonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
sulfate de magnésium, anhydrous, reagent grade, ≥97%
Sigma-Aldrich
Acide oléique, technical grade, 90%
Sigma-Aldrich
Acide phosphorique, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥85%
Sigma-Aldrich
Calcium chloride, anhydrous, BioReagent, suitable for insect cell culture, suitable for plant cell culture, ≥96.0%
Sigma-Aldrich
Iode, flakes, ReagentPlus®, ≥99%
Sigma-Aldrich
Acide phosphorique, puriss. p.a., crystallized, ≥99.0% (T)