A spectrophotometric transesterification-based assay for lipases in organic solvent.
A new method to evaluate lipase activities in nonaqueous conditions using vinyl ester absorbance at ultraviolet (UV) wavelengths is described. The model reaction is the transesterification between vinyl stearate and pentanol in hexane at 30 degrees C or in decane at 50 degrees C. The conversion of vinyl stearate into pentyl stearate is monitored through decreasing UV absorbance at 200 nm. Six commercial lipases were tested with this method, and results were compared with gas chromatography (GC) quantification and a classical spectrophotometric method using p-nitrophenyl palmitate. Results from the new spectrophotometric assay are similar both to results from GC quantification (R(2)=0.999) and to results from p-nitrophenyl palmitate (R(2)=0.989). The proposed method is able to evaluate both high activity from immobilized lipases such as immobilized Candida antarctica B lipase (3060 +/- 350 U g(-1)) and low activity from crude enzymatic extracts such as Carica papaya dried latex (0.1 +/- 0.04 U g(-1)). The method has also been used to measure kinetic parameters of C. antarctica B lipase for vinyl stearate and the correlation between its synthesis activity and its concentration. The method has also proved to be effective in studying the acyl selectivity of a lipase by comparing its activities with increasing chain lengths of vinyl esters.
Goujard
L
L
Equipe d'Ecologie Microbienne, Institut Méditerranéen d'Ecologie et de Paléoécologie (IMEP), UMR CNRS, Université Paul Cézanne Aix-Marseille III, Faculté des Sciences et Techniques de St Jérôme, 13397 Marseille Cedex 20, France. laurent.goujard@yahoo.com
Villeneuve
P
P
Barea
B
B
Lecomte
J
J
Pina
M
M
Claude
S
S
Le Petit
J
J
Ferré
E
E
D004789:Enzyme Activation; D004951:Esterification; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D000439:Pentanols / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013228:Stearates / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D014753:Vinyl Compounds / Q000737:chemistry*
D000439:Pentanols; D012997:Solvents; D013228:Stearates; D014753:Vinyl Compounds; D008049:Lipase