Development of a microtiter plate-based assay for the detection of lipase-catalyzed transesterifications in organic solvents.

Martin Pöhnlein,Christoph Syldatk,Tim Finkbeiner,Rudolf Hausmann

doi:10.1007/s10529-014-1721-0

Martin Pöhnlein, Christoph Syldatk + Show 2 more

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https://doi.org/10.1007/s10529-014-1721-0

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Abstract

A microtiter plate-based assay was developed to evaluate the ability of lipases to perform transesterifications when employed in different organic solvents. A 4-nitrophenol assay was carried out employing seven different lipase formulations and two fatty acid methyl esters with different chain lengths in a total of six organic solvents with logP values approximately between 1 and −1. This assay delivered results within comparatively short times measured by a color reaction and thus facilitates the choice of an enzyme-solvent combination for the synthesis of glycolipids. To validate the findings, glycolipid syntheses were performed using the same lipase formulation in the same solvents. When comparing the results obtained using the microtiter plate-based assay to the results of the glycolipid syntheses using the same lipases and solvents, matching results were obtained.

Highlights

Lipases (E.C. 3.1.1.3, triacylglycerol acyl hydrolases) are hydrolases that act on ester bonds
If the tested lipase exhibited transesterification activity, it could be determined from the colorimetric reaction
Subsequent calculation of the initial transesterification activity allowed the comparison between the different lipase—solvent combinations

Summary

Introduction

Lipases (E.C. 3.1.1.3, triacylglycerol acyl hydrolases) are hydrolases that act on ester bonds. In order to examine lipase activity in organic solvents, several assays have been developed or existing assays have been refined and modified to determine the lipases synthetic activities as well (Furutani et al 1995; Pencreac’h and Baratti 1996) These assays are carried out in unpolar solvents like hexane or heptane (Goujard et al 2009; Teng and Xu 2007). These solvents are not suitable to monitor glycolipid syntheses, since sugars do not dissolve in them To circumvent this problem a 4-nitrophenol (paranitrophenol, pNP) assay has been developed to determine the synthetic activity of different lipases in organic solvents with a lower logP value ranging from approximately 1 to -1, which is described within this work

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