A high‐throughput assay for the determination of acyltransferase activity of lipase CAL‐A

• Published in: European Journal of Lipid Science and Technology
• Main Authors: Müller, Janett, Fredrich, Birte, Kohlmann, Christina, Maksym, Lukas, Bornscheuer, Uwe T.
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2013
• Subjects: Antarc*; Antarctica
• Online Access: http://dx.doi.org/10.1002/ejlt.201300226; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fejlt.201300226; https://onlinelibrary.wiley.com/doi/pdf/10.1002/ejlt.201300226; https://onlinelibrary.wiley.com/doi/full-xml/10.1002/ejlt.201300226

Certain lipases such as Candida antarctica lipase A (CAL‐A) are known to possess acyltransferase activity, which allows ester synthesis even in the presence of water. Discovery of novel lipases and identification of lipase variants with improved acyltransferase activity is laborious using standard methods such as gas chromatography. To overcome this limitation, an enzyme cascade was established based on quantification of the alcohol consumed in fatty acid ester synthesis from plant oils. For this, an alcohol oxidase converts the remaining alcohol to the corresponding aldehyde with concomitant formation of hydrogen peroxide. This is then quantified by the action of horseradish peroxidase leading to the oxidation of the co‐substrate ABTS, which is determined spectrophotometrically at 695 nm. The conversions thus determined correspond well to data measured by off‐line gas chromatography analysis and the assay could be adapted to the microtiterplate format. Practical applications: Fatty acid esters are valuable compounds in various industrial applications, i.e., fatty acid methyl esters are used as biofuels and ethyl esters can serve as fragrance or flavoring compounds in the cosmetic industry. The enzymatic synthesis of these compounds is possible, but often enzymes need to be adapted to meet process requirements. This assay simplifies the identification of suitable enzymes. Moreover, substrate specificities of a lipase for a certain alcohol or acyl acceptor can also be easily studied with this assay.