TY - JOUR

T1 - High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

AU - Schückel, Julia

AU - Kracun, Stjepan Kresimir

AU - Lausen, Thomas Frederik

AU - Willats, William George Tycho

AU - Jørgensen, Bodil

PY - 2017

Y1 - 2017

N2 - A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play an important role in fruit development and ripening processes by modulating the plant cell wall. Knowledge about these enzymes is important for research in fruit development and also important for industry regarding postharvest properties. Although advances in genetic control and cell wall biochemistry have led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy-to-handle, multiplexed and highly reproducible method using CPH assays where different fruits have been screened for enzyme activity. Additionally, the importance and impact of the extraction method and buffer conditions on the assay are investigated. We will show that one experimental setup can be used for testing all enzymes.

AB - A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play an important role in fruit development and ripening processes by modulating the plant cell wall. Knowledge about these enzymes is important for research in fruit development and also important for industry regarding postharvest properties. Although advances in genetic control and cell wall biochemistry have led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy-to-handle, multiplexed and highly reproducible method using CPH assays where different fruits have been screened for enzyme activity. Additionally, the importance and impact of the extraction method and buffer conditions on the assay are investigated. We will show that one experimental setup can be used for testing all enzymes.

U2 - 10.1039/C6AY03431D

DO - 10.1039/C6AY03431D

M3 - Journal article

VL - 9

SP - 1242

EP - 1247

JO - Analytical Methods

JF - Analytical Methods

SN - 1759-9660

ER -