TY  - CHAP
AU  - Fulton, Alexander
AU  - Hayes, Marc R.
AU  - Schwaneberg, Ulrich
AU  - Pietruszka, Jörg
AU  - Jaeger, Karl-Erich
A3  - Bornscheuer, Uwe T.
A3  - Höhne, Matthias
TI  - High-Throughput Screening Assays for Lipolytic Enzymes
VL  - 1685
CY  - New York, NY
PB  - Springer New York
M1  - FZJ-2018-00538
SN  - 978-1-4939-7364-4 (print)
T2  - Methods in Molecular Biology
SP  - 209 - 231
PY  - 2018
AB  - Screening is defined as the identification of hits within a large library of variants of an enzyme or protein with a predefined property. In theory, each variant present in the respective library needs to be assayed; however, to save time and consumables, many screening regimes involve a primary round to identify clones producing active enzymes. Such primary or prescreenings for lipolytic enzyme activity are often carried out on agar plates containing pH indicators or substrates as triolein or tributyrin. Subsequently, high-throughput screening assays are usually performed in microtiter plate (MTP) format using chromogenic or fluorogenic substrates and, if available, automated liquid handling robotics. Here, we describe different assay systems to determine the activity and enantioselectivity of lipases and esterases as well as the synthesis of several substrates. We also report on the construction of a complete site saturation library derived from lipase A of Bacillus subtilis and its testing for detergent tolerance. This approach allows for the identification of amino acids affecting sensitivity or resistance against different detergents.
LB  - PUB:(DE-HGF)7
C6  - pmid:29086311
UR  - <Go to ISI:>//WOS:000684272200013
DO  - DOI:10.1007/978-1-4939-7366-8_12
UR  - https://juser.fz-juelich.de/record/842291
ER  -