This title appears in the Scientific Report :
2018
Please use the identifier:
http://dx.doi.org/10.1007/978-1-4939-7366-8_12 in citations.
High-Throughput Screening Assays for Lipolytic Enzymes
High-Throughput Screening Assays for Lipolytic Enzymes
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 rou...
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Personal Name(s): | Bornscheuer, Uwe T. (Editor) |
---|---|
Höhne, Matthias (Editor) / Fulton, Alexander / Hayes, Marc R. / Schwaneberg, Ulrich / Pietruszka, Jörg / Jaeger, Karl-Erich | |
Contributing Institute: |
Institut für Molekulare Enzymtechnologie (HHUD); IMET Biotechnologie; IBG-1 Institut für Bioorganische Chemie (HHUD); IBOC |
Published in: |
Protein Engineering / Bornscheuer, Uwe T. (Editor) ; New York, NY : Springer New York, 2018, Chapter 12 ; ISSN: 1064-3745=1940-6029 ; ISBN: 978-1-4939-7364-4=978-1-4939-7366-8 ; doi:10.1007/978-1-4939-7366-8 |
Imprint: |
New York, NY
Springer New York
2018
|
Physical Description: |
209 - 231 |
ISBN: |
978-1-4939-7364-4 (print) 978-1-4939-7366-8 (electronic) |
PubMed ID: |
29086311 |
DOI: |
10.1007/978-1-4939-7366-8_12 |
Document Type: |
Contribution to a book |
Research Program: |
Biotechnology |
Series Title: |
Methods in Molecular Biology
1685 |
Publikationsportal JuSER |
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. |