| Chapter title |
A Microtiter Plate-Based Assay to Screen for Active and Stereoselective Hydrolytic Enzymes in Enzyme Libraries.
|
|---|---|
| Chapter number | 11 |
| Book title |
Metagenomics
|
| Published in |
Methods in molecular biology, January 2017
|
| DOI | 10.1007/978-1-4939-6691-2_11 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6689-9, 978-1-4939-6691-2
|
| Authors |
Dominique Böttcher, Patrick Zägel, Marlen Schmidt, Uwe T. Bornscheuer |
| Editors |
Wolfgang R. Streit, Rolf Daniel |
| Abstract |
A procedure for the high-throughput screening (HTS) of esterases is described. This includes a pretest for discrimination of active and inactive clones using an agar plate overlay assay, the enzyme expression in microtiter plates and the measurement of activity and enantioselectivity (E) of the esterase variants using acetates of secondary alcohols as model substrates. Acetic acid released is converted in an enzyme cascade leading to the stoichiometric formation of NADH, which is quantified in a spectrophotometer. The method allows screening of several thousand mutants per day and has already been successfully applied to identify an esterase mutant with an E > 100 towards an important building block for organic synthesis. This protocol can also be used for lipases and possibly other hydrolases that are expressed in soluble form in conventional E. coli strains. |
Mendeley readers
Geographical breakdown
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|---|---|---|
| Unknown | 15 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 3 | 20% |
| Student > Ph. D. Student | 2 | 13% |
| Unspecified | 1 | 7% |
| Student > Doctoral Student | 1 | 7% |
| Librarian | 1 | 7% |
| Other | 2 | 13% |
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| Arts and Humanities | 1 | 7% |
| Unspecified | 1 | 7% |
| Chemical Engineering | 1 | 7% |
| Other | 2 | 13% |
| Unknown | 5 | 33% |