Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications
| Autor: | Evgeny Weinberg, Tamar Ansbacher, Ran Tohar, Maayan Gal, Inbal Sher, Livnat Afriat-Jurnou |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
QH301-705.5
Protein design Article Catalysis Inorganic Chemistry Bacterial collagenase directed enzyme evolution Bacterial Proteins Escherichia coli medicine Physical and Theoretical Chemistry Direct evaluation Biology (General) bacterial lysate screening Molecular Biology QD1-999 protein expression Spectroscopy Collagenase activity chemistry.chemical_classification Organic Chemistry General Medicine Recombinant Proteins molecular dynamics Computer Science Applications collagenase Chemistry Microbial Collagenase Enzyme Biochemistry chemistry Collagenase Collagen enzymatic assay Directed Molecular Evolution Genetic library Clostridium histolyticum Bacterial lysate medicine.drug |
| Zdroj: | International Journal of Molecular Sciences Volume 22 Issue 16 International Journal of Molecular Sciences, Vol 22, Iss 8552, p 8552 (2021) |
| ISSN: | 1422-0067 |
| DOI: | 10.3390/ijms22168552 |
| Popis: | Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity. |
| Databáze: | OpenAIRE |
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