Characterization of ML-005, a novel metaproteomics-derived esterase
- A novel gene encoding for a lipolytic enzyme, designated ML-005, was recently identified using a functional metaproteomics approach. We heterologously expressed this protein in \(\textit {Escherichia coli}\) and biochemically characterized it. ML-005 exhibited lipolytic activity toward short-chained substrates with the preferred substrate being p-nitrophenyl-butyrate, suggesting that ML-005 is an esterase. According to homology analysis and site-directed mutagenesis, the catalytic triad of the enzyme was identified as Ser-99, Asp-164, and His-191. Its optimal pH was determined to be at pH 8. Optimal activity was observed at 45°C. It also exhibited temperature, pH and salt tolerance. Residual relative activity after incubating at 50–60°C for 360 min was above 80% of its initial activity. It showed tolerance over a broad range of pH (5–12) and retained most of its initial activity. Furthermore, incubating ML-005 in 1 – 5M NaCl solution had negligible effect on its activity. DTT, EDTA, and \(\beta\)-mercaptoethanol had no significant effect on ML-005's activity. However, addition of PMSF led to almost complete inactivation consistent with ML-005 being a serine hydrolase. ML-005 remains stable in the presence of a range of metal ions, but addition of \(Cu^{2+}\) significantly reduces its relative activity. Organic solvents have an inhibitory effect on ML-005, but it retained 21% of activity in 10% methanol. SDS had the most pronounced inhibitory effect on ML-005 among all detergents tested and completely inactivated it. Furthermore, the \(V_{max}\) of ML-005 was determined to be 59.8 \(\mu\)M/min along with a \(K_{m}\) of 137.9 \(\mu\)M. The \(k_{cat}\) of ML-005 is 26 \(s^{-1}\) and \(k_{cat}\)/\(K_{m}\) is 1.88 × \(10^{5}\) \(M^{-1}\) \(s^{-1}\).
Author: | Premankur SukulGND, Natalie LupilovGND, Lars I. LeichertORCiDGND |
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URN: | urn:nbn:de:hbz:294-75770 |
DOI: | https://doi.org/10.3389/fmicb.2018.01925 |
Parent Title (English): | Frontiers in microbiology |
Publisher: | Frontiers Media |
Place of publication: | Lausanne |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2020/10/14 |
Date of first Publication: | 2018/08/22 |
Publishing Institution: | Ruhr-Universität Bochum, Universitätsbibliothek |
Tag: | Open Access Fonds biocatalysis; esterase; lipase; metagenomics; metaproteomics |
Volume: | 9 |
Issue: | Article 1925 |
First Page: | 1925-1 |
Last Page: | 1925-14 |
Note: | Corrigendum: "Corrigendum: Characterization of ML-005, a novel metaproteomics-derived esterase" by Sukul, P., Lupilov, N., and Leichert, L. I. (2018). Front. Microbiol. 9:2716. doi: 10.3389/fmicb.2018.02716 |
Note: | Article Processing Charge funded by the Deutsche Forschungsgemeinschaft (DFG) and the Open Access Publication Fund of Ruhr-Universität Bochum. |
Institutes/Facilities: | Medizinisches Proteom-Center |
Institut für Biochemie und Pathobiochemie, Abteilung Biochemie der Mikroorganismen | |
Protein Research Department | |
open_access (DINI-Set): | open_access |
Licence (English): | Creative Commons - CC BY 4.0 - Attribution 4.0 International |