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 × \(10^{5}\) \(M^{-1}\) \(s^{-1}\).

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Metadaten
Author:Premankur SukulGND, Natalie LupilovGND, Lars I. LeichertORCiDGND
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):License LogoCreative Commons - CC BY 4.0 - Attribution 4.0 International