Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense
Abstract
The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0–11.0 after 30 min incuba...tion in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80% of the initial activity after 1 h incubation at 45°C, and 65% of the initial activity after 24 h incubation in 30% dimethyl sulfoxide.
Keywords:
biocatalysis / chiral amines / halophiles / metagenomics / protein expressionSource:
ChemBioChem, 2023, 24, 20Publisher:
- Wiley-VCH GmbH
Funding / projects:
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200026 (University of Belgrade, Institute of Chemistry, Technology and Metallurgy - IChTM) (RS-MESTD-inst-2020-200026)
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200168 (University of Belgrade, Faculty of Chemistry) (RS-MESTD-inst-2020-200168)
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200053 (University of Belgrade, Institute for Multidisciplinary Research) (RS-MESTD-inst-2020-200053)
Collections
Institution/Community
Institut za multidisciplinarna istraživanjaTY - JOUR AU - Kalicanin, Nevena AU - Balaz, Ana Marija AU - Prodanović, Olivera AU - Prodanović, Radivoje PY - 2023 UR - http://rimsi.imsi.bg.ac.rs/handle/123456789/2612 AB - The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0–11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80% of the initial activity after 1 h incubation at 45°C, and 65% of the initial activity after 24 h incubation in 30% dimethyl sulfoxide. PB - Wiley-VCH GmbH T2 - ChemBioChem T1 - Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense IS - 20 IS - e202300414 VL - 24 DO - 10.1002/cbic.202300414 ER -
@article{ author = "Kalicanin, Nevena and Balaz, Ana Marija and Prodanović, Olivera and Prodanović, Radivoje", year = "2023", abstract = "The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0–11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80% of the initial activity after 1 h incubation at 45°C, and 65% of the initial activity after 24 h incubation in 30% dimethyl sulfoxide.", publisher = "Wiley-VCH GmbH", journal = "ChemBioChem", title = "Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense", number = "20, e202300414", volume = "24", doi = "10.1002/cbic.202300414" }
Kalicanin, N., Balaz, A. M., Prodanović, O.,& Prodanović, R.. (2023). Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense. in ChemBioChem Wiley-VCH GmbH., 24(20). https://doi.org/10.1002/cbic.202300414
Kalicanin N, Balaz AM, Prodanović O, Prodanović R. Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense. in ChemBioChem. 2023;24(20). doi:10.1002/cbic.202300414 .
Kalicanin, Nevena, Balaz, Ana Marija, Prodanović, Olivera, Prodanović, Radivoje, "Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense" in ChemBioChem, 24, no. 20 (2023), https://doi.org/10.1002/cbic.202300414 . .