title: Expression, purification and biochemical characterization of a family 6 carboxylesterase from Methylococcus capsulatus (bath).
abstract: The genome of Methylococcus capsulatus (bath) encodes a protein R-est6 that is annotated as a lipase family 3 protein. The phylogenetic and the sequence analyses linked this protein to the family 6 carboxylesterase. The gene encoding R-est6 was cloned and overexpressed in Escherichia coli and the recombinant 6x-His tagged protein was purified by Ni-NTA affinity chromatography. The buffers used in the purification were modified by adding 1% glycerol instead of the salt to prevent the protein aggregation. Far UV-CD spectrum and gel filtration chromatography of the purified R-est6 confirmed that the protein was well folded like a typical α/β hydrolase and had the quaternary structure of a tetramer, in addition to a compact monomer. The optimum pH was in the range of 7.0-9.0 and the optimum temperature was at 55 °C for the hydrolysis of pNP-butyrate. As expected, being a member of the family 6 carboxylesterase, R-est6 hydrolyzed triglycerides, pNP esters of the small and the medium fatty acid chain esters and an aryl ester-phenyl acetate. However, R-est6 was also found to hydrolyze the long-chain fatty acid ester which had never been reported for the family 6 carboxylesterase. Additionally, R-est6 was stable and active in the different water-miscible organic solvents. Therefore, the broad substrate range and the structural stability of R-est6 would be advantageous for its application in industrial processes.
authors.affiliation: DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (East), Mumbai, 400019, Maharashtra, India.
authors.forename: Surabhi
authors.initials: S
authors.lastname: Soni
authors.affiliation: DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (East), Mumbai, 400019, Maharashtra, India.
authors.forename: Annamma A
authors.initials: AA
authors.lastname: Odaneth
authors.affiliation: DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (East), Mumbai, 400019, Maharashtra, India.
authors.forename: Arvind M
authors.initials: AM
authors.lastname: Lali
authors.affiliation: DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (East), Mumbai, 400019, Maharashtra, India. Electronic address: sk.chandrayan@ictmumbai.edu.in.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Chandrayan
mesh_terms: D000595:Amino Acid Sequence; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D020619:Methylococcus capsulatus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D043182:Carboxylesterase
keywords: Family 6 carboxylesterase; Lipase family 3 protein; R-est6; Triglycerides; α/β hydrolase