title: Characterization and immobilization of a novel SGNH hydrolase (Est24) from Sinorhizobium meliloti.
abstract: A novel oligomeric SGNH hydrolase (Est24) from Sinorhizobium meliloti was identified, actively expressed in Escherichia coli, characterized, and immobilized for industrial application. Sequence analysis of Est24 revealed a putative catalytic triad (Ser¹³-Asp¹⁶³-His¹⁶⁹), with moderate homology to other SGNH hydrolases. Est24 was more active toward short-chain esters, such as p-nitrophenyl acetate, butyrate, and valerate, while the S13A mutant completely lost its activity. Moreover, the activity of Est24 toward α- and β-naphthyl acetate, and enantioselectivity on (R)- and (S)-methyl-3-hydroxy-2-methylpropionate were tested. Est24 exhibited optimum activity at mesophilic temperature ranges (45-55 °C), and slightly alkaline pH (8.0). Structural and mutagenesis studies revealed critical residues involved in the formation of a catalytic triad and substrate-binding pocket. Cross-linked enzyme aggregates (CLEAs) of Est24 with and without amyloid fibrils were prepared, and amyloid fibril-linked Est24 with amyloid fibrils retained 83 % of its initial activity after 1 h of incubation at 60 °C. The high thermal stability of immobilized Est24 highlights its potential in the pharmaceutical and chemical industries.
authors.affiliation: Department of Molecular Science and Technology, Graduate School of Interdisciplinary Programs, Ajou University, Suwon 443-749, South Korea.
authors.forename: Song Yi
authors.initials: SY
authors.lastname: Bae
authors.forename: Bum Han
authors.initials: BH
authors.lastname: Ryu
authors.forename: Eunjin
authors.initials: E
authors.lastname: Jang
authors.forename: Seulgi
authors.initials: S
authors.lastname: Kim
authors.forename: T Doohun
authors.initials: TD
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D016962:Sinorhizobium meliloti / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D006867:Hydrolases