Molecular Characterization of Novel Family IV and VIII Esterases from a Compost Metagenomic Library

• Main Authors: Jong-Eun Park, Geum-Seok Jeong, Hyun-Woo Lee, Hoon Kim
• Format: Article
• Language: English
• Published: MDPI AG 2021-07-01
• Series: Microorganisms
• Subjects: compost metagenomic library; family IV esterase; family VIII esterase; glyceryl tributyrate hydrolysis; oligomeric native form; organic solvent stability
• Online Access: https://www.mdpi.com/2076-2607/9/8/1614

Two novel esterase genes, <i>est</i>8L and <i>est</i>13L, were isolated and identified from a compost metagenomic library. The encoded Est8L and Est13L had molecular masses of 33,181 and 44,913 Da consisting of 314 and 411 amino acids, respectively, without signal peptides. Est8L showed the highest identity (32.9%) to a hyper-thermophilic carboxylesterase AFEST from <i>Archaeoglobus</i> <i>fulgidus</i> compared to other esterases reported and was classified to be a novel member of family IV esterases with conserved regions such as HGGG, DY, GXSXG, DPL, and GXIH. Est13L showed the highest identity (98.5%) to the family VIII esterase Est7K from the metagenome library. Est8L and Est13L had the highest activities for <i>p</i>-nitrophenyl butyrate (C4) and <i>p</i>-nitrophenyl caproate (C6), respectively, and Est13L showed a broad substrate specificity for <i>p</i>-nitrophenyl substrates. Est8L and Est13L effectively hydrolyzed glyceryl tributyrate. The optimum temperatures for activities of Est8L and Est13L were identical (40 °C), and the optimum pH values were 9.0 and 10.0, respectively. Est13L showed higher thermostability than Est8L. Sephacryl S-200 HR chromatography showed that the native form of Est8L was a dimer. Interestingly, Est13L was found to be a tetramer, contrary to other family VIII esterases reported. Est8L was inhibited by 30% isopropanol, methanol, and acetonitrile; however, Est13L was activated to 182.9% and 356.1%, respectively, by 30% isopropanol and methanol. Est8L showed enantioselectivity for the <i>S</i>-form, but Est13L showed no enantioselectivity. These results show that intracellular Est8L and/or Est13L are oligomeric in terms of native forms and can be used for pharmaceutical and industrial applications with organic solvents under alkaline conditions.