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

Abstract

Two novel esterase genes, est8L and est13L, 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 Archaeoglobus fulgidus 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 p-nitrophenyl butyrate (C4) and p-nitrophenyl caproate (C6), respectively, and Est13L showed a broad substrate specificity for p-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 S-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.

Characterization of a novel thermostable alkaline lipase derived from a compost metagenomic library and its potential application in the detergent industry

“…Comparisons of their enzymatic properties with Lip54q are shown in Table 5. The results in Table 5 show that the optimum pH of Lip54q was higher than those for EstCS1 (Park et al, 2020a), Est56 (Lu and Daniel, 2021), Est1 (Lu et al, 2019), and EstCS3 (Park et al, 2020b) and was equal to those for Est2 (Lu et al, 2019) and Est8L (Park et al, 2021), but lower than that of Est13L (Park et al, 2021). In addition, the optimum temperature for Lip54q was similar to those of most esterases but significantly lower than those for Est1 and Est2 (Lu et al, 2019).…”

“…In recent years, several lipid hydrolases with unique enzymatic characteristics have been identified from composting habitats (Lu et al, 2019;Park et al, 2020aPark et al, ,b, 2021Lu and Daniel, 2021). Although these enzymes belong to different ester hydrolase families, including family IV (Park et al, 2020a(Park et al, , 2021Lu and Daniel, 2021), family V (Lu et al, 2019), and family VIII (Lu et al, 2019;Park et al, 2020bPark et al, , 2021, the optimal substrates for these enzymes are p-nitrophenyl ester substrates with short carbon chains. Therefore, these enzymes are esterases rather than lipases.…”

Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues

“…Chauhan [ 56 ] and Dhanjal, Chopra [ 57 ] reviewed several novel enzymes discovered using a metagenomic approach, including lipases and esterases obtained from different environmental samples. Recently, a cold-active esterase PMGL3 was obtained from the metagenomic DNA library of Siberian permafrost, and other lipase genes have also been isolated and identified from various metagenomic libraries [ 36 , 58 , 59 ]. These studies showed that metagenomics is an effective technique for identifying, extracting, and discovering novel lipolytic enzymes.…”

Biochemical characterization of a family IV esterase with R-form enantioselectivity from a compost metagenomic library

“…They were mixed, digested with a restriction enzyme, cloned with plasmid pUC19, and 18 positive subclones were obtained [19]. By sequencing, nine different lipolytic enzymes were identified, and some of them were reported [19,23,24]. In this study, a positive clone YH-E15 was selected for further study.…”

“…Compost has been selected as an object of metagenomic study because it contains various useful enzymes, such as endoglucanase, xylanase, and esterase [20][21][22]. We selected the compost metagenome and reported the properties of some lipolytic enzymes obtained from the compost metagenomic library, such as Est2K, Est7K, Est8L, and Est13L [19,23,24]. Recently, a family IV esterase from another compost metagenome was reported, and it showed organic solvent stability [25].…”