Characterization of a Novel Moderately Thermophilic Solvent-Tolerant Esterase Isolated From a Compost Metagenome Library

Park, Jimin; Kang, Chul-Hyung; Won, Sung‐Min; Oh, Ki-Hoon; Yoon, Jung‐Hoon

doi:10.3389/fmicb.2019.03069

Cited by 28 publications

(23 citation statements)

References 55 publications

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“…In comparison with family IV esterases from similar compost metagenomic sources, Est8L had poor organic solvents stabilities, whereas EstCs1 from a compost metagenomic library was stable at 30% isopropanol, ethanol, acetonitrile, acetone, methanol, dimethyl formamide, and dimethyl sulfoxide [10]. In addition, Est8L was optimally active at pH 10.0, whereas EstCs1 was optimally active at pH 8.0 [10]. In enantioselectivity, Est8L hydrolyzed the S-form with 10.1% higher activity than the R-form, suggesting preferences for the S-form, whereas Est13L showed no selectivity for the enantiomers (Figure 12).…”

Section: Discussionmentioning

confidence: 97%

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

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 97%

“…Family IV and VIII esterases have been practically interested in the usage of ester synthesis, transesterification reactions, and hydrolysis of antibiotics [ 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…As a result of industrial bioconversion catalyzed by lipolytic enzymes are usually conducted in non-aqueous medium (Oh et al 2012), researchers are dedicated to search for solvent-tolerant enzymes (Zarafeta et al 2016;Park et al 2020) or adopt directed evolutionary approach to acquire stable enzymes in hydrophilic organic solvents (Hyun et al 2012). EstQ7 showed moderate tolerance to a range of organic solvents (Table 2).…”

Section: Discussionmentioning

confidence: 99%

“…Metagenomic strategy provides an access to genomes of the uncultured microorganisms skipping culturing microorganisms and have been applied to screen novel biocatalysts for industrial applications in the past decades (Handelsman 2004). Various novel biocatalysts have been successfully identi ed from soil metagenomic library, such as lipolytic enzymes (Li et al 2018;Park et al 2020), proteases (Gong et al 2017), cellulases (Garg et al 2016;Yang et al 2016), and novel natural products (Katz et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of a novel carboxylesterase EstQ7 from a soil metagenomic library

et al. 2021

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A novel lipolytic gene, estq7, was identi ed from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42 kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174-Asp306-His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17 mM and 1910 S -1 , respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Homology modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/β hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking reveled the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property, and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.

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“…It can catalyze the breaking of ester bonds to form the corresponding alcohols and acids with water molecules [10]. Esterases are distributed in plants, animals, and microorganisms, and among them the microbial esterases are found to be more stable and favourable for production at a large scale [11][12][13][14][15]. Esterase is also used to resolve (R, S)-DMPM.A study reported that the enantiomeric excess of lipase MC 16-3 and lipase 99-2-1 resolution (R, S) -DMPM from Burkholderia sp.were 91.8% (conversion rate 25%) and 91%(conversion rate 30%).…”

Section: Introduction (R)-mentioning

confidence: 99%

Enantioselective Resolution of (R, S)-DMPM by a Adsorption-Covalent Crosslinked Esterase PAE07 from Pseudochrobactrum Asaccharolyticum WZZ003

Zhou

Xia

Zhang

et al. 2021

Preprint

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(R)-N-(2,6-dimethylphenyl) alanine ((R)-MAP-acid) is an important chiral intermediate of the Fungicide (R)-Metalaxyl. In this study, ten kinds of immobilized resins(XAD1180N, H103, HAD7HP, D3520, NKA, D101 , DM11,850 JinKai, Primary amino resin and 850 synthetic resin) were used to adsorption-covalent crosslinked esterase PAE07 for splitting (R, S)-DMPM. The resin D3520 with porous structure and hydrophobic polystyrene was selected for immobilization as the carrier, after optimization of the immobilization conditions, the enzyme load is 20:1 (mg/g), the adsorption time is 4h, and the adsorption buffer pH is 7.0 . The Km and Vmax of the free esterases were 35.66 mM and 4.46 mM/mg·min, respectively, The Km and Vmax of the immobilized PAE07 were 19.05 mM and 2.84 mM/mg·min. The SEM analysis showed that the immobilized esterase PAE07 had higher thermal stability, pH stability and substrate specifity than those from the free esterase. Under the optimal conditions,the reaction was carried out at 35°C and 200 rpm for resolution of 350 mM substrate for 14 hours, the conversion rate reached 48%, and the e.e.p was 99.5%.The repeatability of immobilized esterase PAE07 was evaluated by continuous catalytic resolution of (R, S)-DMPM. The results showed that after 15 times of repeated use, 86.2% of the relative enzyme activity was retained. These results proved that immobilized esterase PAE07 as a new catalyst had great potential for the application and industrial enzymatic resolution of (R, S)-DMPM to prepare (R)-metalaxyl.

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Characterization of a Novel Moderately Thermophilic Solvent-Tolerant Esterase Isolated From a Compost Metagenome Library

Cited by 28 publications

References 55 publications

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

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

Identification and characterization of a novel carboxylesterase EstQ7 from a soil metagenomic library

Enantioselective Resolution of (R, S)-DMPM by a Adsorption-Covalent Crosslinked Esterase PAE07 from Pseudochrobactrum Asaccharolyticum WZZ003

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