Mechanism and Structural Insights Into a Novel Esterase, E53, Isolated From Erythrobacter longus

Esterases are a class of enzymes that split esters into an acid and an alcohol in a chemical reaction with water, having high potential in pharmaceutical, food and biofuel industrial applications. To advance the understanding of esterases, we have identified and characterized E53, an alkalophilic esterase from a marine bacterium Erythrobacter longus. The crystal structures of wild type E53 and three variants were solved successfully using the X-ray diffraction method. Phylogenetic analysis classified E53 as a member of the family IV esterase. The enzyme showed highest activity against p-nitrophenyl butyrate substrate at pH 8.5–9.5 and 40°C. Based on the structural feature, the catalytic pocket was defined as R1 (catalytic center), R2 (pocket entrance), and R3 (end area of pocket) regions. Nine variants were generated spanning R1–R3 and thorough functional studies were performed. Detailed structural analysis and the results obtained from the mutagenesis study revealed that mutations in the R1 region could regulate the catalytic reaction in both positive and negative directions; expanding the bottleneck in R2 region has improved the enzymatic activity; and R3 region was associated with the determination of the pH pattern of E53. N166A in R3 region showed reduced activity only under alkaline conditions, and structural analysis indicated the role of N166 in stabilizing the loop by forming a hydrogen bond with L193 and G233. In summary, the systematic studies on E53 performed in this work provide structural and functional insights into alkaliphilic esterases and further our knowledge of these enzymes.

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

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 Family IV Esterase Containing a Predicted CzcO Domain and a Family V Esterase with Broad Substrate Specificity from an Oil-Polluted Mud Flat Metagenomic Library

Two novel esterase genes, est2L and est4L, were identified from a previously constructed metagenomic library derived from an oil-polluted mud flat sample. The encoded Est2L and Est4L were composed of 839 and 267 amino acids, respectively, without signal peptides. Est2L was a unique fusion type of protein composed of two domains: a domain of the CzcO superfamily, associated with a cationic diffusion promoter with CzcD, and a domain of the acetylesterase superfamily, belonging to family IV with conserved motifs, such as HGG, GXSAG, and GXPP. Est2L was the first fused esterase with a CzcO domain. Est4L belonged to family V with GXS, GXSMGG, and PTL motifs. Native Est2L and Est4L were found to be in dimeric and tetrameric forms, respectively. Est2L and Est4L showed the highest activities at 60 °C and 50 °C, respectively, and at a pH of 10.0. Est2L preferred short length substrates, especially p-nitrophenyl (pNP)-acetate, with moderate butyrylcholinesterase activity, whereas Est4L showed the highest activity with pNP-decanoate and had broad specificity. Significant effects were not observed in Est2L from Co2+ and Zn2+, although Est2L contains the domain CzcD. Est2L and Est4L showed high stabilities in 30% methanol and 1% Triton X-100. These enzymes could be used for a variety of applications, such as detergent and mining processing under alkaline conditions.

Identification of Detoxification Esterase StrH Initiating Strobilurin Fungicides Degradation in Hyphomicrobium sp. DY-1

Strobilurin fungicides are widely used in agricultural production due to their broad-spectrum and fungal mitochondrial inhibitory activities. However, their massive application has detained the growth of eukaryotic algae and increased the collateral damage in freshwater systems, notably the harmful cyanobacterial blooms (HCBs). In this study, a strobilurin fungicide-degrading strain Hyphomicrobium sp. DY-1 was isolated and characterized successfully. Moreover, a novel esterase gene strH responsible for the de-esterification of strobilurin fungicides was cloned, and the enzymatic properties of StrH were studied. For trifloxystrobin, StrH displayed the maximum activity at 50°C and pH 7.0. The catalytic efficiency (kcat/Km) of StrH for different strobilurin fungicides were 196.32±2.30 μM−1·s−1 (trifloxystrobin), 4.64±0.05 μM−1·s−1 (picoxystrobin), 2.94±0.02 μM−1·s−1 (pyraclostrobin), and (2.41±0.19)×10−2 μM−1·s−1 (azoxystrobin). StrH catalyzed the de-esterification of a variety of strobilurin fungicides generating the corresponding parent acid to achieve the detoxification of strobilurin fungicides and relieve strobilurin fungicides growth inhibition on Chlorella. This research will provide insight into the microbial remediation of strobilurin fungicides-contaminated environments. IMPORTANCE Strobilurin fungicides have been widely acknowledged as an essential group of pesticides worldwide. So far, their residues and toxic effects on aquatic organisms have been reported in different parts of the world. Microbial degradation could eliminate xenobiotics from the environment. Therefore, the degradation of strobilurin fungicides by microorganisms has also been reported. However, little is known about the involvement of enzyme or gene in strobilurin fungicides degradation. In this study, a novel esterase gene strH responsible for the detoxification of strobilurin fungicides was cloned in the newly isolated strain Hyphomicrobium sp. DY-1. This degradation process detoxifies the strobilurin fungicides and relieves their growth inhibition on Chlorella.

A novel esterase from a soil metagenomic library displaying a broad substrate range

A novel esterase gene was isolated from a soil metagenomic library. The gene encoded a protein of 520 amino acids which contained a 21 aa signal peptide. Primary structure analysis of the protein sequence revealed that it contained a conserved active site motif (SxSxG) and a structural motif (CS-D-HC). Then the esterase gene was cloned and expressed in Escherichia coli BL21(DE3). SDS-PAGE analysis of the purified esterase showed that it was expressed in a highly soluble form and its molecular mass was estimated to be 55 kDa. Characterization of the esterase revealed that it exhibited high activity toward p-nitrophenyl esters with short acyl chains and especially p-nitrophenyl acetate, suggesting that it was a typical carboxylesterase rather than a lipase. With p-nitrophenyl acetate as substrate, the enzyme showed its optimal activity at pH 7.0 and 30 °C, and it was stable at a broad pH range from 4.5 to 10.0 and temperature not higher than 50 °C. Furthermore, the enzyme showed different substrate specificity from known esterase, it was not only hydrolyzing against p-nitrophenyl esters, but also hydrolyzing all hydroxybenzoic esters and hydroxycinnamic ester assayed. As it was an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase, chlorogenate esterase and tannase activities, it could serve as a valuable candidate for applications in biotechnology.