Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain): a metagenomic approach

Abstract

The esterases and lipases from the α/β hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46'N, 2°59'W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated.

Fig 1

Effect of NaCl concentration (upper), pH (middle), and temperature (lower) on the hydrolytic activity of Lake Arreo enzymes. The heat map colors represent the relative percentages of specific activity (U/mg) compared to the maximum (100%) within each enzyme, using pNP butyrate as the substrate. The effect of NaCl concentration was measured in 20 mM HEPES buffer (pH 7.0) at 30°C and varied from 0 to 1.2 M; the pH dependence was tested in the range of pH 4.0 to 9.5 at 30°C in 20 mM buffers; the temperature dependence in the range of 4 to 70°C at pH 7.0 was also determined. Reaction conditions were as described in Table 1 and Materials and Methods.

Fig 2

Substrate profiles of the wild-type LAE2, LAE5, and LAE7 α/β hydrolases from Lake Arreo against a set of structurally diverse ester substrates. LAE2, LAE5, and LAE7 were the α/β hydrolases characterized by a restricted substrate spectrum. Reactions (30°C and pH 7.2) were performed as described for the α-naphthyl and triacylglycerol esters (Table 1) and were monitored during 15 min, and the absorbance of p-nitrophenol at 404 nm was recorded and plotted. For best and/or representative substrates, specific activities (U/mg) were calculated as described in Table 1 and Materials and Methods and are shown on the top of bars. In all cases, three independent experiments were performed for each parameter, and graphs were plotted using mean values; the standard deviations were less than 5%.

Fig 3

Substrate specificity of the wide-substrate-spectrum LAE1, LAE3, LAE4, and LAE6 α/β hydrolases from Lake Arreo against a set of structurally diverse ester substrates. LAE1, LAE3, LAE4, and LAE6 were the hydrolases characterized by the widest substrate spectrum. Reaction conditions (30°C and pH 7.2) and activity parameter determinations were as described in Table 1 and the legend to Fig. 2. M2Cl3HP, methyl-2-chloro-3-hydroxypropionate.

Fig 4

Clustering of substrate spectrum profile of Lake Arreo enzymes and commercial preparations, applying the Pearson's correlation to calculate the distances. Hierarchical clustering was derived from binomial distribution based on the presence or absence of activity against particular substrates (activity data not shown), measured as described in Table 1, using 2-μg protein extracts.

Fig 5

Dendrogram of protein sequence similarity relationships between newly identified and reference esterases/lipases. Enzyme families are depicted according to the Arpigny and Jaeger classification (48).