Characterization of a novel family VIII esterase EstM2 from soil metagenome capable of hydrolyzing estrogenic phthalates

Abstract

Background: Microbes are rich sources of enzymes and esterases are one of the most important classes of enzymes because of their potential for application in the field of food, agriculture, pharmaceuticals and bioremediation. Due to limitations in their cultivation, only a small fraction of the complex microbial communities can be cultured from natural habitats. Thus to explore the catalytic potential of uncultured organisms, the metagenomic approach has turned out to be an effective alternative method for direct mining of enzymes of interest. Based on activity-based screening method, an esterase-positive clone was obtained from metagenomic libraries.

Results: Functional screening of a soil metagenomic fosmid library, followed by transposon mutagenesis led to the identification of a 1179 bp esterase gene, estM2, that encodes a 392 amino acids long protein (EstM2) with a translated molecular weight of 43.12 kDa. Overproduction, purification and biochemical characterization of the recombinant protein demonstrated carboxylesterase activity towards short-chain fatty acyl esters with optimal activity for p-nitrophenyl butyrate at pH 8.0 and 37 °C. Amino acid sequence analysis and subsequent phylogenetic analysis suggested that EstM2 belongs to the family VIII esterases that bear modest similarities to class C β-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C β-lactamases but did not exhibit β-lactamase activity. Guided by molecular docking analysis, EstM2 was shown to hydrolyze a wide range of di- and monoesters of alkyl-, aryl- and benzyl-substituted phthalates. Thus, EstM2 displays an atypical hydrolytic potential of biotechnological significance within family VIII esterases.

Conclusions: This study has led to the discovery of a new member of family VIII esterases. To the best of our knowledge, this is the first phthalate hydrolase (EstM2), isolated from a soil metagenomic library that belongs to a family possessing β-lactamase like catalytic triad. Based on its catalytic potential towards hydrolysis of both phthalate diesters and phthalate monoesters, this enzyme may find use to counter the growing pollution caused by phthalate-based plasticizers in diverse geological environment and in other aspects of biotechnological applications.

Keywords: Biocatalyst; Esterase; Metagenome; Phthalate; Unculturable; β-lactamase.

Fig. 1

Phylogenetic tree based on the protein sequences of bacterial lipolytic enzymes belonging to different enzyme families. Numbers at the nodes indicate the levels of bootstrap support based on neighbour joining analysis of 100 resampled data sets. Bootstrap values below 50% are not shown. The scale bar represents 0.1 substitutions per nucleotide position. GenBank accession numbers of the sequences are indicated within parentheses. Phthalate ester hydrolases are denoted with an asterisk

Fig. 2

a Phylogenetic analysis of EstM2 with reported family VIII esterases. Numbers at the nodes indicate the levels of bootstrap support based on neighbor joining analysis of 100 resampled data sets. Bootstrap values below 50% are not shown. The scale bar represents 0.1 substitutions per nucleotide position. GenBank accession numbers are indicated within parentheses. AmpC, a class C β-lactamase from Aeromonas enteropelogenes (ABW05394) was used as an outgroup. b Multiple sequence alignment of EstM2 and other family VIII carboxylesterases showing the conserved sequence motifs. Amino acid residues responsible for the formation of catalytic triad for ester bond hydrolysis are shaded. Protein sequences with classical pentapeptide G-x-S-x-G motif are shown within a box. Alignment of two class C β-lactamases from Aeromonas enteropelogenes (AmpC, ABW05394) and Enterobacter cloacae (Blac, Q59401) respectively were also shown for sequence comparisons

Fig. 3

Plots showing relative activity of EstM2 at different pH (a), temperature (b) and on thermostability (c)

Fig. 4

A Surface topology of EstM2 showing the binding of dimethyl phthalate within the catalytic pocket (pink) via electrostatic interaction with active site residues Ser68 and Trp343 based on docking analysis; B Lucid view showing hydrophobic interactions of dimethyl phthalate with different residues at the catalytic pocket of EstM2; C Alignment of other phthalate esters within the catalytic pocket of EstM2, as obtained from docking studies: diethyl phthalate (a), di-n-butyl phthalate (b), butyl benzyl phthalate (c), diphenyl phthalate (d), monomethyl phthalate (e), monoethyl phthalate (f), monobenzyl phthalate (g) and monophenyl phthalate (h)