A novel family VII esterase with industrial potential from compost metagenomic library

Abstract

BackgroundAmong the vast microbial genomic resources now available, most microbes are unculturable in the laboratory. A culture-independent metagenomic approach is a novel technique that circumvents this culture limitation. For the screening of novel lipolytic enzymes, a metagenomic library was constructed from compost, and the clone of estCS2 was selected for lipolytic properties on a tributyrin-containing medium.ResultsThe estCS2 sequence encodes a protein of 570 amino acid residues, with a predicted molecular mass of 63 kDa, and based on amino acid identity it most closely matches (45%) the carboxylesterase from Haliangium ochraceum DSM 14365. EstCS2 belong to family VII, according to the lipolytic enzyme classification proposed by Arpigny and Jaeger, and it retains the catalytic triad Ser245-Glu363-His466 that is typical of an α/β hydrolase. The Ser245 residue in the catalytic triad of EstCS2 is located in the consensus active site motif GXSXG. The EstCS2 exhibits strong activity toward p-nitrophenyl caproate (C6), and it is stable up to 60°C with an optimal enzymatic activity at 55°C. The maximal activity is observed at pH 9, and it remains active between pH 6-10. EstCS2 shows remarkable stability in up to 50% (v/v) dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). The enzyme has the ability to cleave sterically hindered esters of tertiary alcohol, as well as to degrade polyurethanes, which are widely used in various industries.ConclusionsThe high stability of EstCS2 in organic solvents and its activity towards esters of ketoprofen and tertiary alcohols, and in polyurethane suggests that it has potential uses for many applications in biotransformation and bioremediation.

The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes

“…For example, the activity of the EstA esterase from Arthrobacter nitroguajacolicus was slightly stimulated (10-20 %) by the addition of DMSO (30 %) or acetonitrile (30 %), whereas lower concentrations (10 %) of these solvents reduced (20 to 90 %) the activity of esterases from Sulfolobus solfataricus (EST1) and Archaeoglobus fulgidus (Est-AF) (Kim et al 2008;Schutte and Fetzner 2007;Sehgal et al 2002). Similarly, several metagenomic esterases have been reported to be inhibited, unaffected or stimulated by the addition of DMSO (10-30 %), whereas all these enzymes were sensitive to acetonitrile and detergents (Triton X-100 and Tween-20) (Elend et al 2006;Fu et al 2011;Jiang et al 2012;Kang et al 2011). Since the metagenomic enzymes cannot be protected from the effect of low temperatures, the evolution of cold-adapted enzymes is the main mechanism of cold resistance in marine microbes.…”

“…It has been reported that purified microbial and metagenomic esterases exhibit different sensitivities to organic solvents and detergents (Elend et al 2006;Fu et al 2011;Jiang et al 2012;Kang et al 2011). For example, the activity of the EstA esterase from Arthrobacter nitroguajacolicus was slightly stimulated (10-20 %) by the addition of DMSO (30 %) or acetonitrile (30 %), whereas lower concentrations (10 %) of these solvents reduced (20 to 90 %) the activity of esterases from Sulfolobus solfataricus (EST1) and Archaeoglobus fulgidus (Est-AF) (Kim et al 2008;Schutte and Fetzner 2007;Sehgal et al 2002).…”

Classification of lipolytic enzymes and their biotechnological applications in the pulping industry

“…The units at the bottom of the tree indicate the number of substitution events. Reproduced with permission from figure 1b in Kang et al (2011). acylglycerols (Ellaiah et al 2004;Bornscheuer 2002;Litthauer et al 2002;Verger 1997;Ollis et al 1992).…”

A feruloyl esterase derived from a leachate metagenome library

“…Multiple sequence alignment of Fae6 with a recently published metagemomic derived esterase (25) from family VII and the other related sequence from the BLASTp hits showed that the following residues were highly conserved: Ser253 within the G-E-S-A-G sequence motif, as well as the C-terminus located Glu307 and His507 ( Fig. S1; Supplementary material).…”

“…The classical examples include esterase from Arthrobacter oxydans which is active against phenylcarbamate herbicides (32), a Bacillus subtilis esterase which hydrolyses p-nitrobenzyl esters (33) and a compost metagenome derived esterase, which in addition to hydrolyzing polyurethane and tertiary alcohol esters, also shows enantioselectivity against (R/S)-ketoprofen ethyl ester (25). Thus esterases belonging to family VII constitute one of the most versatile lipolytic enzyme groups.…”