Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes

Abstract

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40 °C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.

Fig 1. Sequence alignment of Est10 with its major closest homologs.

The amino acid sequences correspond to Est10 (GI accession number KM042178), EstZ3 (ADE28720.1, 41% identity with Est10), EstGK1 (ADE28720.1, 39% identity), Est5S (ABI17943.1, 92% identity), EstD2 (ADN26553, 29% identity), and EstWSD (AFY63009, 29% identity). The conserved pentapeptide is shown with a rectangle. Residues that are 100% conserved are shadowed in black, and those between 75% and 100% are shadowed grey. The residues that encompass the putative signal peptide on Est10 are marked with a black line.

Fig 2. Maximum Likelihood inference of the phylogenetic relationships between members of family XV based on amino acid sequences.

Alignment was obtained with MAFFT with the E-INS-I strategy [52]. The numbers of interior branches represent estimated SH-like support values. ESTHER database accession numbers of the sequences used are included in the tree next to the genus of the organisms of origin. GI accession number is included when the protein sequence was retrieved from GenBank database. The position of Est10 is indicated with a black arrow, characterized members of family XV with black circles, and characterized members of the domain family DUF3089 with white circles.

Fig 3. SDS-PAGE analysis of purified 6xHis-Est10 protein stained with Coomassie blue.

Recombinant 6xHis-Est10 was purified by affinity chromatography on a Ni²⁺-NTA matrix. Lane PM: protein molecular weight marker. Lanes E1-4: consecutive eluted fractions with 250 mM of imidazol. The position of recombinant Est10 is indicated by a black arrowhead. The calculated molecular weight of Est10 is 40.2 kDa.

Fig 4. Characterization of Est10 esterase activity.

(A) Determination of chain length specificity using pNP esters of fatty acids: acetate (C2), butyrate (C4), and decanoate (C10. (B) Effect of the pH on esterase activity of Est10. (C) Thermal stability of Est10. Est10 was pre-incubated for 30 min at temperatures ranging from 30°C to 65°C before determining its residual activity. The control was not pre-incubated. (D) Effect of the temperature on the esterase activity of Est10. The reaction was carried at temperatures ranging from 30°C to 55°C. Except when noted, reactions were performed at 40°C using pNP butyrate as substrate. In all cases averages of triplicate assays are shown and error bars represent standard deviation.

Fig 5. Determination of Est10 substrate specificity and tolerance to metals, detergents, chelating agents and additives.

(A) Salts, detergents, chelating agents, and additives were added at a concentration of 1mM to a reaction mix containing 50nM Est10 and 1mM pNP butyrate. Reactions were carried out at 40°C during 15 min. (B) Est10 activity was determined with different esters as substrates. In all cases averages of triplicate assays are shown and error bars represent standard deviation.