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.
Highlights
Lipolytic enzymes, such as carboxylesterases (EC 3.1.1.1) and triacylglycerol lipases (EC 3.1.1.3), have been extensively used in the manufacturing and processing of detergents, foodstuffs, drugs, paper, textiles, leathers, and fine chemicals, demonstrating their versatility for biotechnological applications [1, 2]
Lipolytic enzymes belong to the α/β-hydrolase superfamily and contain a catalytic triad that usually consists of a nucleophilic serine in a GXSXG pentapeptide motif and an acidic residue that is hydrogen bonded to a histidine residue [9, 10]
The pentapeptide motif is usually located between a βstrand and a α-helix, and assumes an extremely sharp turn called the nucleophilic elbow [11]
Summary
Lipolytic enzymes, such as carboxylesterases (EC 3.1.1.1) and triacylglycerol lipases (EC 3.1.1.3), have been extensively used in the manufacturing and processing of detergents, foodstuffs, drugs, paper, textiles, leathers, and fine chemicals, demonstrating their versatility for biotechnological applications [1, 2]. Est a Novel Alkaline Esterase from Cow Rumen they can be highly selective and even stereo-selective. They do not require cofactors and are stable in various organic solvents [3,4,5,6,7,8]. Lipolytic enzymes belong to the α/β-hydrolase superfamily and contain a catalytic triad that usually consists of a nucleophilic serine in a GXSXG pentapeptide motif and an acidic residue (aspartic acid or glutamic acid) that is hydrogen bonded to a histidine residue [9, 10]. The pentapeptide motif is usually located between a βstrand and a α-helix, and assumes an extremely sharp turn called the nucleophilic elbow [11]
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