Detection of a new enzyme for stereoselective hydrolysis of linalyl acetate using simple plate assays for the characterization of cloned esterases from Burkholderia gladioli.

Applying an agar plate assay, a novel gene encoding an esterase from Bacillus subtilis Est4B was cloned and highly overexpressed in E. coli . The protein sequence revealed high homology to the srf D gene, which encodes the fourth open reading frame, a putative thioesterase gene from the surfactin synthetase gene cluster. It was almost identical to an unpublished sequence from a putative surfactin synthetase cluster from B. subtilis A13. The enzyme (Est4B1) was produced in E. coli , purified to homogeneity and crystallized. Computational prediction of the protein fold showed high structural similarity of Est4B1 to haloperoxidases and dehalogenases and much lower similarity to lipases and esterases. However, by primary sequence analysis we found a typical esterase/thioesterase/lipase motif. Biocatalytic activity on several model esterase substrates was detected and quantified. This is the first time enzymatic activity could be shown for this type of independent putative thioesterases and this enzyme may serve as a model protein to solve the structure of this type of hydrolytic enzymes, which is commonly found in gene clusters of non-ribosomal peptide synthetases.

Cloning, expression and characterization of a new 2-Cl-propionic acid ester hydrolase from B. subtilis

A new esterase gene from Xanthomonas vesicatoria (formerly X. campestris) DSM 50861 was identified, cloned from a chromosomal gene library and overexpressed in Escherichia coli. The corresponding DNA fragment contains an ORF of 1,818 bp, encoding a hydrolase of the GDSL esterase family. A protein of about 67 kDa, named Xv_EstE, was expressed from this fragment. A N-terminal signal peptide was processed under low-expression conditions, yielding a 63-kDa mature protein. The predicted amino acid sequence showed distinct homology to esterases of the GDSL family. Based on homology, a catalytic triad Gly-Asp-Ser could be defined. Amino acid sequence alignments and computer-assisted structure prediction indicated the presence of a carboxyl-terminal β-barrel membrane domain which might facilitate binding of Xv_EstE to the outer membrane. This could be verified by differential cell fractionation experiments, in which Xv_EstE was exclusively found in the outer membrane fraction. Xv_EstE showed preferential hydrolytic activity on short chain (up to C8) and para-substituted nitrophenylesters as substrates. However, only long-chain 1-hydroxy-pyrene-3,6,8-trisulfonic acid (HPTS)-fatty acid esters were hydrolyzed. Xv_EstE was also found to be active on a series of substrates of industrial interest, such as 1-methylprop-2-ynyl acetate, for which an enantioselectivity up to 93% ee could be recognized.

Esterase EstE from Xanthomonas vesicatoria (Xv_EstE) is an outer membrane protein capable of hydrolyzing long-chain polar esters

The two new esterases, EP6 and EP10, from Pseudomonas marginata were studied with respect to their substrate specificities and selectivities. Esters of aliphatic and cyclic secondary alcohols were shown to be substrates for EP10, which could be accepted stereoselectively in hydrolysis and transesterification reactions. EP6 showed no selectivity in hydrolysis of the studied substrates.

New Pseudomonas esterases by genetic engineering

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New Pseudomonas esterases by genetic engineering