Cloning and Characterization of an Enantioselective l-Menthyl Benzoate Hydrolase from Acinetobacter sp. ECU2040.

A new esterase gene abmbh, encoding a benzoate hydrolase which can enantioselectively hydrolyze l-menthyl benzoate to l-menthol, was recently identified from the genomic library of a soil isolate Acinetobacter sp. ECU2040. The abmbh gene contains a 1080-bp open reading frame encoding a protein of 360 amino acids with a calculated molecular mass of 40.7 kDa. The corresponding enzyme AbMBH was functionally expressed in Escherichia coli BL21 (DE3), purified, and characterized. The AbMBH displayed the maximum activity towards p-nitrophenyl butyrate at 50 °C, and an optimum pH of 8.5. A K M of 2.6 mM and a k cat of 0.26 s(-1) were observed towards dl-menthyl benzoate. The AbMBH exhibited a moderate enantioselectivity (E = 27.5) towards dl-menthyl benzoate. It can also catalyze the enantioselective hydrolysis of a variety of racemic menthyl esters, including dl-menthyl acetate, dl-menthyl chloroacetate, and dl-menthyl butyrate.

Enantioselective hydrolysis of dl-menthyl benzoate by cell-free extract of newly isolated Acinetobacter sp. ECU2040.

Production of l-menthol by bioprocesses attracts increasing attention nowadays. Herein, we attempted to develop a bioresolution process for production of l-menthol through enantioselective hydrolysis of dl-menthyl benzoate using a newly isolated bacterium from soil samples. Among 129 active soil isolates screened rapidly by thin-layer chromatography, an outstanding bacterial strain numbered ECU2040, which was subsequently identified as Acinetobacter species, was finally selected as our target enzyme producer due to its highest activity and the best enantioselectivity toward l-substrate as confirmed by chiral gas chromatography. The catalytic performance of the cell-free extract from Acinetobacter sp. ECU2040 was preliminarily examined, indicating that its optimal pH and temperature for the reaction were 7.5 and 37 °C, respectively. Under the optimal conditions, the enzymatic reaction was performed on a 1-L scale, affording l-menthol in 48 % yield and 71 % ee.

Cloning of a novel arylamidase gene from Paracoccus sp. strain FLN-7 that hydrolyzes amide pesticides.

The bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-amino-acid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with K(m) and k(cat) values of 29.5 µM and 49.2 s(-1), respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments.