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

Abstract

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.

Enhancing the Enantioselectivity and Catalytic Efficiency of Esterase from Bacillus subtilis for Kinetic Resolution of l-Menthol through Semirational Design

“…7 The esterase AbMBH from Acinetobacter sp. ECU2040 exhibited only low enantioselectivity (E = 27.5) toward D,L-menthyl benzoate, 4 and the lipase from Burkholderia cepacia ATCC 25416 showed a 96% optical purity for L-menthol production, with 15% (v/v) dimethyl sulfoxide (DMSO) as the cosolvent and 50% conversion, the tolerable substrate concentrations of which were low (0.23 mol/L). 11 Here, we cloned esterase pnbA from B. subtilis for the L-menthol synthesis.…”

“…l -Menthol was originally extracted and isolated from peppermint and is extensively used in the food, medicine, cosmetics, and tobacco industries. Because the yield of natural l -menthol extracted from plants is no longer able to meet market demand, chemical synthesis of l -menthol has been well developed and used for industrial production by Symrise (Germany), Takasago (Japan), and BASF SE (Germany). − Among these chemical synthesis routes, asymmetric synthesis of l -menthol or chemical resolution of d,l -menthyl esters requires a large amount of heavy metal catalyst and organic solvents, which are hazardous to the environment and human health. Therefore, alternative methods using biocatalysts have attracted increasing interest.…”

A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli

“…In recent research , Escherichia coli ( E. coli ) has established itself as one of the most extensively used systems in the industrial production of recombinant proteins, which owes much to the high yield of production that is possible with this system, the low manufacturing cost, a well-characterized expression system, and amenability to high cell-density fermentation biotechnology [ 1 , 2 ]. In addition, due to a lack of appropriate folding-assistance proteins and cofactors that can mediate post-translational modifications, the likelihood of inclusion body formation is considerately high when heterologous proteins are expressed in E. coli [ 3 ].…”