Enzymatic Production of l‐Menthol by a High Substrate Concentration Tolerable Esterase from Newly Isolated Bacillus subtilis ECU0554

Abstract

Enzymatic preparation of l-menthol has been attracting much attention in the flavor and fragrance industry. A new ideal strain, Bacillus subtilis ECU0554, which exhibited high hydrolytic activity and excellent enantioselectivity towards l-menthyl ester, has been successfully isolated from soil samples through enrichment culture and identified as Bacillus subtilis by 16S rDNA gene sequencing. The esterase extracted from B. subtilis ECU0554 (BSE) showed the best catalytic properties (E > 200) for dlmenthyl acetate among the five menthyl esters examined. Enantioselective hydrolysis of 100 mM dlmenthyl acetate at 308C and pH 7.0, using crude BSE as biocatalyst and 10% ethanol (v/v) as cosolvent, resulted in 49.0% conversion (3 h) and 98.0% ee for the l-menthol produced, which were much better than those using commercial enzymes tested.Moreover, BSE exhibited strong tolerance against high substrate concentration (up to 500 mM), and the concentration of l-menthol produced could reach as high as 182 mM, and more importantly, the optical purity of l-menthol produced was kept above 97% ee, which were not found in previous reports. These results imply that BSE is a potentially promising biocatalyst for the large-scale enzymatic preparation of l-menthol. Using this excellent biocatalyst, the enzymatic production of l-menthol will become a mild, efficient, inexpensive and easy-to-use "green chemistry" methodology.

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

“…Glycerin tributyrate and Tween-80 were purchased from TCI (Tokyo, Japan). dl-Menthyl benzoate, dl-menthyl acetate, dl-menthyl butyrate, dl-menthyl chloroacetate, and p-nitrophenyl butyrate (pNPB) were synthesized in our laboratory [9,16]. The other chemicals used in this work were of analytical grade from local sources.…”

Efficient production of l-menthol in a two-phase system with SDS using an immobilized Bacillus subtilis esterase

“…Materials dl-Menthol was purchased from Alfa Aesar (Tianjin, China), and dl-menthyl acetate was synthesized as described before [23]. All other reagents were obtained commercially and of analytic grade.…”

“…, 0.5 M) and excellent enantioselectivity (E > 100) [23]. The esterase (BsE) catalyzing the enantioselective hydrolysis of dl-menthyl acetate was cloned and overexpressed in Escherichia coli [24].…”

“…The solubility of dl-menthyl acetate in mono-aqueous phase was very low, and ethanol was added as cosolvent to improve the substrate solubility [23]. Although BsE showed relatively high substrate tolerance, the conversion decreased sharply with increase of substrate concentration from 1 to 3 M in mono-aqueous system, which might be due to the severe inhibition of enzyme activity at extremely high concentration of substrate or product.…”

Improving Pseudomonas alcaligenes lipase’s diastereopreference in hydrolysis of diastereomeric mixture of menthyl propionate by site-directed mutagenesis

“…Lipases (glycerol ester hydrolases, EC 3.1.1.3) exhibit high levels of activity and stability, as well as excellent asymmetric selectivity, and have hence been widely employed in asymmetric syntheses for the production of chiral chemicals [2][3][4][5]. Intensive studies of the chiral recognition mechanism of lipases have made important contributions to our understanding of their structure-activity relationship, have aided the rational design of enzymes with improved functions, and have broadened the applications of these enzymes in chiral chemical manufacturing [6][7][8][9][10].…”