Biocatalytic Preparation Of L-Menthol Through Enantioselective Hydrolysis Of Dl-Menthyl Benzoate

l-Menthol is an extremely important flavor chemical used extensively in Pharmaceuticals, foods, tobacco products, cosmetics and oral products because of its well-known, unique cooling characteristics. In the last few decades, scientists have shown great interest in biocatalytic processes for production of l-menthol because of their advantages such as lower energy consumption, environmental friendliness, lower costs, less waste and shorter synthetic route.dl-Menthyl benzoate is a practical starting compound to produce l-menthol since it had been used in industry for the chemical manufacture of l-menthol in thousands tons scale per year. Herein, we attempted to develop a biocatalytic process for production of l-menthol through enantioselective hydrolysis of dl-menthyl benzoate using a strain newly isolated from soil with good enantioselectivity and high hydrolysis degree. The dissertation is composed of two parts as follows:The first part of the dissertation aimed to search for novel biocatalysts with excellent enantioselectivity and high hydrolysis activity through the strategies of target reaction-oriented screening. From soil samples,341 single strains were screened through thin layer chromatography (TLC) analysis, and among them 129 strains were capable of enantioselectively hydrolyzing dl-menthyl benzoate to form l-menthol. Through further screening and confirmation with gas chromatography (GC), the strain number 40 exhibited the good hydrolytic activity and enantioselectivity as compared to others. So it was selected as the best enzyme producer and subsequently identified as Acinetobacter sp. based on the 99% similarity of its 16S rDNA sequence, and named Acinetobacter sp. ECU2040.The second part of the dissertation is the studies on catalytic properties of Acinetobacter sp. ECU2040, and optimization and scale-up of the biocatalytic process. The results showed that the optimum pH and temperature of reaction were 7.5 and 37℃, respectively. The optimum surfactant for helping the substrate dispersion was Tween-80, which was employed at 0.5% concentration (w/v), followed by homogenization of the mixture with ultrasonic wave to form a well-dispersed emulsion. Effect of substrate concentration on enzyme tolerance was examined, indicated that the reaction had relatively good results at 10 mM of substrate concentration. Finally, a larger-scale reaction (1-L) was performed under the optimum conditions. The conversion of substrate reached 48%, which was similar to the result of reaction at 10 mL scale. This result indicates that further scale-up of the reaction volume is achievable.