Chiral Resolution Of Dl-menthol By Esterases From Burkholderia Cepacia

The yield and quality of natural menthol are affected seriously by weather and region, etc., synthetic menthol can overcome all these drawbacks. Therefore, there is a great interest in separation of dl-menthol to prepare l-menthol by microbial methods.A strain named Burkholderia cepacia ATCC 25416 with high enantioselectivity and hydrolysis activity toward dl-menthyl acetate was screened through combined screening strategy of hydrolysis ability with stereoselectivity. The cultivation conditions and the enantioselective hydrolysis conditions of B. cepacia whole cells were studied in this work. In order to extend the usage and clone the enzyme in a large scale, the key enzymes were also purified and characterized. The main research contents were shown as follows:(1) To increase the enzyme activity, the cultivation conditions of B. cepacia were optimized using response surface methodology (RSM). The optimum culture medium components were 2.27% glucose, 1.58% beef extract, 1.83% peptone, 0.42% K₂HPO₄, 0.02% MgSO₄·7H₂O, 0.01% CaCl₂, 0.5% NaCl. The optimum environment conditions were 6% inoculation size, 15% medium volume, initial pH 7.0⁷.5, 30℃and 48 h. Under these conditions, the enzyme activity was increased by 2.3 times.(2) The key enzymes were purified from B. cepacia by a four-step purification, i.e., ammonium sulfate precipitation, butyl-Sepharose, DEAE-Sepharose, and Superdex G-75 column chromatography. Est1 was obtained with activity of 0.77 U/mg and molecular weight of 66 kDa. Est2 was obtained with activity of 5.91 U/mg and molecular weight of 37 kDa. The optimum pH and the optimum temperature of Est1 and Est2 were pH 7.0 and 30℃, respectively. These two enzymes belonged to the class of serine hydrolases, but not metalloenzyme. The enantioselectivities of the two enzymes were not temperature depended, but pH depended, and decreased sharply under harsh pH conditions. The enantioselectivities were also droped when different detergents or inhibitors used in the reaction systems. There was not position specificity about Est1, while Est2 had a 2-position specificity. The two enzymes showed higher hydrolytic activity toward short acyl chain p-nitrophenol esters and short acyl chain menthol esters. Except menthyl formate, they all preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity, which indicated that the two purified enzymes were short-chain fatty acid esters hydrolase. The N-terminal amiano acid sequence of Est1 had been determined as MHITTT, and the N-terminal sequence of Est2 was MGARTDA. There were no homologies of the two sequences with other Burkholderia sp. hydrolase (esterase/lipase). Considering the N-terminal sequences and their high stereospecificity for short chain menthyl esters, these two esterases were concluded as new enzymes belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family.(3) The catalytic reaction process about enzntioselective hydrolysis of dl-menthyl acetate to l-menthol catalyzed by B. cepacia whole cells was studied. The results showed that the permeability of the whole cells and the interface area between substrate and enzyme were changed after addition of co-solvents, which influenced the hydrolysis conversion and ee-value (enantiomeric excess) of product. High hydrolysis rate and ee-value of product were achieved when 15% (v/v) dimethyl sulfoxide (DMSO) was added. The optimum hydrolysis conditions catalyzed by B. cepacia whole cells were 1 mL Na₂HPO₄-KH₂PO₄ (pH 7.0, 0.067 mol/L), 15% dimethyl sulfoxide (DMSO), 1% dl-menthyl acetate, 15 g/L dry cells, 30℃and 24 h. Under these conditions, l-menthol could be obtained with the optical purity higher than 95%. The product l-menthol was separated with a recovery of 85%, and the total yield of l-menthol was 66% in each cycle. These results proved that it is reasonable to split dl-menthyl acetate in preparation l-menthol using B. cepacia whole cells as biocatalyst after chemical esterification of dl-menthol with acetic anhydride.