| (2S,5S)-2,5-hexandiol is an important intermediate for the synthesis of chiral pharmaceuticals and various chiral phosphine ligands. The existing production of (2S,5S)-2,5-hexandiol is through the resolution of the racemic mixture (R, S)-2,5-hexandiol from chemical synthesis. The production of (2S,5S)-2,5-hexandiol has not been reported in China. In this work, Saccharomyces cerevisiae sp. strain HD-5 was screened for asymmetric reduction of 2,5-hexandione to (2S,5S) -2,5-hexandiol.A determined method of quantitative analysis of the substrate 2,5-hexandione and the product 2,5-hexandiol in the biotransformational medium was established by gas chromatography(GC). Glycol as an internal standard, the determinations of 2,5-hexane -dione and 2,5-hexanediol by GC were linear well at the range of 0.5～40 mmol?L-1.A method of the separation of 2,5-hexanediol enantiomers via derivatization -capillary column gas chromatography was established. 2,5-hexanediol enantiomers were derivatized with S-(-)-phenylethyl isocyanate and the baseline separation of the resulted derivatives was achieved by GC. The main factors including the flow rate of carrier gas and the programmed temperature to influence the separation were also discussed. The analytical method of reproducibility and accuracy was verified. The optimum separation condition was the vaporization temperature of 270℃and the detector temperature of s290℃, the diffluence ratio of 4.9:1 and the carrier gas velocity of 6.2 mL·L-1.The strain HD-5 with high catalytic activity, from 9 strains of Saccharomyces cerevisiae, was screened for asymmetric reduction of 2,5-hexanedione. Deducing from GC-MS, the reduction of 2,5-hexanedione proceeded in a two-step reaction. In the course of reaction first the 5-hydroxyhexane -2-one was formed. Subsequently, this intermediate was further reduced to 2,5-hexanediol. The effects of the substrate and the product on the growth characteristics of yeast HD-5 were investigated. The yeast HD-5 cells can grow well at the substrate concentrations less than 40 mmol·L-1 and the product concentrations less than 50 mmol·L-1.The influence of the surfactant Tween 80, cetyltrimethylammonium bromide (CTAB) and tetrahydrofuran (THF) on the asymmetric reduction of 2,5-hexanedione with yeast HD-5 were investigated . The result was that the addition (3g·L-1) of Tween-80 was helpful for increasing the product yield, not affecting the the enantiomeric excess (e.e.) value of the product (2S,5S)-2,5-hexandiol.The effects of culture components, temperature, pH, cell concentration, initial glucose concentration, initial substrate and product concentration on the catalytic bioreduction activity and enantioselectivity of Saccharomyces cerevisiae HD-5 were studied systematically. The optimal reaction condition was organic culture, 32℃, pH 6.0, cell concentration of 50 g?L-1(cell dry weight), initial glucose concentration of 20 g?L-1. With the increase of the initial substrate concentration, the average accumulation rate of the intermediate increased gradually. When this intermediate concentration was higher than 30 mmol·L-1, the production rate of the object product 2,5-hexandiol decreased. When the product concentrations was higher than 70 mmol·L-1, the production rate of the object product decreased obviously.Under the optimal reaction condition, while the substrate concentration was adjusted at about 5.0 mmol·L-1(at first period of reduction 12h), the concentrations of the substrate fed into the reactor were added up to 56.5 mmol·L-1. When the reaction lasted for 72h, the object product concentration reached 53.8 mmol·L-1, and the product yield was 95.2%, the selectivity for product purity was 94.38 % , the e.e. value was 93.76 % for (2S,5S)-2,5-hexandiol.This work, including the screen of the special strain with high bioreduction activity of (2S,5S)-2,5-hexandiol and the optimization of catalytic reduction conditions, provide an important foundation for the further scale-up experiments.
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