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Bacillus sp. DL-2 was isolated from the deep sea of the Western Pacific and further utilized as novel biocatalysts to efficiently asymmetrically hydrolyze (±)-1-phenylethyl acetate. After the optimization of hydrolytic reactions, chiral chemicals (R)-1-phenylethanol and (S)-1-phenylethyl acetate were obtained with high optical purities (96% and 99.8%, respectively). Our research is about the asymmeric hydrolysis of (±)-1-phenylethyl acetate using whole-cell biocatalysts. In addition, the optical purity of (S)-1-phenylethyl acetate generated through the kinetic resolution of (±)-1-phenylethyl acetate using the whole-cells of Bacillus sp. DL-2 was the highest report so far. Using the whole cells of deep sea bacterium Bacillus sp. DL-2 as the biocatalysts is an environmentally friendly method and will play critical roles in industrial asymmetric synthesis.

Chiral 1-phenylethanol and its ester derivative are important chiral chemicals in diverse industries and the preparation of those optically pure enantiomers is of great importance. One bacterium, Bacillus sp. DL-2, whose extracellular proteases could efficiently asymmetrically hydrolyse (±)-1-phenylethyl acetate, was isolated from the deep sea of the Western Pacific. After the immobilization of extracellular proteases and the optimization of enzymatic reactions, (R)-1-phenylethanol was prepared with the enantiomeric excess (e.e.) being 97% and the yield being 41%, respectively. The optimal resolution reaction condition for the preparation of (R)-1-phenylethanol using immobilized extracellular proteases was found to be 5-mM (±)-1-phenylethyl acetate, 360 mg/mL immobilized extracellular proteases, pH 6.5, and 20 °C for 2 h. (S)-1-phenylethyl acetate was generated through enzymatic kinetic resolution with the e.e. being as high as 99% and the yield being 71%, respectively. The optimal resolution reaction condition for the preparation of (S)-1-phenylethyl acetate was found to be 2.5-mM (±)-1-phenylethyl acetate, 440 mg/mL immobilized extracellular proteases, pH 7.5, and 35 °C for 10 h. Our report is the first report about the kinetic resolution of (±)-1-phenylethyl acetate using proteases and the enantio-preference of the proteases was found to be the same as those of most other esterases/lipases. Also notably, the optical purity of (S)-1-phenylethyl acetate generated through kinetic resolution using the proteases of Bacillus sp. DL-2 was the highest report so far. Proteases from deep-sea Bacillus sp. DL-2 are new contributions to the biocatalyst library for the preparation of valuable chiral alcohols and chiral esters through kinetic resolution.

We previously identified and characterized 1 novel deep-sea microbial esterase PHE21 and used PHE21 as a green biocatalyst to generate chiral ethyl (S)-3-hydroxybutyrate, 1 key chiral chemical, with high enantiomeric excess and yield through kinetic resolution. Herein, we further explored the potential of esterase PHE21 in the enantioselective preparation of secondary butanol, which was hard to be resolved by lipases/esterases. Despite the fact that chiral secondary butanols and their ester derivatives were hard to prepare, esterase PHE21 was used as a green biocatalyst in the generation of (S)-sec-butyl acetate through hydrolytic reactions and the enantiomeric excess, and the conversion of (S)-sec-butyl acetate reached 98% and 52%, respectively, after process optimization. Esterase PHE21 was also used to generate (R)-sec-butyl acetate through asymmetric transesterification reactions, and the enantiomeric excess and conversion of (R)-sec-butyl acetate reached 64% and 43%, respectively, after process optimization. Deep-sea microbial esterase PHE21 was characterized to be a useful biocatalyst in the kinetic resolution of secondary butanol and other valuable chiral secondary alcohols.