Aijun Sun

Enantio-selective preparation of (S)-1-phenylethanol by a novel marine GDSL lipase MT6 with reverse stereo-selectivity

Abstract We previously functionally characterized a novel marine microbial GDSL lipase MT6 and identified that the stereo-selectivity of MT6 was opposite to that of other common lipases in trans-esterification reactions. Herein, we have investigated the use of MT6 in stereo-selective biocatalysis through direct hydrolysis reactions. Notably, the stereo-selectivity of MT6 was also demonstrated to be opposite to that of other common lipases in hydrolysis reactions. Parameters, including temperature, organic co-solvents, pH, ionic strength, catalyst loading, substrate concentration, and reaction time, affecting the enzymatic resolution of racemic 1-phenylethyl acetate were further investigated, with the e.e. of the final ( S )-1-Phenylethanol product and the conversion being 97% and 28.5%, respectively, after process optimization. The lengths of side chains of 1-phenylethyl esters greatly affected the stereo-selectivity and conversion during kinetic resolutions. MT6 is a novel marine microbial GDSL lipase exhibiting opposite stereo-selectivities than other common lipases in both trans-esterification reactions and hydrolysis reactions.

Characterization of a novel marine microbial esterase and its use to make D-methyl lactate

A novel marine microbial esterase PHE14 was cloned from the genome of Pseudomonas oryzihabitans HUP022 isolated from the deep sea of the western Pacific Ocean. Esterase PHE14 exhibited very good tolerance to most organic solvents, surfactants and metal ions tested, thus making it a good esterase candidate for organic synthesis that requires an organic solvent, surfactants or metal ions. Esterase PHE14 was utilized as a biocatalyst in the asymmetric synthesis of D-methyl lactate by enzymatic kinetic resolution. D-methyl lactate is a key chiral chemical. Contrary to some previous reports, the addition of an organic solvent and surfactants in the enzymatic reaction did not have a beneficial effect on the kinetic resolution catalyzed by esterase PHE14. Our study is the first report on the preparation of the enantiomerically enriched product D-methyl lactate by enzymatic kinetic resolution. The desired enantiomerically enriched product D-methyl lactate was obtained with a high enantiomeric excess of 99% and yield of 88.7% after process optimization. The deep sea microbial esterase PHE14 is a green biocatalyst with very good potential in asymmetric synthesis in industry and can replace the traditional organic synthesis that causes pollution to the environment.

Functional characterization of a novel microbial esterase identified from the Indian Ocean and its use in the stereoselective preparation of ( R )-methyl mandelate

Genomic mining has identified a novel microbial alkaline esterase from the Indian Ocean. This esterase was overexpressed in E. coli BL21 (DE3) and further functionally characterized. Under optimal conditions (10 mmol/L substrate, pH 6.0, 2 h at 40 °C), this esterase can hydrolyze racemic methyl mandelate to (R)-methyl mandelate with very high optical purity (e.e. >99%) and yield (nearly 90%). Interestingly, the stereoselectivity of this esterase is opposite to that of two previously reported lipases that can generate (S)-methyl mandelate through the hydrolysis of racemic methyl mandelate. No organic solvents or other additives were required to optimize the optical purity and production of the final chiral product (R)-methyl mandelate, which can potentially simplify the production procedure of (R)-methyl mandelate catalyzed by esterase.

Utilization of one novel deep-sea microbial protease sin3406-1 in the preparation of ethyl ( S )-3-hydroxybutyrate through kinetic resolution

One novel protease sin3406-1 was identified from Streptomyces niveus SCSIO 3406, which was isolated from the deep sea of the South China Sea, and heterologously expressed in E. coli BL21(DE3). Protease sin3406-1 was further used as a green biocatalyst in the kinetic resolution of racemic ethyl-3-hydroxybutyrate. After careful process optimization, chiral product ethyl (S)-3-hydroxybutyrate was generated with an enantiomeric excess of over 99% and a conversion rate of up to 50% through direct hydrolysis of inexpensive racemic ethyl-3-hydroxybutyrate catalyzed by sin3406-1. Interestingly, protease sin3406-1 exhibited the same enantio-preference as that of esterase PHE21 during the asymmetric hydrolysis of the ester bonds of racemic ethyl-3-hydroxybutyrate. Through mutation studies and molecular docking, we also demonstrated that the four residues close to the catalytic center, S85, A86, Q87 and Y254, played key roles in both the hydrolytic activity and the enantioselectivity of protease sin3406-1, possibly through forming hydrogen bonds between the enzyme and the substrates. Deep-sea microbial proteases represented by sin3406-1 are new contributions to the biocatalyst library for the preparation of valuable chiral drug intermediates and chemicals through enzymatic kinetic resolution.

Utilization of deep-sea microbial esterase PHE21 to generate chiral sec-butyl acetate through kinetic resolutions

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.

Functional characterization of salt-tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)-3-hydroxybutyrate

The two enantiomers of ethyl 3-hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)-3-hydroxybutyrate. Herein, we also functionally characterized one novel salt-tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)-3-hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio-selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates.

Characterization of a novel deep-sea microbial esterase EstC10 and its use in the generation of (R)-methyl 2-chloropropionate

A novel esterase EstC10 from Bacillus sp. CX01 isolated from the deep sea of the Western Pacific Ocean and the functionalities of EstC10 was characterized. At present, the reports about the kinetic resolution of racemic methyl 2-chloropropionate were quite rare. So we developed deep-sea microbial esterase EstC10 as a novel biocatalyst in the kinetic resolution of racemic methyl 2-chloropropionate and generate (R)-methyl 2-chloropropionate with high enantiomeric excess (>99%) after the optimization of process parameters such as pH, temperature, organic co-solvents, surfactants, substrate concentration and reaction time. Notably, the optimal substrate concentration (80 mmol/L) of esterase EstC10 was higher than the kinetic resolution of another esterase, Est12-7 (50 mmol/L). The novel microbial esterase EstC10 identified from the deep sea was a promising green biocatalyst in the generation of (R)-methyl 2-chloropropionate as well of many other valuable chiral chemicals in industry.