Resolution of rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester by immobilized cell

Abstract:

[Objective] We screened and identified a strain capable of enantioelectively hydrolyzing rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester, followed by using immobilized cells to catalyze the racemic ester.[Methods] We used rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester as esterase induction in the medium inoculated with soils to enrich the target microorganism. The strain with high activity for enantioselective hydrolysis of rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester was identified by 16S rRNA sequence analysis, morphological observation, physiological and biochemical properties. We optimized the immobilization conditions of the strain, studied the catalytic properties and operated stability of immobilized cells.[Results] We isolated a gram-negative bacterial strain which was identified as Methylopila. The optimal immobilization conditions were shown as follows:0.15% (V/V) of polymine, 0.2% (V/V) of glutaraldehyde, 6 g/L diatomite and 100 g/L cells. Compared with free cells, the optimum pH of immobilized cells changed from 8.0 to 8.5, the optimum temperature changed from 35℃ to 40℃, the stability of pH and temperature was improved. Cu²⁺, Mn²⁺ and Ca²⁺ can improve enzyme activity, but Zn²⁺ and Fe²⁺ inhibited it. The organic solvent tolerance of immobilized cells was higher than that of free cells. Affinity of immobilized cells with substrates decreased with the increased K_m value. We used the immobilized cells to catalyze rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester at the concentration of 200 g/L for 20 h, the remaining substrate was (S)-ester with ee_s>99.4% and the yield reached 47.8%. The relative activity of the immobilized cells remained 80% after 12 batches.[Conclusion] We established a process to produce (S)-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester from rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester by Methylopila sp. cxzy-L013 immobilized cells as the catalyst, which has a great potential for industrialization.