RESUMO
Esterases (EC 3.1.1.X) have been used as biocatalysts due to their good stability, high chemo-, regio- and stereoselectivity. In our previous studies, Bacillus megaterium WZ009 harboring esterase displayed the unique capability to convert (S)-4-Chloro-3-hydroxyethylbutyrate (CHBE) in the racemate to (S)-3-hydroxy-γ-butyrolactone (HL) through stereoselective hydrolysis, dechlorination, and lactonization. The remaining (R)-CHBE and formed (S)-HL could be obtained in a one-pot enzymatic reaction. An esterase from B. megaterium WZ009 was purified and was found to have 466 encoded amino acids and an apparent molecular mass of 55 kDa. The purified esterase exhibited maximal activity at a temperature of 25 °C and at a pH of 11.5 towards 100 mM CHBE. When the stereoselective biocatalysis of rac-CHBE was performed using the recombinant Escherichia coli BL21 (DH3) cells harboring the esterase, the catalytic activity increased by 20-fold compared with the original strain B. megaterium WZ009. With the addition of activated carbon (62 g/L) in the reaction system, the conversion was increased from 39% to 45% at a substrate concentration of 750 mM. Another remarkable advantage is that both of the obtained residual (R)-CHBE and the formed (S)-HL had high optical purities (e.e.s > 99.9%, e.e.p > 99.9%), thereby making this esterase a usable biocatalyst for industrial application.
Assuntos
Bacillus megaterium , Proteínas de Bactérias , Clonagem Molecular , Esterases , Expressão Gênica , Bacillus megaterium/enzimologia , Bacillus megaterium/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Esterases/biossíntese , Esterases/química , Esterases/genética , Esterases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A new esterase-producing strain (Bacillus cereus WZZ001) which exhibiting high hydrolytic activity and excellent enantioselectivity on rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester (R, S-1) has been isolated from soil sample by our laboratory. In this study, the stereoselective hydrolysis of (R, S-1) was performed using the recombinant Bacillus cereus esterase which expressed in Escherichia coli BL21 (DE3). Under the optimized conditions of pH 8.0, 35 °C, and concentration of substrate 400 mM, a successful enzymatic resolution was achieved with an e.e. s of 99.5 % and conversion of 49 %. Immobilization considerably increased the reusability of the recombinant esterase; the immobilized enzyme showed excellent reusability during 6 cycles of repeated 2 h reactions at 35 °C. Thereby, it makes the recombinant B. cereus esterase a usable biocatalyst for industrial application.
