Title : A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: Purification and characterization - Beloti_2013_Protein.Expr.Purif_91_175 |
Author(s) : Beloti LL , Costa BZ , Toledo MA , Santos CA , Crucello A , Favaro MT , Santiago AS , Mendes JS , Marsaioli AJ , Souza AP |
Ref : Protein Expr Purif , 91 :175 , 2013 |
Abstract :
A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an alpha/beta hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45 degrees C); showing optimum performance at pH 6.0 and 30 degrees C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495muM and 0.24muM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols. |
PubMedSearch : Beloti_2013_Protein.Expr.Purif_91_175 |
PubMedID: 23973866 |
Beloti LL, Costa BZ, Toledo MA, Santos CA, Crucello A, Favaro MT, Santiago AS, Mendes JS, Marsaioli AJ, Souza AP (2013)
A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: Purification and characterization
Protein Expr Purif
91 :175
Beloti LL, Costa BZ, Toledo MA, Santos CA, Crucello A, Favaro MT, Santiago AS, Mendes JS, Marsaioli AJ, Souza AP (2013)
Protein Expr Purif
91 :175