A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: Purification and characterization

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• 作者：Lilian L. Beloti ; Bruna Z. Costa ; Marcelo A.S. Toledo ; Clelton A. Santos ; Aline Crucello ; Marianna T.P. F¨¢varo ; Andr¨¦ S. Santiago ; Juliano S. Mendes ; Anita J. Marsaioli ; Anete P. Souza
• 刊名：Protein Expression and Purification
• 出版年：2013
• 出版时间：October, 2013
• 年：2013
• 卷：91
• 期：2
• 页码：175-183
• 全文大小：1388 K

文摘

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 ¦Á/¦Â 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 ¡ãC); showing optimum performance at pH 6.0 and 30 ¡ãC. The Michaelis constant (K<sub>Msub>) and maximum rate (V<sub>maxsub>) values were 495 ¦ÌM and 0.24 ¦ÌM/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 5 min 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 24 h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.