Improved Catalytic Performance of Bacillus megaterium Epoxide Hydrolase in a Medium Containing Tween-80 Peng-Fei Gong, Jian-He Xu,* Yan-Fa Tang, and Hui-Yuan Wu Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China A new epoxide hydrolase with high enantioselectivity toward (R)-glycidyl phenyl ether (R-GPE) was partially purified from Bacillus megaterium strain ECU1001. The maximum activity of the isolated enzyme was observed at 30 °C and pH 6.5 in a buffer system with 5% (v/v) of DMSO as a cosolvent. The enzyme was quite stable at pH 7.5 and retained full activity after incubation at 40 °C for 6 h. Interestingly, when the cosolvent DMSO was replaced by an emulsifier (Tween-80, 0.5% w/v) as an alternative additive to help disperse the water-insoluble substrate, the apparent activity of the epoxide hydrolase significantly increased by about 1.8-fold, while the temperature optimum shifted from 30 to 40 °C and the half-life of the enzyme at 50 °C increased by 2.5 times. The enzymatic hydrolysis of rac-GPE was highly enantioselective, with an E-value (enantiomeric ratio) of 69.3 in the Tween-80 emulsion system, which is obviously higher than that (41.2) observed in the DMSO-containing system. Introduction Enantiopure epoxides and vicinal diols are extensively employed as useful chiral building blocks for synthesis of various bioactive products in the pharmaceutical and agrochemical industries. For example, glycidyl phenyl ether (GPE) is one of the potentially useful aryloxy epoxides for the synthesis of chiral amino alcohols (1) and -blockers (2). One of the most promising ways for preparing such chiral synthons under environmentally gentle conditions is the enantioselective hydrolysis of racemic epoxides using cofactor-independent epoxide hydrolases [EHs; EC 3.3.2.3] (3). In our laboratory, a newly isolated bacterial strain, Bacillus megaterium ECU1001, producing an epoxide hydrolase with high enantioselectivity toward (R)-GPE, has been identified and proved to be very useful for chiral synthesis in preparative scale (4). More importantly, this strain exhibited a complementary enantioselectivity as compared with those strains so far described (5, 6), affording the unreacted epoxide of (S)-configuration, which is the solely useful enantiomer for synthesis of bioactive -blockers (2). However, the intact cell-mediated reaction may be hampered by some technical limitations such as lower specific activity of the cells and difficulties in separation of the products. In addition, the reaction kinetics is almost impossible to be measured accurately because the insoluble substrate and product are adsorbed onto the cells. In this work, the reaction was catalyzed by the use of an isolated enzyme instead of the intact cell of B. megaterium ECU1001. We have investigated the catalytic performance of the isolated EH, getting some interesting results since no satisfactory EH had been discovered previously for this special type of substrate (GPE). Further study showed that the addition of Tween-80 instead of DMSO, which was frequently employed by previous researchers, could significantly improve the catalytic performance of the EH enzyme, including activ- ity, stability and enantioselectivity. Materials and Methods Chemicals and Microbial Strain. rac-GPE was purchased from ACROS Co., Ltd.; butyl-Toyopearl was from Tosoh Corp., Japan; DEAE-cellulose and Sephadex G-75 were products of Pharmacia Biotech Co.; and all other chemicals were also obtained commercially. Bacil- lus megaterium ECU1001 was isolated from soil (4) and cultivated under previously described conditions (7). Partial Purification of Enzyme. All purification steps were performed at 0-5 °C, and Tris-HCl buffer (with indicated concentration, pH 7.5, containing 1 mM cysteine and 1 mM EDTA) was used as a standard buffer. The crude extract was prepared by sonication for 30 min of cells (45 g, wet weight) suspended in 150 mL of 100 mM Tris-HCl buffer. The disrupted cells were centrifuged at 30,000 × g for 25 min. The supernatant was collected and fractionated with ammonium sulfate (55-85% satu- ration). The active precipitate was dialyzed overnight against buffer A (10 mM Tris-HCl) before being applied onto a DEAE-cellulose column (φ2 × 25 cm) equilibrated with buffer A. Active fractions were eluted at 0.25-0.40 M gradient of NaCl and pooled, and ammonium sulfate was added up to a concentration of 1.8 M. The solution was then applied onto a butyl-Toyopearl 650 column (φ1 × 15 cm) that had been equilibrated with buffer A containing 1.8 M ammonium sulfate. The enzyme was eluted with a linear gradient of 1.8 to 0 M ammonium sulfate in buffer A. The fractions displaying high enzyme activity were pooled and dialyzed against buffer A. After concentrated with PEG-2000, the enzyme solution was applied onto a Sephadex G-75 column (φ2 × 95 cm) equilibrated with buffer A. The active fractions were eluted with buffer A at a flow rate of 1 mL min -1 and collected in fractions of 3 mL. The resultant preparation was stored at 4 °C. * To whom correspondence should be addressed. Fax: +86-21- 6425-3904; E-mail: jianhexu@ecust.edu.cn. 652 Biotechnol. Prog. 2003, 19, 652-654 10.1021/bp020293v CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 02/14/2003