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Poster Presentation 6-22
Cloning and Purification of an Esterase from Pseudomonas fluorescens KCTC 1767 with High Activity Toward (S)-Ketoprofen
Ji-Heui Kim, Gi-sub Choi, Ji-youn Kim, Geun-joong Kim, Yeon-woo Ryu
Ajou University Department of Molecular Science & Technology 5 Wonchon Paldal, Suwon, Kyounggi 442-749 Korea
Telephone: (82) 31-219-2455; Fax: (82) 31-216-8777; E-mail: jiheui_kim@hanmail.net
The comparative study of enzymes that catalyze similar reactions but have a different substrate spectrum and/or stereospecificity is a powerful approach to understanding the reaction mechanism between the relative enzymes, and is also a useful tool in cloning the related enzyme without typical cloning from the DNA library of genomic pools. For this purpose, we conducted an approach that compared the molecular and protein level of esterases from various sources including a previously identified (S)-stereospecific esterase of Pseudomonas sp. ES1. As expected, we found esterase-family genes which shared a high similarity at the protein and genetic level in the identical genus Pseudomonad. Based on esterase sequences, we constructed degenerate primers, which were used for direct PCR-cloning of the Pseudomonas flourescens KCTC 1767 esterase gene. The PCR product was cloned to three vectors. The expression level of pTrc99A clone was very low, so it was subcloned to pMAL-c2X and pQE30 expression vector. The pMAL-c2X was made of MBP (maltose binding protein) fusion protein used for single-step purification. However, it degraded very quickly. MBP-esterase fusion protein could hydrolyze ketoprofen ethyl ester and the pMAL-c2X expression system exhibited the highest expression level of the three vectors. Esterase size was estimated to be 41 kDa by gel filtration chromatography. The purified enzyme was stable between pH 7.0 and pH 10. The enzyme had residual activity for 2 h, preincubation up to 40oC--but over 50oC, lost its activity completely. The highest activity was found for p-nitrophenyl acetate. Esters of longer fatty acids (>C8) were hydrolyzed at significantly lower rates. The purified esterases exhibited very high enantioselectivity in the resolution of racemic ketoprofen ethyl ester (E >99) and the specific activity was calculated at 27,000 units. The specificity of purified esterase toward ketoprofen ethyl ester was confirmed by determining kinetic data; Km and Vmax were measured to be 3.26 mM and 56.82 mM/mg-protein/min, respectively.
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