| Summary: | 碩士 === 逢甲大學 === 化學工程學所 === 90 === Abstract
In this study, an intracellular carboxylesterase (EC 3.1.1.1) from Psudomonas sp. was purified and further characterized. The protein with a predicted isoelectric point of 9.49 consists of 365 amino acids and assumes a molecular weight of 40.68 kDa. Overproduction of the carboxylesterase in Escherichia coli BL21(DE3) resulted in a large formation of inclusion body was. A protocol of the protein renaturation was successfully developed. The insoluble inclusions were first solublized with the solution comprising 6M Gdn-HCl and 32mM DTT, followed by allowing the protein refolding in a 1.5M Tris-buffer. With the use of immobilized metal affinity chromatography, the renatured protein was purified 58-fold to homology. The protein functioned optimally at 20℃-40℃and pH 7.4-9.0 and showed preference toward the short-chain fatty acids. The determination of Vmax/Km indicated that the protein had the highest substrate specificity on para-nitropheny butyrate. It was found that the enzyme activity was seriously inhibited by Hg2+, Ag2+, Pb2+, Ba2+ and NBS. EDTA exerted no inhibition on the enzyme activity, suggesting that the protein had no requirements of metal ions for active function.
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