Title Purification and characterization of an exo-polygalacturonase from botrytis cinerea
Author Lee, Tae Ho · Kim, Byung Young · Chung, Young Ryun · Lee, Sang Yeol¹ · Lee, Chang Won · Kim, Jae Won *
Address Department of Microbiology and Biochemistry¹, College of Natural Science and Research Institute of Natural Sciences, Gyeongsanf National University
Bibliography Journal of Microbiology, 35(2),134-140, 1997,
Key Words Botrytis cenerea, Enzyme production, exo-polygalacturonase
Abstract Botrytis cinerea T81-1 has been shown to produce at least four different polygalacturonases into a liquid medium containing citrus pectin, a carbon sousrce. One of the enzymes, which had an apparent molecular weight of 66 kDa estimated by denatured polyacrylamide gel electrophoresis, was purified to electrophoretic homogeneity by a series of procedures including a cetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. The molecular weight of native enzyme was determined to be 64 kDa by gel permeation chromatography indicating the enzyme to be a single polypeptide chain. By viscometric analysis, the enzyme was revealed as exo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as Ca^2+, Mg^2+, and Cu^2+. Km and Vmax for polygalacturonic acid hydrolysis were 0.33 mg/ml and 28.6 nM/min, respectively. The optimum temperature for enzymatic activity was 50℃. And the enzyme showed optimal pH values between 4.0 and 5.0. The enzyme was stable upto 12 hours in the range of pH 3 to 8 and at temperature below 30℃.
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