Purification of Cyclic β-1,2-Glucan Synthetase from Agrobacterium radiobacter

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A cyclic β-1,2-glucan synthetase was solubilized by sonicating 100,000×g pellets of the crude extract of Agrobacterium radiobacter IFO 12665 A1-5 in the presence of ethylenediaminetetraacetic acid. The enzyme was separated by DEAE-Sephadex column chromatography into adsorbed and nonadsorbed fractions (cyclic β-1,2-glucan synthetase I and II, respectively). Both enzymes were further purified by Toyo-pearl HW55 gel filtration and high performance liquid chromatography on Shodex WS-803. Neither enzyme could penetrate into 4% polyacrylamide gel in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of synthetase I was 9.5×10^<-4> units/mg and it was purified 26-fold from the crude extract with a yield of 1.9%. Synthetase I was optimal at 33℃ and pH8-9 for the enzyme reaciton. The activity was almost stable up to 40℃ for 30 min, and mostly inactivated at 50℃ for 30 min. The enzyme activity was stimulated about three times with the addition of 10-20 mM FeCl_3. The enzyme produced the same cyclic β-1,2-glucan with a polymerization degree of 17-24 from UDP-glucose that the cell produced. The specific activity of synthetase II was 23.6×10^<-4> units/mg and it was purified 60-fold from the crude extract with a yield of 2.3%.
公益社団法人日本生物工学会の論文
1991-12-25