Purification of cyclic β-1,2-glucan synthetase from Agrobacterium radiobacter

Abstract

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 synthetases 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°C and pH 8–9 for the enzyme reaction. The activity was almost stable up to 40°C for 30 min, and mostly inactivated at 50°C 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%.