CYCLOMALTODEXTRIN GLUCANOTRANSFERASE FROM BACILLUS-CIRCULANS E-192 .1. PURIFICATION AND CHARACTERIZATION OF THE ENZYME

The cyclomaltrodextrin glucanotransferase (CGTase) [1,4‐alpha‐D‐glucan:4‐alpha‐D‐(1,4‐alpha‐D‐glucano)‐transferase (cyclizing), EC 2.4.1.19] from Bacillus circulans E 192 has been purified to homogeneity by Cetavlon treatment, ammonium sulfate precipitation, DEAE Trisacryl M chromatography, Q Fast Flow chromatography, and affinity on beta‐cyclodextrin‐Sepharose 4B. Two isoenzymes were separated by FPLC on a Mono Q column. Their isoelectric points were estimated as 6.7 and 6.9 and they represented 13 and 87%, respectively, of the initial activity. Their molecular weight, pH, and temperature optima were estimated as 78,000, 5.5, and 60 degrees C, respectively. Kinetic parameters indicated that both enzymes had the same properties; they preferentially modified high‐molecular‐weight substrates to produce cyclodextrins. The apparent Vmax and Km values for soluble starch were 43 mumol of beta‐cyclodextrin/min/mg of protein and 0.57% (w/v), respectively. Although this CGTase is not markedly thermostable, it is protected against heat denaturation by substrate, product, and/or calcium ions. The ratios of alpha‐, beta‐, and gamma‐cyclodextrins produced have been determined as 1/7/2 in the initial phase of the reaction and 3/3/1 at equilibrium. 1992 The Swiss Political Science Review