Cyclomaltodextrin glucanotransferase from Bacillus circulans E 192. I. 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.
Bovetto
L J
LJ
Laboratoire de Chimie Biologique, Université des Sciences et Techniques de Lille Flandres Artois, Villeneuve D'Ascq, France.
Backer
D P
DP
Villette
J R
JR
Sicard
P J
PJ
Bouquelet
S J
SJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D013696:Temperature
D000596:Amino Acids; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase