title: Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22.
abstract: A novel raw starch degrading alpha-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5-9.0 whereas it was most stable in the pH range 6-9. The CGTase was most active in the temperature range 35-50 degrees C. This CGTase is inherently temperature labile and rapidly loses activity above 30 degrees C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40 degrees C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30 degrees C for a month. The K(m) and k(cat) values for the pure enzyme were 1.35 mg ml(-1) and 249 &mgr;M mg(-1) min(-1), respectively, with soluble starch as the substrate. The enzyme predominantly produced alpha-cyclodextrin without addition of any complexing agents. The conditions employed for maximum alpha-cyclodextrin production were 100 g l(-1) gelatinized soluble starch or 125 g l(-1) raw wheat starch at an enzyme concentration of 10 U g(-1) of starch. The alpha:beta:gamma-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.
authors.affiliation: Chemical Engineering Division, National Chemical Laboratory, 411 008, Pune, India
authors.forename: B N.
authors.initials: BN
authors.lastname: Gawande
authors.forename: A Y.
authors.initials: AY
authors.lastname: Patkar