title: Analysis of cyclomaltodextrin glucanotransferase isoenzymes by isoelectric focusing in immobilized pH gradients.
abstract: The catalytically active subforms of cyclomaltodextrin glucanotransferase (CGTase; EC 2.4.1.19) from Bacillus circulans var. alkalophilus and from a strain in which the CGTase expressing gene had been cloned were studied by using isoelectric focusing (IEF) in immobilized and in conventional pH gradients. Even with high protein loads the best resolution was achieved in immobilized pH gradients (IPG). Native CGTase, focused on IPG 4.5-5.4, was resolved into more than 6 subforms, a major one with pI 4.97 and the others between pH 4.75 and 4.99. Cloned CGTase focused on the same IPG gel was resolved into more than 7 subforms over the pH range 4.78-5.22, with one major component of pI 5.20. A sensitive assay for the detection of CGTase on analytical gel slices was developed. The different behaviour between native and cloned CGTases was most probably a consequence of the presence--in the latter--of four extra amino acids at the N-terminus. A study of the reaction products by the enzymatic subforms confirmed that they are catalytically identical, hence small differences outside the active site are responsible for the presence microheterogeneity of the protein.
authors: Department of Biochemistry, University of Turku, Finland.
authors: P
authors: P
authors: Mattsson
authors: S
authors: S
authors: Meklin
authors: T
authors: T
authors: Korpela
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase