Conversion of the maltogenic alpha-amylase Novamyl into a CGTase

Novamyl is a thermostable five-domain maltogenic alpha-amylase that shows s equence and structural homology with the cyclodextrin glycosyltransferases (CGTases), Comparing X-ray crystal structures of Novamyl and CGTases, two m ajor differences in the active site cleft were observed: Novamyl contains a loop insertion consisting of five residues (residues 191-195) and the loca tion of an aromatic residue known to be essential to obtain an efficient cy clization reaction. To convert Novamyl into a cyclodextrin (CD)producing en zyme, the loop was deleted and two substitutions, F188L and T189Y, were int roduced. Unlike the parent Novamyl, the obtained variant is able to produce beta-CD and showed an overall conversion of starch to CD of 9%, compared w ith CGTases which are able to convert up to 40%. The lower conversion compa red with the CGTase is probably due to additional differences in the active site cleft and in the starch-binding E domain. A variant with only the fiv e-residue loop deleted was not able to form beta-CD.