Production and immobilization of a proteinase-reduced cyclodextrin glycosyltransferase preparation

Cyclodextrin glycosyltransferase (CGTase) was produced by a 3-day cultivation of Bacillus macerans growing in a natural medium containing grated-potatoes. Besides CGTase the culture supernatant contained a mixture of serine proteinases with a predominant subtilisin-like activity. By fractionated precipitation with ammonium sulphate the CGTase (molecular mass approximately 70 kDa) was concentrated and largely separated from the proteinases (molecular mass approximately 28 kDa). Among the various immobilization methods and carrier materials tested the enriched CGTase was covalently bound preferably onto porous glass beads using glutardialdehyde as cross-linker. The discontinuous conversion of soluble starch into beta-cyclodextrin was carried out with native as well as immobilized CGTase over 24 h. The batch re-usability of the fixed enzyme proved to be at least 20 times with a residual CGTase activity of 65%.