Abstract
β-Cyclodextrin glycosyltransferase (β-CGTase) belongs to the α-amylase family of enzymes and converts starch to cyclic oligosaccharides called β-cyclodextrins (β-CD). The β-CGTase from alkalophilic Bacillus sp. N-227 was separately mutagenized to give three site-directed β-CGTase mutants, Y127F, R254F and D355R, that showed enhanced cyclization activity towards a starch substrate from 1.64 to 2.1-folds. Kinetic studies indicate that the mutants had higher affinity towards the substrate than the wild type β-CGTase. The Y127F mutant had the highest affinity which was indicated by the lowest Km of 15.30 mM and the highest catalytic activity. Increasing hydrophobicity around the catalytic center appeared to favor the cyclization activity of the mutants. The β-CGTase and the three mutants showed optimal enzyme activity at 60 °C and pH 6.0. All the enzymes were stable for at least 60 min across a wide pH range (5.0–7.0).
Highlights
Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) belongs to the a-amylase family of enzymes (Bautista et al 2012; Janecek and Sevcik 1999; MacGregor et al 2001), which catalyzes three type reactions towards starch, namely cyclization to yield cyclodextrins (CDs), hydrolysis, and disproportionation
Abstract b-Cyclodextrin glycosyltransferase (b-CGTase) belongs to the a-amylase family of enzymes and converts starch to cyclic oligosaccharides called b-cyclodextrins
Previous studies have shown that mutations in the amino residues of b-CGTase influence the enzyme activity of bCGTase at different levels (Leemhuis et al 2002a, 2003b, 2004a, b; Penninga et al 1995; van der Veen et al 2000a)
Summary
Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) belongs to the a-amylase family of enzymes (Bautista et al 2012; Janecek and Sevcik 1999; MacGregor et al 2001), which catalyzes three type reactions towards starch, namely cyclization to yield cyclodextrins (CDs), hydrolysis, and disproportionation (van der Veen et al 2000b, c). N-227 was separately mutagenized to give three site-directed bCGTase mutants, Y127F, R254F and D355R, that showed enhanced cyclization activity towards a starch substrate from 1.64 to 2.1-folds. Kinetic studies indicate that the mutants had higher affinity towards the substrate than the wild type b-CGTase.
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