A Novel Glucanotransferase that Produces a Cyclomaltopentaose Cyclized by an .ALPHA.-1,6-Linkage

Abstract

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an α-1,6-linkage [ICG5; cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS-PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50°C, and stable from pH 4.5 to 9.0 at up to 35°C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 40°C. It acted on maltooligosaccharides that have degrees of polymerization (DP) of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular α-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular α-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme. The gene for IGTase was cloned from the genome of B. circulans AM7. The IGTase gene, designated igtY, consisted of 2985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The four conserved regions common in the α-amylase family enzymes were found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme (IgtZ) was purified. The enzyme acted on maltooligosaccharides that have a DP of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme, which has a novel amino-acid sequence, should be assigned to α-amylase. It is notable that both IGTase and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.