Enzymes Involved in the Biosynthesis and Degradation of Cyclic Maltosyl-maltose in Arthrobacter globiformis M6

Abstract

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide from starch. This oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate α-1,4 and α-1,6 linkages. The cyclic tetrasaccharide, cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}, was designated cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as α-amylase, β-amylase, glucoamylase, isoamylase, pullulanase, maltogenic α-amylase and α-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. A glycosyltransferase involved in the synthesis of CMM from starch was purified to homogeneity from the culture supernatant of A. globiformis M6. The enzyme acted on maltooligosaccharides that have degrees of polymerization more than 3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan and dextran. The CMM-forming enzyme catalyzed both intermolecular and intramolecular α-1,6-maltosyl transfer reaction and found to be a novel maltosyltransferase (6MT). To reveal the degradation pathway of CMM, we identified two enzymes, CMM hydrolase (CMMase) and α-glucosidase, as the responsible enzymes from the cell-free extract of the strain. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates; however, it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the α-1,6-linkage of CMM. α-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates; however, it did not degrade CMM. Furthermore, when CMMase and α-glucosidase existed simultaneously in the reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by synergistic action of CMMase and α-glucosidase. The genes for 6MT, CMMase and α-glucosidase were cloned from the genomic library of A. globiformis M6. The four conserved regions common in the α-amylase family enzymes were also found in 6MT, CMMase and α-glucosidase, indicating that these enzymes should be assigned to this family. In the cloning experiments, three other open reading frames (ORFs) were found. These ORFs were expected to encode proteins concerned with incorporation of CMM via cell membrane. The genes for CMMase and α-glucosidase and three ORFs were located downstream of the gene for 6MT, and expected to form gene cluster. The results of gene analysis suggested that A. globiformis M6 has a unique starch utilization pathway via CMM.