Purification and Characterization of Cyclic Maltosyl-(1→6)-Maltose Hydrolase and α-Glucosidase from an Arthrobacter globiformis Strain

Abstract

Cyclic maltosyl-maltose [CMM, cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate α-1,4 and α-1,6 linkages. CMM is synthesized from starch by the action of 6-α-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and α-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 °C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 °C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the α-1,6-linkage of CMM. The molecular mass of α-glucosidase was determined to be 60.1 kDa by SDS–PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for α-glucosidase activity were 7.0 and 35 °C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 °C. α-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and α-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and α-glucosidase.

• cyclic maltosyl-maltose
• α-1,6-linkage
• Arthrobacter globiformis
• CMM hydrolase
• α-glucosidase