POTATO D-ENZYME CATALYZES THE CYCLIZATION OF AMYLOSE TO PRODUCE CYCLOAMYLOSE, A NOVEL CYCLIC GLUCAN

Potato D-enzyme was purified from recombinant Escherichia coli, and it s action on synthetic amylose (average M(r) of 320,000) was analyzed. D-enzyme treatment resulted in a decrease in the ability of the amylos e to form a blue complex with iodine. Analysis of the products indicat ed that the enzyme catalyzes an intramolecular transglycosylation reac tion on amylose to produce cyclic alpha-1,4-glucan (cycloamylose). Con firmation of the cyclic structure was achieved by demonstrating the ab sence of reducing and nonreducing ends, resistance to hydrolysis by gl ucoamylase (an exoamylase), and by ''time of flight'' mass spectrometr y. The degree of polymerization of cycloamylose products was determine d by time of flight mass spectrometry analysis and by highperformance anion-exchange chromatography following partial acid hydrolysis of pur ified cycloamylose molecules and was found to range from 17 to several hundred. The yield of cycloamylose increased with time and reached >9 5%, D-enzyme did not act upon purified cycloamylose, but if glucose wa s added as an acceptor molecule, smaller cyclic and linear molecules w ere produced. The mechanism of the cyclization reaction, the possible role of the enzyme in starch metabolism, and the potential application s for cycloamylose are discussed.