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Purification, Characterization, and Gene Cloning of a Novel Maltosyltransferase from an Arthrobacter globiformis Strain That Produces an Alternating α-1,4- and α-1,6-Cyclic Tetrasaccharide from Starch

ABSTRACT A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{→6)-α-d-Glcp(1→4)-α-d-Glcp(1→6)-α-d-Glcp(1→4)-α-d-Glcp(1→}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50°C and was stable from pH 5.0 to 9.0 and up to 30°C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 45°C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of ≥3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose → 64-O-α-maltosyl-maltotetraose + maltose and (ii) 64-O-α-maltosyl-maltotetraose → CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular α-1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to α-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the α-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.