Aspergillus niger alpha-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of alpha-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of alpha-(1 -> 6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(alpha 1-4)Glc] yields both alpha-(1 -> 6)- and alpha-(1 -> 4)-glucosidic linkages, the latter constituting similar to 25% of the total transfer reaction product. The maltotriose [Glc(alpha 1-4)Glc(alpha 1-4)Glc], alpha-(1 -> 4)-glucosyl product disappears quickly, whereas the alpha-(1 -> 6)-glucosyl products panose [Glc(alpha 1-6)Glc(alpha 1-4)Glc], isomaltose [Glc(alpha 1-6)Glc], and isomaltotriose [Glc(alpha 1-6)Glc(alpha 1-6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the alpha-(1 -> 4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W).