Role of Glu720 mutation in transglycosylating α-glucosidase derived from Aspergillus neoniger NCIM 1400

Transglycosylating α-glucosidase (tAG) is used as processing aid for the industrial production of isomaltooligosaccharides (IMO). Glutamate residue at 720 position (E720) was rationally selected and mutated to elucidate its role in the catalytic activity (hydrolytic and transglycosylation) and conformational changes of the enzyme. Seven mutant libraries of the wtAG (wild-type tAG) were successfully constructed, and amongst them, E720Y showed a 1.9-fold increase in the specific hydrolytic activity over the wtAG. The thermostability of E720Y was 11 % higher than that of wtAG. The E720Y mutation significantly prolonged storage stability over a 45-day period with 71.6 % and 63 % of its relative activity at 4 °C and 30 °C, respectively, whereas wtAG retained only 46.3 % and 33.6 % of its relative activity at those temperatures. Circular dichroism (CD) and steady-state fluorescence studies suggested that E720Y mutation had a subtle effect on the secondary structure (reduced random coil and higher α-helical content) but led to no major changes in the tertiary structure of wtAG. The Molecular dynamics simulation results indicate that E720Y induces structural changes, which align with the findings from the CD analysis. The total IMO yield of wtAG and E720Y mutant was found to be 0.29 and 0.38 g g^-1 of maltose, respectively.