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 iso-
maltooligosaccharides (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.