Glucose tolerance, an enzymatic performance in the presence of glucose, was improved with Geobacillus thermoglucosidasius oligo-1,6-glicosidase (GTAGL) by site-directed mutagenesis. The quadruple mutant of GTAGL (qGTAGL: M203W/Q216E/G259E/R298I) produced by this work resulted in an increase of the glucose tolerance. Although GTAGL lost its enzyme activity in the presence of 2.2% glucose, qGTAGL retained the activity in the presence of 2.7% glucose. Notably, enzymatic properties including thermostability and optimal temperature were not severely affected by the mutations.