Abstract
β-Galactosidase is widely used for hydrolysis of whey lactose. However, galactose inhibition has acted as a major constraint on the catalytic process. Thus, it is sensible to improve upon this defect in β-galactosidase through protein modification. To reduce the galactose inhibition of Aspergillus candidus β-galactosidase (LACB), four amino acid positions were selected for mutation based on their molecular bindings with galactose. Four mutant libraries (Tyr96, Asn140, Glu142, and Tyr364) of the LACB were constructed using site-directed mutagenesis. Among all of the mutants, Y364F was superior to the wild-type enzyme. The Y364F mutant has a galactose inhibition constant (K (i)) of 282 mM, 15.7-fold greater than that of the wild-type enzyme (K (i) = 18 mM). When 18 mg/ml galactose was added, the activity of the wild-type enzyme fell to 57% of its initial activity, whereas Y364F activity was maintained at over 90% of its initial activity. The wild-type enzyme hydrolyzed 78% of the initial lactose (240 mg/ml) after 48 h, while the Y364F mutant had a hydrolysis rate greater than 90%. The β-galactosidase Y364F mutant with reduced galactose inhibition may have greater potential applications in whey treatment compared to wild-type LACB.