α-Galactosidase has potential applications, and attempts to improve proteolytic resistance of enzymes have important values. We use a novel strategy for genetic manipulation of a pepsin-sensitive region specific for a pepsin-sensitive but trypsin-resistant high-temperature-active Gal27B from Neosartorya fischeri to screen mutants with enhanced pepsin resistance. All enzymes were produced in Pichia pastoris to identify the roles of loop 4 (Gal27B-A23) and its key residue at position 156 (Gly156Arg/Pro/His) in pepsin resistance. Gal27B-A23 and Gly156Arg/Pro/His elevated pepsin resistance, thermostability, stability at low pH, activity toward raffinose (5.3-6.9-fold) and stachyose (about 1.3-fold), and catalytic efficiencies (up to 4.9-fold). Replacing the pepsin cleavage site Glu155 with Gly improved pepsin resistance but had no effect on pepsin resistance when Arg/Pro/His was at position 156. Thus, pepsin resistance could appear to occur through steric hindrance between the residue at the altered site and neighboring pepsin active site. In the presence of pepsin or trypsin, all mutations increased the ability of Gal27B to hydrolyze galactosaccharides in soybean flour (up to 9.6- and 4.3-fold, respectively) and promoted apparent metabolizable energy and nutrient digestibility in soybean meal for broilers (1.3-1.8-fold). The high activity and tolerance to heat, low pH, and protease benefit food and feed industry in a cost-effective way.
Keywords: catalytic performance; hydrolysis of soybean galactosaccharides; loop replacement; pepsin resistance; residue substitution in proximity to pepsin cleavage site; stability at low pH; thermostability; trypsin resistance; α-galactosidases.