In this study, the activity of an alpha-galactosidase obtained from Penicillium janczewskii zalesk was improved via modifying its gene by error-prone PCR and DNA shuffling. The mutated DNA was ligated to pBGP1, an autonomous-replicating vector, which was subsequently transformed into Pichia pastoris X-33. The expressed enzyme activities were measured after single colonies were cultured in yeast-peptone-dextrose medium in deep-well plates. After two rounds of screening, two mutants with higher activity were obtained. By PCR analysis, four mutation sites (S167G, P455L, N637S, and P490L/P490H) were found in these two variants (mutant-59 and mutant-8). Mutant-59 showed the highest activity at pH 5.0 and 40 A degrees C with an increased V (max) value of 769 mu mol/min and the specific activity of 667 U/mg against p-nitrophenyl alpha-D-galactopyranoside. The two mutant enzymes also showed similar resistance to the metal ions of Cu2+, Fe2+, and Zn2+. In a 10-L fermenter, the supernatant enzyme activity reached the maximum of 550.2 U/mL upon the methanol induction for 96 h. This fermentation activity of the mutant was improved approximately two more folds than the wild type alpha-galactosidase. This mutant of alpha-galactosidase is prospective in feed manufacturing as feed additives to improve nutrient digestibility in monogastric animals.