Journal
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
Volume 168, Issue 3, Pages 638-650Publisher
SPRINGER
DOI: 10.1007/s12010-012-9806-5
Keywords
Directed evolution; alpha-Galactosidase; Improved activity; Pichia pastoris
Funding
- Program for the New Century Excellent Talents in University [NCET-07-0807]
- Scientific and Technical Supporting Programs [2011BAD26B02-2]
- Bud Program of Beijing Academy of Science and Technology [2011-5]
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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.
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