Directed Evolution, High-Level Expression and Application of the β-Galactosidase from Aspergillus oryzae

Abstract:

The β-galactosidase (AoBgal35A) from Aspergillus oryzae was modified by directed evolution to improve its hydrolysis efficiency of lactose. A mutation library of AoBgal35A was constructed by error-prone PCR. The mutants with high hydrolysis efficiency of lactose were screened by high-throughput screening and efficiently expressed in Pichia pastoris to prepare lactose-free milk. A positive mutant (mAoBgal35A) was selected and its enzyme activity reached 4 760 U/mL after high cell density fermentation. Purified mAoBgal35A showed the highest activity at pH 5.0 and 55 ℃. The optimum pH and temperature of mAoBgal35A were increased by 0.5 and decreased by 5 ℃ compared with the wild-type AoBgal35A, respectively. The mAoBgal35A could effectively hydrolyze the lactose in milk in 72 h at room temperature with the enzyme dosage of 2 U/mL, and the content of lactose in milk was 0.38 g/dL, which was within the standard range of lactose-free milk. However, for the wild-type enzyme, the content of lactose in milk was 0.6 g/dL with the enzyme dosage of 20 U/mL under the same conditions. The results showed that directed evolution significantly improved the hydrolysis efficiency of AoBgal35A, and the positive mutant mAoBgal35A showed great application potential in lactose-free milk.