定向进化米曲霉β-半乳糖苷酶（AoBgal35A），以提高其水解乳糖的效率，通过易错PCR构建米曲霉β-半乳糖苷酶的突变体文库，高通量筛选水解乳糖效率高的突变体，将其在毕赤酵母中高效表达，并用于制备无乳糖牛奶。筛选得到一个正向突变体（mAoBgal35A），经高密度发酵酶活力达4 760 U/mL。纯化后，mAoBgal35A的最适pH和温度分别为5.0和55 ℃，比野生型AoBgal35A分别提高了0.5和降低了5 ℃。mAoBgal35A在室温下能高效水解牛奶中的乳糖，加酶量为2 U/mL时，水解72 h后牛奶中的乳糖质量浓度为0.38 g/dL，达到无乳糖牛奶标准。而野生型AoBgal35A的加酶量为20 U/mL时，在相同条件下水解后，乳糖质量浓度为0.6 g/dL。由此表明，定向进化明显提高了米曲霉β-半乳糖苷酶水解乳糖的效率，所得正向突变体在制备无乳糖乳品中具有很大的应用潜力。
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.