β-半乳糖苷酶是乳制品加工中最重要的酶之一。它将乳糖转化为葡萄糖和半乳糖，还催化半乳糖形成低聚半乳糖（GOS），即所谓的益生元。然而，来自发酵剂培养物中的大多数 β-半乳糖苷酶具有低的转半乳糖基化活性，这一过程会导致半乳糖在酸奶中积累。在这里，尝试了一种定点突变策略，对嗜热链球菌的 β-半乳糖苷酶进行基因修饰。出 28链球菌。thermophilus菌株是一种名为bgaQ的 β-半乳糖苷酶基因，编码高 β-半乳糖苷酶水解活性 (BgaQ)，是从Strep 菌株中克隆出来的。嗜热菌SDMCC050237。它的大小为 3,081 bp，有 1,027 个推断的氨基酸残基，属于 GH2 家族。用 His 801和 Gly 802替换 BgaQ 活性位点周围的 Tyr 801和 Pro 802后，生成的突变蛋白 BgaQ-8012 的 GOS 合成在 5% 乳糖下从 20.5% 提高到 26.7%，水解活性没有明显变化. 随后，将纯化的 BgaQ 或 BgaQ-8012 添加到接种了 2 个来自Strep 的发酵剂的灭菌牛奶中。嗜热菌SDMCC050237 和德氏乳杆菌ssp。保加利亚ATCC11842。添加 BgaQ 或 BgaQ-8012 的 GOS 产量分别增加到 5.8 和 8.3 g/L，而未添加酶的产量为 3.7 g/L。同时，Bga​​Q或BgaQ-8012的添加使发酵酸奶中的乳糖含量降低了49.3%和54.4%，缩短了凝乳时间。因此，本研究为提高链球菌β-半乳糖苷酶的转半乳糖基化活性提供了一种定点突变策略。嗜热菌用于制作富含 GOS 的酸奶。

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β-Galactosidase is one of the most important enzymes used in dairy processing. It converts lactose into glucose and galactose, and also catalyzes galactose to form galactooligosaccharides (GOS), so-called prebiotics. However, most of the β-galactosidases from the starter cultures have low transgalactosylation activities, the process that results in galactose accumulation in yogurt. Here, a site-directed mutation strategy was attempted, to genetically modify β-galactosidase from Streptococcus thermophilus. Out of 28 Strep. thermophilus strains, a β-galactosidase gene named bgaQ, encoded for high β-galactosidase hydrolysis activity (BgaQ), was cloned from the strain Strep. thermophilus SDMCC050237. It was 3,081 bp in size, with 1,027 deduced amino acid residuals, which belonged to the GH2 family. After replacing the Tyr801 and Pro802 around the active sites of BgaQ with His801 and Gly802, the GOS synthesis of the generated mutant protein BgaQ-8012 increased from 20.5% to 26.7% at 5% lactose, and no hydrolysis activity altered obviously. Subsequently, the purified BgaQ or BgaQ-8012 was added to sterilized milk inoculated with 2 starters from Strep. thermophilus SDMCC050237 and Lactobacillus delbrueckii ssp. bulgaricus ATCC11842. The GOS yields with added BgaQ or BgaQ-8012 increased to 5.8 and 8.3 g/L, respectively, compared with a yield of 3.7 g/L without enzymes added. Meanwhile, the addition of the BgaQ or BgaQ-8012 reduced the lactose content by 49.3% and 54.4% in the fermented yogurt and shortened the curd time. Therefore, this study provided a site-directed mutation strategy for improvement of the transgalactosylation activity of β-galactosidase from Strep. thermophilus for GOS-enriched yogurt making.