Department of Food Science and Engineering, Ningbo University, Ningbo 315211, PR China; Institute of Quality Standards and Testing Technology for Agro-Products, Fujian Academy of Agricultural Sciences, Fuzhou 350003, PR China.
Department of Food Science and Engineering, Ningbo University, Ningbo 315211, PR China.
Int J Biol Macromol. 2021 Jan 1;166:760-770. doi: 10.1016/j.ijbiomac.2020.10.233. Epub 2020 Nov 1.
A phospho-β-galactosidase gene (BsGal1332) was cloned from Bacillus velezensis and successfully expressed in Escherichia coli BL21(DE3). The active BsGal1332 was identified to be a homodimer with a combined molecular mass of approximately 113 kDa, and it belonged to the glycoside hydrolase family 1. The BsGal1332 displayed relative strict substrate specificity for galactosyl compounds compared with the other phospho-β-galactosidases. The purified BsGal1332 showed the maximum activity at pH 8.0 and 50 °C for 2-nitrophenyl-β-d-galactopyranoside (oNPGal) and at 40 °C for lactose. BsGal1332 was slightly activated by K and Na, but not strongly affected by Ca, and was stable at pH 6.0-7.0 and 40 °C or below it. The activity of BsGal1332 decreased quickly after incubation at 50 °C or higher temperature, suggesting it was a cold-adapted enzyme. Moreover, BsGal1332 could hydrolyze lactose and oNPGal with K values of 23.68 and 2.36 mM and k values of 117.55 and 155.61 s at 4 °C, respectively. Additionally, 1 U of the BsGal1332 could thus be capable of hydrolyzing about 38% of the lactose in 1 mL of milk after incubating at 4 °C for 4 h. Taken together, these properties of BsGal1332 made it a new promising industrial biocatalyst for efficient lactose hydrolysis in milk.
从解淀粉芽孢杆菌中克隆得到一个磷酸-β-半乳糖苷酶基因（BsGal1332），并在大肠杆菌 BL21（DE3）中成功表达。鉴定具有活性的 BsGal1332 为大约 113 kDa 的同源二聚体，属于糖苷水解酶家族 1。与其他磷酸-β-半乳糖苷酶相比，BsGal1332 对半乳糖基化合物表现出相对严格的底物特异性。纯化的 BsGal1332 在 pH 8.0 和 50°C 下对 2-硝基苯-β-D-半乳糖吡喃糖苷（oNPGal）显示最大活性，在 40°C 下对乳糖显示最大活性。BsGal1332 被 K 和 Na 轻微激活，但不受 Ca 强烈影响，在 pH 6.0-7.0 和 40°C 或以下时稳定。BsGal1332 在 50°C 或更高温度孵育后活性迅速下降，表明它是一种耐冷酶。此外，BsGal1332 在 4°C 下可以分别以 23.68 和 2.36 mM 的 K 值和 117.55 和 155.61 s 的 k 值水解乳糖和 oNPGal。此外，1 U 的 BsGal1332 在 4°C 孵育 4 h 后可以水解约 1 mL 牛奶中 38%的乳糖。总之，BsGal1332 的这些特性使其成为在牛奶中高效水解乳糖的有前途的新型工业生物催化剂。
