多粘类芽孢杆菌适冷性新型<i>β</i>-半乳糖苷酶的重组表达和酶学性质

向芷璇; 关乐颖; 李敬; 闫巧娟; 江正强

doi:10.13386/j.issn1002-0306.2023010185

多粘类芽孢杆菌适冷性新型β-半乳糖苷酶的重组表达和酶学性质

Recombinant Expression and Characterization of A Novel Cold-Adapted β-Galactosidase from Paenibacillus polymyxa

摘要

摘要: 目的：本实验旨在挖掘一个适冷性新型β-半乳糖苷酶，为低温生产低乳糖乳制品或合成低聚半乳糖（Galactooligosaccharides，GOS）提供基础。方法：从多粘类芽孢杆菌（Paenibacillus polymyxa）基因组中克隆一个β-半乳糖苷酶（PpBgal42A）基因，构建重组表达质粒pET-28a-PpBgal42A在大肠杆菌BL21（DE3）中表达，经亲和层析纯化后研究重组β-半乳糖苷酶的酶学性质，利用高效液相色谱检测各糖分浓度，以GOS转化率为指标，评价PpBgal42A的转糖苷能力。结果：PpBgal42A与酸热脂环酸芽孢杆菌（Alicyclobacillus acidocaldarius）来源的糖苷水解酶42家族β-半乳糖苷酶同源性最高，为59.9%。纯化后，PpBgal42A的比活力为163.7 U/mg，分子量约为79 kDa。其最适pH和温度分别为pH7.5和35 ℃，在pH7.0~9.5范围内和4~35 ℃以下保持稳定，50 ℃时快速失活，35 ℃时的半衰期为1777 min。PpBgal42A利用350 g/L乳糖于30 ℃反应6 h后合成GOS的转化率为31.6%。结论：PpBgal42A是一个适冷性新型β-半乳糖苷酶，可水解乳糖并合成低聚半乳糖，具有潜在应用价值。

Abstract: Objective: To analyze a novel cold-adapted β-galactosidase, which provided the basis for producing low lactose dairy products or synthesizing galactooligosaccharides (GOS) at low temperatures. Methods: The β-galactosidase (PpBgal42A) gene was cloned from the genome of Paenibacillus polymyxa. The recombinant plasmid (pET-28a-PpBgal42A) was constructed and successfully expressed in E.coli BL21(DE3). After purification by affinity chromatography, the enzymatic properties of recombinant β-galactosidase were studied. To evaluate the transglycosylation ability of PpBgal42A, the concentration of each component was determined by high performance liquid chromatography with the conversion rate of GOS as evaluation index. Results: PpBgal42A shared the highest identity of 59.9% with the GH family 42 β-galactosidase from Alicyclobacillus acidocaldarius. After purification, the specific activity of PpBgal42A was 163.7 U/mg and the molecular weight was about 79 kDa. Its optimal pH and temperature were pH7.5 and 35 ℃, respectively. It was stable in the range of pH7.0~9.5 and 4~35 ℃. It was rapidly inactivated at 50 ℃. The half-life of PpBgal42A at 35 ℃ was 1777 min. PpBgal42A synthesized 31.6% of GOS using 350 g/L lactose as the substrate at 30 ℃ for 6 h. Conclusion: PpBgal42A is a novel cold-adapted enzyme. It can hydrolyze lactose and synthesize galactooligosaccharides, which has potential application value.