【摘要】 本研究旨在为低聚半乳糖（galactooligosaccharide,GOS）的合成提供新的双歧杆菌酶源，对Bifidobacterium bifidum ATCC 29521的β-半乳糖苷酶(β-gal^ATCC29521)进行生物信息学分析，并在Escherichia coli BL21（DE3）中实现异源表达。研究β-gal^ATCC29521的异源表达条件和发酵优化条件、酶学性质及其在乳清体系合成GOS的应用。生物信息学分析表明β-gal^ATCC29521属于糖苷水解酶家族2，不存在信号肽和跨膜区。发酵优化实验结果表明，E.coli BL21（DE3）-pET28a-β-gal^ATCC29521重组菌在含有10 g/L蔗糖、12 g/L胰蛋白胨、24 g/L酵母粉的培养基中，且0.05 mmol/L的异丙基-β-D-硫代半乳糖苷在27℃诱导培养16 h后，酶活力可达318.03 U/mL。粗酶液经过镍柱亲和层析得到电泳纯蛋白，其最适pH值和最适温度分别为7.5和40℃。β-gal^ATCC29521纯化酶在温度10～30℃和pH 6.5～9.0范围内表现出良好的稳定性。Mn²⁺和Mg²⁺可显著提高酶活性，Cu²⁺和Fe³⁺则显著抑制酶活性。以乳清为底物合成GOS时，该酶的产率为29.83%。研究结果表明β-gal^ATCC29521在GOS的制备领域中具有潜在的应用价值。更多还原

【Abstract】 This study introduced a new source of bifidobacterial enzyme for the synthesis of galactooligosaccharide （GOS）.We investigated the bioinformatics analysis,heterologous expression,enzymatic properties and application in the synthesis of GOS of a newly identified β-galactosidase (β-gal^ATCC29521) derived from Bifidobacterium bifidum ATCC 29521.Bioinformatic analysis showed that β-gal^ATCC29521 belonged to the glycoside hydrolase family 2,without a signal peptide or a transmembrane region.The optimal medium for β-gal^ATCC29521 expression was composed of 10 g/L sucrose,12 g/L tryptone,and 24 g/L yeast powder,and after 16 h of culture induced by 0.05 mmol/L isopropyl-β-D-thiogalactopyranoside （IPTG） at 27℃,the activity of β-gal^ATCC29521 was 318.03 U/mL.The crude enzyme was purified to electrophoretic homogeneity through Ni-IDA aff inity chromatography.The optimal pH and temperature for β-gal^ATCC29521 were 7.5 and 40℃,respectively.The purified enzyme was stable within the pH range of 6.5–9.0 and the temperature range of 10–30℃.Its activity was significantly enhanced in the presence of Mn²⁺and Mg²⁺but inhibited in the presence of Cu²⁺and Fe³⁺.When using whey as the substrate,the yield of GOS synthesized by β-gal^ATCC29521 was 29.83%.The results indicated that β-gal^ATCC29521 holds potential in the synthesis of GOS.更多还原