乳酸克鲁维酵母乳糖酶基因在大肠杆菌中的表达及酶学性质

Expression and Enzymatic Properties of Lactase Gene from Kluyveromyces lactis in Escherichia coli

通过PCR扩增从乳酸克鲁维酵母基因组获得乳糖酶基因lac4,将其克隆至大肠杆菌表达载体pET-28a(+),并在大肠杆菌BL21中获得表达,酶活性达(44.78±2.84)U/mL。利用Ni-ResinHP亲和层析技术纯化该酶,SDS-PAGE分析表明,重组酶分子质量约为122ku。酶学性质研究表明,该酶最适反应温度为43℃,37℃时半衰期为30min,最适反应pH为7.0,在pH4.0～10.0范围内有良好的稳定性,Ca2+、Zn2+、Fe2+、Co2+、Mg2+、Mn2+对酶有非常明显的激活作用,比酶活性为(255.55±2.32)U/mg,酶反应常数Km为3.49mmol/L,最大反应速率vmax为4.22mmol/(min.mg)。

The lactase gene（lac4） was amplified from the genome of Kluyveromyces lactis through polymerase chain reaction（PCR） and then cloned into Escherichia coli expression vector pET-28a（＋）,and the lac4 gene was successfully expressed in Escherichia coli 21,enzyme activity reached （44.78±2.84） U/mL.The recombinant lactase was purified with affinity chromatography technology,then analyzed by SDS-PAGE,its molecular weight was approximate 122 ku.The optimal reaction conditions of lactase such as temperate,pH were 43 ℃ and 7.0,its half-life was 30 min at 37℃.It was very stable on the condition of pH 4.0 to10.0,and can be greatly activated by Ca2＋,Zn2＋,Fe2＋,Co2＋,Mg2＋,Mn2＋,its specific activity was （255.55±2.32） U/mg,the response constant Km was 3.49 mmol/L,the maximum reaction velocity vmax was 4.22 mmol/（min·mg）.