摘要
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).
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2010年第2期175-180,共6页
Journal of Huazhong Agricultural University
基金
浙江省重大科技攻关项目(2004C12010)资助
关键词
乳酸克鲁维酵母
乳糖酶
基因表达
酶学性质
Kluyveromyces lactis
lactase
gene expression
enzymatic properties