【摘要】 采用PCR方法,分别从大肠杆菌和嗜热链球菌基因组DNA中扩增获得glyA基因,分别克隆入载体pET-28 a(+)中并进行表达,分离和纯化得到两种不同来源的SHMT,分别检测两种SHMT的逆向酶活。比较来源于大肠杆菌K12与嗜热链球菌AS1.2471中的glyA基因表达的丝氨酸羟甲基转移酶(SHMT)的活性,以获得高活性的SHMT。结果成功获得两种菌中的glyA基因,并表达出具有较高活性的SHMT,其中嗜热链球菌中glyA基因表达出的SHMT的酶活性大约为大肠杆菌的两倍。从嗜热链球菌中克隆表达的SHMT具有更高的催化活性及良好的工业应用前景。更多还原

【Abstract】 The activities of serine hydroxymethyl transferase(SHMT) encoded by glyA gene isolated from E.coli K12 and S.thermophilus AS1.2471 were compared.The complete coding sequence of glyA gene from E.coli K12 and S.thermophilus AS1.2471 were obtained directly from the genomic DNAs by PCR amplification.The PCR fragments of two kind were inserted into pET-28a(+) respectively.The expression vector pET28-K12-glyA and pET28-AS1.2471glyA were expressed in E.coli BL21 and the purified protein was obtained by nickel affinity gel column chromatography,and activities of the two kind of SHMT were measured.The activity of SHMT encoded by glyA gene isolated from S.thermophilus was almost twice as high as that from E.coli.SHMT encoded by the glyA gene isolated from S.thermophilus possessed efficiency of catalytic activity,which will have a better application for industry.更多还原