摘要
用生物工程技术将萤火虫荧光素酶基因转移到大肠杆菌,在大肠杆菌中合成荧光素酶。这种工程菌已可通过发酵大量培养,并从菌体分离得到接近纯化的荧光素酶。这种酶的分子量是103kD;巯基试剂5,5’-巯基-2(2-硝基苯甲酸)“DTNB”能抑制酶的活性;对于底物荧光素的K_m为1.2μmol/L;酶反应最适pH为7.77;酶催化的生物发光峰在560nm。
Firefly luciferase was produced in Escherichia coli harbouring a recombinant plasmid containing the firefly luciferase gene. The enzyme was purified approximately 29.8 fold from Escherichia coli by means of chromatography on DEAE-Sepharose column (Table 1, Fig. 1). Two protein bands were shown by polyacrylamide gel electrophoresis for the purified enzyme (Fig. 2). The enzyme had an approximate molecular weight of 103 kD (Fig. 8) and consisted of two identical subunits (Fig. 6). The chemical and physical properties of the enzyme compared well with the native one isolated from fireflies. The enzyme was inhibited by sulfhydryl reagant DTNB (Fig. 11), the Km for luciferin was 1.2 μmol / L (Fig.9) and the optimal pH was 7.77 (Fig. 5). The bioluminescence had a peak emission at 560 nm (Fig. 10).
关键词
生物发光
萤火虫
荧光素
酶
bioluminescence
firefly
luciferase
Escherichia coli
