Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila
Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila
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DOI：
10.1128/jb.178.11.3270-3274.1996
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发表时间：
1996-06-01
影响因子：
3.2
通讯作者：
Ferry, JG
中科院分区：
文献类型：
--
作者：
Alber, BE;Ferry, JG
嗜热甲烷单胞菌碳酸酐酶基因在大肠杆菌中高效表达，异源表达的酶纯化了14倍，达到了表观均一。从大肠杆菌中纯化的酶具有与从嗜热分枝杆菌中分离的真正酶相似的性质(比活性、抑制剂敏感性和热稳定性)；然而，分子质量上的差异表明碳酸氢酶在大肠杆菌或嗜热分枝杆菌中是翻译后修饰的。正品酶和异源酶在55℃下加热15min都是稳定的，但在更高的温度下会失活，以对硝基苯乙酸酯为底物没有检测到酯酶活性，等离子体发射光谱显示每个亚基大约有0.6个锌。根据估计的天然分子质量判断，该酶要么是三聚体，要么是四聚体。免疫印迹分析表明，嗜热支原体细胞提取物蛋白的水平受生长底物的调节，醋酸盐中的蛋白水平高于甲醇或三甲胺培养的细胞。
The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity. The enzyme purified from E. coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M. thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E. coli or M. thermophila. Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures, No esterase activity was detected with p-nitrophenylacetate as the substrate, Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit. As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer. Western blot (immunoblot) analysis of cell extract proteins from M. thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.