Institute of Molecular Biotechnology, RWTH-Aachen University, Forckenbeckstraße 6, Aachen, Germany.
Extremophiles. 2012 Jan;16(1):45-55. doi: 10.1007/s00792-011-0404-1. Epub 2011 Oct 19.
The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a β-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αββα fold.
来自超嗜热菌 Aquifex aeolicus 的 CphAII 蛋白具有金属β-内酰胺酶（MBL）超家族的五个保守基序，与气单胞菌属 subsp. B2 CphA MBL 具有 28%的同源性。CphAII 基因通过 PCR 从 A. aeolicus 基因组 DNA 中扩增，并使用基于 pLex 的表达系统在大肠杆菌中过表达。通过加热（使大肠杆菌蛋白变性）和两步固定化金属亲和层析的组合对重组 CphAII 蛋白进行纯化。纯化的酶制剂没有表现出β-内酰胺酶活性，但对双对硝基苯磷酸酯和胸苷 5'-单磷酸对硝基苯酯表现出金属依赖性的磷酸二酯酶活性，最佳温度为 85°C。圆二色性光谱与 MBL αββα 折叠的特征二级结构百分比一致。
