Mueller Patrick, Egorova Ksenia, Vorgias Constantinos E, Boutou Effrosini, Trauthwein Harald, Verseck Stefan, Antranikian Garabed
Hamburg University of Technology, Institute of Technical Microbiology, Germany.
Protein Expr Purif. 2006 Jun;47(2):672-81. doi: 10.1016/j.pep.2006.01.006. Epub 2006 Feb 3.
Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py. abyssi. This recombinant nitrilase was purified by heat treatment of E. coli crude extract followed by anion-exchange chromatography with a yield of 88% and a specific activity of 0.14 U/mg. The recombinant enzyme, which represents the first archaeal nitrilase, is a dimer (29.8 kDa/subunit) with an isoelectric point of pI 5.3. The nitrilase is active at a broad temperature (60-90 degrees C) and neutral pH range (pH 6.0-8.0). The recombinant enzyme is highly thermostable with a half-life of 25 h at 70 degrees C, 9 h at 80 degrees C, and 6 h at 90 degrees C. Thermostability measurements by employing circular dichroism spectroscopy and differential scanning microcalorimetry, at neutral pH, have shown that the enzyme unfolds up to 90 degrees C reversibly and has a T(m) of 112.7 degrees C. An inhibition of the enzymatic activity was observed in the presence of acetone and metal ions such as Ag(2+) and Hg(2+). The nitrilase hydrolyzes preferentially aliphatic substrates and the best substrate is malononitrile with a K(m) value of 3.47 mM.
在嗜热古菌深渊栖热球菌、堀越栖热球菌、激烈栖热球菌和嗜火栖热菌（生长温度为90-100摄氏度）的基因组中鉴定出四个编码假定腈水解酶的开放阅读框。将编码腈水解酶的基因克隆并在大肠杆菌中过表达。仅在深渊栖热球菌的情况下检测到酶活性。通过对大肠杆菌粗提物进行热处理，然后进行阴离子交换色谱法，纯化了这种重组腈水解酶，产率为88%，比活性为0.14 U/mg。该重组酶是首个古菌腈水解酶，为二聚体（29.8 kDa/亚基），等电点为pI 5.3。该腈水解酶在较宽的温度范围（60-90摄氏度）和中性pH范围（pH 6.0-8.0）内具有活性。该重组酶具有高度的热稳定性，在70摄氏度下半衰期为25小时，80摄氏度下为9小时，90摄氏度下为6小时。在中性pH条件下，采用圆二色光谱法和差示扫描量热法进行的热稳定性测量表明，该酶在高达90摄氏度时可逆展开，熔解温度为112.7摄氏度。在丙酮和金属离子如Ag(2+)和Hg(2+)存在的情况下，观察到酶活性受到抑制。该腈水解酶优先水解脂肪族底物，最佳底物是丙二腈，K(m)值为3.47 mM。
