Mizunashi W, Nishiyama M, Horinouchi S, Beppu T
Department of Biotechnology, University of Tokyo, Japan.
Appl Microbiol Biotechnol. 1998 May;49(5):568-72. doi: 10.1007/s002530051214.
High-molecular-mass nitrile hydratase (H-NHase, 530 kDa) is a cobalt-containing enzyme produced by Rhodococcus rhodochrous J1. For efficient production of H-NHase in R. rhodochrous ATCC12674, several plasmids were constructed. The enzyme was produced in the recombinant Rhodococcus cells only in the presence of an upstream region (approximately 4 kb) of the H-NHase gene under the control of the promoter for the amidase-NHase gene cluster from Rhodococcus sp. N-774. Although H-NHase was produced as a soluble protein in the cells, the protein did not show NHase activity. However, when the recombinant R. rhodochrous ATCC12674 cells were cultured in the presence of amide compounds, such as crotonamide and methacrylamide, markedly high NHase activity was detected, Gel-filtration chromatography revealed that the NHases produced by the cells grown in the presence and absence of the amide compounds had a molecular mass of more than 500 kDa and 50-80 kDa respectively. These results suggest that the amide compounds are essential for subunit assembly to form an enzymatically active multimer. By the use of the recombinant expression system, NHase activity 1.7 times higher than that of the original strain, R. rhodochrous J1, was achieved.
高分子量腈水合酶（H-NHase，530 kDa）是由红平红球菌J1产生的一种含钴酶。为了在红平红球菌ATCC12674中高效生产H-NHase，构建了几种质粒。该酶仅在来自红球菌属N-774的酰胺酶-NHase基因簇启动子控制下的H-NHase基因上游区域（约4 kb）存在时，才在重组红球菌细胞中产生。尽管H-NHase在细胞中以可溶性蛋白形式产生，但该蛋白不显示NHase活性。然而，当重组红平红球菌ATCC12674细胞在酰胺化合物（如巴豆酰胺和甲基丙烯酰胺）存在下培养时，检测到明显较高的NHase活性。凝胶过滤色谱显示，在有和没有酰胺化合物存在下生长的细胞产生的NHase分子量分别大于500 kDa和50-80 kDa。这些结果表明，酰胺化合物对于亚基组装形成具有酶活性的多聚体至关重要。通过使用重组表达系统，获得了比原始菌株红平红球菌J1高1.7倍的NHase活性。
