Nagasawa T, Mauger J, Yamada H
Department of Agricultural Chemistry, Kyoto University, Japan.
Eur J Biochem. 1990 Dec 27;194(3):765-72. doi: 10.1111/j.1432-1033.1990.tb19467.x.
A new type of nitrilase, arylacetonitrilase, has been purified from isovaleronitrile-induced cells of Alcaligenes faecalis JM3 in four steps. The purity of the enzyme was confirmed by SDS/polyacrylamide gel electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 275 kDa and consists of six subunits of identical molecular mass. The purified enzyme exhibits a pH optimum of 7.5 and a temperature optimum of 45 degrees C. The enzyme is specific for arylacetonitriles such as 2-thiophenacetonitrile, p-tolyacetonitrile, p-chlorobenzylcyanide, p-fluorobenzylcyanide and 3-pyridylacetonitrile. The enzyme stoichiometrically catalyzes the hydrolysis of arylacetonitrile to arylacetic acid and ammonia, no formation of amide occurring. However, the enzyme does not attack nitrile groups attached to aromatic and heteroaromatic rings, which are hydrolyzed preferably by the nitrilases known previously. The enzyme requires thiol compounds such as dithiothreitol and 2-mercaptoethanol to exhibit its maximum activity.
一种新型腈水解酶——芳基乙腈酶，已通过四个步骤从粪产碱菌JM3的异戊腈诱导细胞中纯化出来。通过SDS/聚丙烯酰胺凝胶电泳、两性电解质电聚焦和琼脂糖双向免疫扩散确认了该酶的纯度。该酶的分子量约为275 kDa，由六个分子量相同的亚基组成。纯化后的酶最适pH为7.5，最适温度为45℃。该酶对芳基乙腈具有特异性，如2-噻吩乙腈、对甲苯乙腈、对氯苄腈、对氟苄腈和3-吡啶乙腈。该酶以化学计量方式催化芳基乙腈水解为芳基乙酸和氨，不生成酰胺。然而，该酶不作用于连接在芳环和杂芳环上的腈基，这些腈基优先被先前已知的腈水解酶水解。该酶需要硫醇化合物如二硫苏糖醇和2-巯基乙醇来展现其最大活性。
