Abstract:

A strain of Alcaligenes sp., DN25, isolated and preserved in the authors’ lab, shows a high cyanide-degrading activity and thus has a potential of industrial application.The cyanide-degrading routes were determined based on the analysis of degradation products and time curves.The experiments on optimization of culture conditions for the cyanide-degrading enzyme were carried out according to the characteristics of enzyme active site.The results demonstrated that the reaction was through a hydrolytic pathway co-functioned by a cyanide hydratase, an amidase and a cyanide hydrolase, among which cyanide hydrolase played a major role.Four sulfur-containing substances were added to the medium to test the effect on specific activity of culture.DL-cysteine was found to be beneficial for the enzyme production and DL-methionine for both enzyme production and cell yield, resulting in increased specific activity of culture of 1.26 U·ml^-1 and 2.10 U·ml^-1，when 0.8% DL-cysteine and DL-methionine was added into fermentation culture respectively.The experiments on optimization of liquid culture conditions in 1L shake flask gave the optimal conditions as 100ml of liquid loading, pH of 9.0, inoculation size of 8%, and 0.8% DL-methionine added into the medium.Under such conditions, the specific activity of culture could reach 2.18 U·ml^-1, increased by 3.46 fold compared to that before optimization.The cell growth curve had a similar trend to that of enzyme production and the stabilized phase could be reached within 40 h.The current work could lay the basis for further application and purification of the cyanide-degradating enzyme.

Key words: 产碱杆菌, 氰水解酶, 氰水合酶, 降解代谢途径, 产酶条件优化

摘要：

本实验室分离保藏的一株产碱杆菌Alcaligenes sp.DN25具有较高降氰活性，通过分析氰降解代谢产物确定了其降解途径，并根据降氰酶催化活性部位特征对产酶条件进行了优化。结果显示，氰的降解代谢途径可推断为由氰水解酶、氰水合酶和酰胺水解酶共同作用的水解途径，而其中氰水解酶的活性占主要作用；在培养基中分别添加4种含硫物质，发现DL-半胱氨酸对菌体产酶有明显促进作用，DL-甲硫氨酸能同时提高菌体的产酶水平和细胞生长量。此两种物质添加量为0.8%时，单位发酵液酶活力可分别达到1.26 U·ml^-1和2.10 U·ml^-1。在1L三角瓶中进行扩大培养优化，得到最佳培养条件：装液量100 ml，pH 9.0，接种量8%，在基本培养基中添加0.8%的DL-甲硫氨酸。菌株的生长曲线和产酶曲线趋势基本一致，培养40 h后进入稳定期，单位发酵液酶活力平均值为2.18 U·ml^-1，是优化前的3.46倍。

关键词: 产碱杆菌, 氰水解酶, 氰水合酶, 降解代谢途径, 产酶条件优化