Application Potential of Cyanide Hydratase from Exidia glandulosa: Free Cyanide Removal from Simulated Industrial Effluents

Abstract

Industries such as mining, cokemaking, (petro)chemical and electroplating produce effluents that contain free cyanide (fCN = HCN + CN−). Currently, fCN is mainly removed by (physico)chemical methods or by biotreatment with activated sludge. Cyanide hydratases (CynHs) (EC 4.2.1.66), which convert fCN to the much less toxic formamide, have been considered for a mild approach to wastewater decyanation. However, few data are available to evaluate the application potential of CynHs. In this study, we used a new CynH from Exidia glandulosa (protein KZV92691.1 designated NitEg by us), which was overproduced in Escherichia coli. The purified NitEg was highly active for fCN with 784 U/mg protein, kcat 927/s and kcat/KM 42/s/mM. It exhibited optimal activities at pH approximately 6–9 and 40–45 °C. It was quite stable in this pH range, and retained approximately 40% activity at 37 °C after 1 day. Silver and copper ions (1 mM) decreased its activity by 30–40%. The removal of 98–100% fCN was achieved for 0.6–100 mM fCN. Moreover, thiocyanate, sulfide, ammonia or phenol added in amounts typical of industrial effluents did not significantly reduce the fCN conversion, while electroplating effluents may need to be diluted due to high fCN and metal content. The ease of preparation of NitEg, its high specific activity, robustness and long shelf life make it a promising biocatalyst for the detoxification of fCN.

Design and development of a spectrophotometric cyanide assay based on a cascade reaction catalyzed by cyanide hydratase, formamidase and formate dehydrogenase

“…These effects have to be investigated for various types of samples. While studying the degradation of fCN by CynHs, we observed that wastewater components such as sul des or thiocyanates did not signi cantly inhibit the enzyme [25], which is encouraging. Sul des and sul tes (both from 1 mg/L) strongly interfere with the picric acid method, and thiocyanates in a concentration of over 0.05 mg/L affect the results obtained with the above kit, along with other ions [8].…”

“…CynH (GenBank: KZV9269) from Exidia glandulosa was produced in Escherichia coli Origami B(DE3) as a fusion protein with a C-terminal His 6 -tag. Cultivation and puri cation were carried out and the activity was determined as described previously [25]. Brie y, cultivation proceeded in 2xYT medium at 37°C until optical density measured at 600 nm reached 1.0.…”

“…CynHs are known for their excellent speci c activities at alkaline pH, which is a key factor for the success of a cyanide assay (avoidance of HCN vapor losses at lower pH). For example, a CynH from Exidia glandulosa exhibited a speci c activity of ≈ 780 U/mg for 25 mM KCN at pH 9.0 and 30°C [25]. The enzyme still retained some activity at pH 10.5.…”

Enzymes and Biocatalysis

“…A simulated electroplating effluent was composed of 100 mM fCN and 1 mM AgNO 3 or 1 mM CuSO 4 . The results showed that the NitEg at a concentration of 14 µg enzyme mL −1 was sufficient to remove more than 97% fCN in the simulated electroplating effluent after 1 h. The NitEg demonstrated excellent performance in fCN solutions of up to 100 mM concentrations under alkaline conditions [29].…”

“…Cyanide hydratases (CynHs) (EC 4.2.1.66) belong to the nitrilase family and catalyze the hydration of cyanide to formamide. Sedova et al [29] used a new CynH from Exidia glandulosa (protein KZV92691.1, namely, NitEg), which was overproduced in Escherichia coli. The cell-free extract had a specific activity of 280 U mg protein −1 , which was increased to 697 U mg protein −1 after purification via cobalt ion affinity chromatography.…”