Probing The Catalytic Function Of Cyanide-degrading Enzyme From Alcaligenes Sp. DN25

Cyanidase and cyanide hydratase belong to nitrilase superfamily. Both of them which come from different strains use cyanide as substrate, but their products are different (formate, ammonia and formamide respectively).We successfully cloned and purified a cyanide-degrading enzyme from Alcaligenes sp. DN25(naming cdE) in our early work. The products of cyanide hydrolysis included formate, ammonia, and formamide. As a result, we attempt to probe the catalytic action of duble-fuction cdE in this study.First of all, We obtain cdE with the purity of 97% via gradient elution. The purified cdE has an optimal activity at a temperature of 30℃ and a pH of 7.0 and a good pH tolerance. The relative content of the formate and formamide was influenced by pH and temperature. We proved that the His tag have no influence on the catalytic properties of cdE, and in turn, we presumed that cdE exhibited both cyanide hydratase activity and cyanidase activity. Although the cyanidase from Pseudomonas stutzeri AK61 shows a sequence identity of 99% to cdE, the point mutations experiment have proved that the different catalytic function between them is not caused by gene mutations.A reaction mechanism of cyanide hydrolysis by cdE was proposed. The formed tetrahedral intermediate between substrate and the catalytic triad (Cys163-Glu47-Lys129) can lead to two different reaction routes (pathway A, showing cyanidase activity; pathway B, showing cyanide hydratase activity) in cyanide hydrolysis. The selectivity of the reaction can be changed. Base on homology modeling and molecular docking,We obtained several mutants which play an importance role on the catalytic properties of cdE. Among these mutants, Trp164Ala, Met162His, and Glu136Ala can be conducive to the formation of formamide and the formamide/formate ratios increased 5.5-,1.24-, and 1.76-fold. The others (Met162Ala, Met162Cys, Met162Ser, Met162Glu, Met162Asn, Met162Phe, His166Asn, SerlOlAla) were beneficial to the formation of formate and the formamide/formate ratios increased 2.9-,2.8-,7.8-,3.2-,1.8-,1.3-,1.7-, and 2.8-fold. The combination mutants (Met162Ala/Ser101Ala, Met162Cys/ SerlOlAla) has a certain increase in the formation of formate compared with wild-type cdE and the formamide/formate ratios increased 4.9-fold and 5.1-fold respectively. The carbon terminal of cdE mutants(△310 and △307) led to a increase in formamide/formate ratios (1.27-fold and 1.35-fold respectively).The treatment of cyanide-containing wastewater from a concentrator was investigated by using Alcaligenes sp. DN25. The degradation efficiency of total cyanide could reach 100% in 23 hours. While the degradation efficiency was 95% with 8.8mg/L total cyanide of slag in the leached liquid in 25 hours. With an addition of carbon and nitrogen sources (glucose and ammounium chloride, respectively), the growth of cells as well as their detoxification process carried out simultaneously in the range of total cyanide concentration of 5mg/L to 20mg/L, and in this case, the degradation efficiency of free cyanide was higher than 87% at culture 67h.