Biodegradation of cyanide using recombinant Escherichia coli expressing Bacillus pumilus cyanide dihydratase

Abstract

Despite its high toxicity, cyanide is used in several industrial processes, and as a result, large volumes of cyanide wastewater need to be treated prior to discharge. Enzymatic degradation of industrial cyanide wastewater by cyanide dihydratase, which is capable of converting cyanide to ammonia and formate, is an attractive alternative to conventional chemical methods of cyanide decontamination. However, the main impediment to the use of this enzyme for the biodegradation of cyanide is the intolerance to the alkaline pH at which cyanide waste is kept and its low thermoresistance. In the present study, the catalytic properties of whole E. coli cells overexpressing a cyanide dihydratase gene from B. pumilus were compared to those of the purified enzyme under conditions similar to those found in industrial cyanide wastewater. In addition, the capacity of whole cells to degrade free cyanide in contaminated wastewater resulting from the gold mining process was also determined. The characteristics of intracellular enzyme, relative to purified enzyme, included increased thermostability, as well as greater tolerance to heavy metals and to a lesser extent pH. On the other hand, significant enzymatic degradation (70%) of free cyanide in the industrial sample was achieved only after dilution. Nevertheless, the increased thermostability observed for intracellular CynD suggest that whole cells of E. coli overexpressing CynD are suited for process that operate at elevated temperatures, a limitation observed for the purified enzyme.

Isolation of a Bacillus safensis from mine tailings in Peru, genomic characterization and characterization of its cyanide-degrading enzyme CynD

“…3A, S3). The K m value is similar to that previously reported for CynD C1 and from other species (2.56 mM to 7.3 mM) similar but k cat is lower (433 s -1 to 61600 s -1 ) Crum et al, 2016;Jandhyala et al, 2005;Vargas-Serna et al, 2020).…”

“…Figure 3B shows that recombinant CynD PER-URP-08 carrying a Cterminal 6x-His tag is active up to pH 9 and inactive at pH 10 and 11. Other wild-type CynDs have been shown to be active only up to pH 8 (Crum et al, 2016;Jandhyala et al, 2005) and CynD C1 with C-terminal 6x-His tag had its activity compromised at pH 9 (Vargas-Serna et al, 2020). The CynD C1 and CynD PER-URP-08 sequences only differ at five positions: are I18V, S25T, E155D, H305Q and N307Y (first letter correspond to CynD C1 ) with the last two substitutions H305Q and N307Y near the C-terminus.…”

Cyanide Bioremediation by Bacillus subtilis under Alkaline Conditions

“…stronger empirical fit with a higher coefficient of determination (R 2 ), agreeing with the literature in showing second-order dependence on the rate-limiting cyanide dihydratase enzyme [26,34,65]. Cyanide dihydratase catalyzes the conversion of cyanide to ammonia and formate via Michaelis-Menten kinetics as an inducible enzyme requiring cyanide as a substrate [65][66][67][68]. The higher coefficient of determination (R 2 ) and lower second-order rate constant (k2) compared to the first-order rate constant (k1) indicate that the secondorder model more accurately reflects the enzymatic rate-limiting mechanism.…”

Aislamiento y adaptación de Pseudomonas pseudoalcaligenes en un efluente de proceso de cianuración de minerales auríferos

“…La acción metabólica de las bacterias del género Pseudomonas son consideradas un paradigma clave en el reciclado de materia orgánica de diversos ecosistemas; por tanto, juega un papel importante en la mejora y mantenimiento de la calidad del medio ambiente (Escribano, 2016;Panay et al, 2020). Teniendo en cuenta que la extracción del oro y la plata se realiza generalmente mediante procesos de cianuración, generándose grandes volúmenes de efluentes con cianuro de forma iónica simple y complejo; los que al no ser tratados impactan negativamente al medio ambiente, afectando el suelo y el agua próximos al proceso de tratamiento y a la atmósfera, por la emanación de gases cianhídricos (Gordillo, 2018); requiriéndose su tratamiento en la etapa final del procesamiento, hasta conseguir la conversión en productos no contaminantes y que la concentración final esté por debajo de los límites permitidos para el vertido al medio ambiente (Johnson, 2014;Alvillo et al, 2021).…”