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

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

https://doi.org/10.21203/rs.3.rs-4616809/v1

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Cyanide assays are required for the analyses of industrial, environmental, food and clinical samples. Enzymatic methods are not widely used in this field despite their selectivity and other promising properties. Therefore, we present here a proof of concept for a new enzymatic assay of free cyanide (fCN) which consists of HCN and CN^- at a pH dependent ratio. The assay is based on the transformation of HCN into the readily detectable NADH. The reaction is catalyzed by a multienzyme cascade consisting of cyanide hydratase, formamidase and formate dehydrogenase. The last step is a spectrophometric quantification of NADH with a tetrazolium salt, WST-8. The new assay offers selectivity (with the exception of formic acid interference) and mild operational conditions; in addition, fCN is detoxified during the reaction. The assay has been miniaturized to a microtiter plate format, making consumption of enzymes and chemicals very low. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated to be 9.1 µM fCN and 27.6 µM fCN, respectively. An analogous assay without CynH can be used for the determination of formamide with LOD and LOQ of 7.0 µM and 21.4 µM, respectively. Further measures to improve sensitivity are possible.

Free cyanide assay

Cyanide hydratase

Formamidase

Formate dehydrogenase

Spectrophotometry

Water-soluble tetrazolium salt

Free cyanide (fCN) is the sum of cyanide ions (CN⁻) and hydrogen cyanide (HCN). The latter tends to form highly toxic vapors, especially at pH ≤ 8.5 [1]. fCN is the most toxic among the different types of cyanides. Some cyanide species also form complexes with various metals and differ in stability [1]. Cyanide is one of highly threatening environmental contaminants and originates from several industries (gold and silver mining, petrochemistry, coal coking, fine chemical production, work-up of cyanogenic food plants etc.) but also other resources such as motor vehicle exhaust gases and smoking. Particularly, extraction of gold and silver ores requires bulk amounts of simple cyanide (NaCN) and leaves significant fCN residues in the wastes (“tailings”) [2–4].

Determination of fCN is required for various types of samples − industrial wastewaters, natural water, drinking water, cyanogenic plants and foods produced from them, body fluids and tissues etc. Routine fCN tests are primarily based on colorimetric reactions. A well-known example is the reaction of HCN with picric acid (2,4,6-trinitrophenol) [5–7]. The commercial Spectroquant® Cyanide Reagent Test is based on chlorinating fCN to cyanogen chloride (CNCl) followed by the reaction of CNCl and 1,3-dimethylbarbituric acid [8]. Recently, new colorimetric methods were developed using a cobalamin (vitamin B₁₂) derivative [9] or Ag and Ag-Au nanoparticles [10, 11]. Other methods include, e.g., a chromatographic separation of CN⁻ followed by amperometric detection [12], GC-MS [13], or ion-selective electrodes [14].

Enzymes have been proposed for use in fCN biosensors. The transformation of fCN was catalyzed by cyanide dihydratase (CynD), which is a type of nitrilase (EC 3.5.5.1) and hydrolyzes HCN to ammonia and formate. fCN was quantified on the basis of pH change [15], ammonia production [16, 17], or NADH production by formate dehydrogenase (FDH, EC 1.17.1.9) [18]. NADH was determined amperometrically or utilized in a coupled reaction catalyzed by salicylate hydroxylase (SHL, EC 1.14.13.1.). SHL recycled NADH along with oxygen consumption. The concomitant decrease in current was measured using the Clarke electrode (Pt working electrode, Ag/AgCl reference electrode) [19].

Enzymatic assays based on a spectrophotometric measurement of NADH are selective and sensitive. They have been developed e.g. for L- or D-lactic acid or formic acid, and commercial kits are available for some of them [20, 21]. In addition, the sensitivity of NADH detection and quantitation can be significantly enhanced by the addition of an electron mediator and a water-soluble tetrazolium (WST) salt that is reduced by NADH to formazan [22, 23]. However, assays of this type have been lacking for fCN. Therefore, we investigated whether this type of assay could be used for this purpose.

In this work, we have designed and developed a chemoenzymatic assay of fCN based on formazan formation. It consists of a multienzyme cascade including cyanide hydratase (CynHs; EC 4.2.1.66), formamidase (AmiF; EC 3.5.1.49) and FDH. The product of the cascade, NADH, is determined colorimetrically after reacting with a WST salt, 2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium dye (WST-8): NADH is oxidized, while WST-8 is reduced to color formazan (Fig. 1). This colorimetric method is much more sensitive than the conventional detection of NADH at 340 nm [22, 23].

Thus, the CynD used is the above-mentioned biosensors has been replaced by a two-step hydrolysis catalyzed by CynH and AmiF. CynH and AmiF exhibit higher specific activities than CynDs. In addition, CynH retains its activity up to high pH values (≈ 10) unlike CynD. The high pH is crucial to avoid losses of fCN. The combination of CynH and AmiF was already shown to be feasible in our recent work that focused on the bioremediation of fCN [24].

Chemicals

The standards and reaction mixture ingredients were purchased from standard suppliers: Formamide (99.5%) for analysis (Acros Organics (Thermo Scientific Chemicals), U.S.A.), KCN (≥ 96%) - ACS reagent (Sigma-Aldrich, U.S.A.); Nicotinamide adenine dinucleotide free acid (NAD) for biochemistry (Merck, U.S.A.); NADH disodium salt (Abcam, U.S.A.); 4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-l,3-benzene disulfonate sodium salt (WST-8; MedChemExpress, U.S.A.); 1-methoxy-5-methylphenazinium methyl sulfate (Cayman Chemical, U.S.A.).

Enzymes

FDH from Candida boidinii was purchased from Megazyme Ltd.

CynH (GenBank: KZV9269) from Exidia glandulosa was produced in Escherichia coli Origami B(DE3) as a fusion protein with a C-terminal His₆-tag. Cultivation and purification were carried out and the activity was determined as described previously [25]. Briefly, cultivation proceeded in 2xYT medium at 37°C until optical density measured at 600 nm reached 1.0. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a concentration of 0.02 mM and temperature was decreased to 20°C. The cells were harvested after 20 h cultivation at 20°C and cell-free extract was obtained by cell sonication. The enzyme was purified in one step using Talon® Metal Affinity Resin (Clontech, U.S.A.). One unit of the CynH activity is the amount of enzyme that converts 1 µmol of HCN per min at pH 9.0 and 30°C.

AmiF (pdb code: 5G3O_A) from Bacillus cereus (enzyme BceAmiF) was produced in E. coli BL21(DE3) as a fusion protein with a C-terminal His₆-tag. Cultivation and purification were carried out according to [26] with modifications as described previously [24]. The cultivation and purification methods were as described for CynH above but temperature was decreased to 16°C after adding IPTG. The activity of AmiF was determined as described previously [24], but with 100 mM Tris/HCl buffer, pH 7.6. Here, one unit of AmiF activity is the amount of enzyme that converts 1 µmol of formamide per min at pH 7.6 and 30°C.

CynD, CynH and AmiF were stored on ice and could be used within several months under these conditions.

Enzyme reactions

The first reaction of the cascade was performed using a Thermomixer Eppendorf Compact (30°C, 350 rpm). The reaction mixture (0.5 mL) contained 0.1 M glycine/NaOH buffer, pH 9.5, a KCN standard (0.01–0.10 mM of 96% KCN) and 10 U of purified CynH. Samples were withdrawn after 20 min and directly used for the next reaction which was performed in a 96-well microplate using a microtitration plate reader Tecan Sunrise™ controlled by the software Magellan™ (Tecan). The reaction mixture (0.22 mL) contained a sample (0.1 mL) from fCN hydrolysis (first reaction), 0.02 mL of 1 M Tris/HCl buffer, pH 7.6, purified AmiF (1 U), FDH (0.2 U), 5 mM NAD⁺, 0.2 mM WST-8, and 0.008 mM 1-methoxy PMS. The reaction was started with FDH. The reaction proceeded at 37°C after a 5-min preincubation at the same temperature. The plates were shaken for 10 s prior to each read-out. The production of formazan was monitored at 460 nm for 50 min. Optionally, formamide standards (0.010–0.20 mM) in 0.1 M glycine/NaOH buffer, pH 9.5, were used instead of samples from the first reaction.

Determination of detection and quantification limits

Calibration curves were constructed by plotting the absorbance at 460 nm against the concentration of standard (KCN or formamide). Limit of detection (LOD) and limit of quantification (LOQ) were calculated according to the following equations [27]:

LOD = 3.3 σ/S,

LOQ = 10 σ/S,

where σ is the standard deviation of response (STEYX function in MS Excel), and S is the slope of the calibration curve (SLOPE function in MS Excel).

Enzyme selection

CynHs are known for their excellent specific 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 specific 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. Thus, CynHs surpass CynDs in terms of specific activities and pH profiles. Wild-type CynDs are suitable for use at pH 8 but not pH 9 or higher [28]. Although some mutants of CynDs were active at a pH of 9.0, they were much less active at pH 9.5 and inactive at pH 10.0 [28]. In addition, their specific activities appear to be much lower compared to CynH: an activity of ≈ 22 U/mg was reached in the best CynD mutant at pH 9.0, although it must be noted that the conditions of the CynD assay were different (4 mM KCN, room temperature).

Using a CynH for the first step of the proposed cascade required to use AmiF to convert the product of CynH, formamide, to formic acid. One of the previously characterized AmiF enzymes, originating from B. cereus [26], was recently used by us for a two-step degradation of fCN [24]. The maximum activity of 2,800 ± 500 U/mg was reached at pH 6.0 and 50°C [26]. Recently, we investigated the activity of the enzyme at pH 8.0−10.0 and 30°C. The activity at pH 8.0 was 1,440 ± 200 U/mg and ≈ 55% and ≈ 40% of this activity was retained at pH 9.0 and 9.5, respectively [24].

The last enzyme in the enzymatic cascade was FDH, which is available commercially. The employed enzyme originated from C. boidinii and its optima were pH 7.6 and 37°C according to manufacturer [28].

Assay design

Based on the pH preferences of the used enzymes, we divided the cascade (Fig. 1) into two parts. AmiF is principally compatible with both CynH and FDH. We decided to combine it with FDH due to their similar pH optima. In addition, fCN was found to decrease the activity of AmiF [24]. This was another reason for using AmiF in the second step, which is presumably void of significant levels of fCN. Therefore, the assay was designed as follows: In the first step, CynH was hydrolyzed to formamide by the CynH at pH 9.5. This step was performed separately, while the rest of the cascade is performed as a “one-pot” reaction. This reaction involves formamide hydrolysis by AmiF and dehydrogenation of the product, formic acid, by FDH, followed by the colorimetric reaction of the formed NADH. A high pH is not necessary in this “one-pot” reaction where the substrate is formamide. Thus, pH in this reaction mixture of around 8.2 was appropriate for all three steps including the final reduction of WST-8 to formazan.

Determination of formamide concentration

First, we investigated the performance of the AmiF − FDH cascade using standard solutions of formamide (0.005-0.1 mM). The absorbance at 460 nm reached its maximum after 20 to 30 min (Fig. 2a) and the response was almost linear for 0.005 mM to 0.05 mM formamide (Fig. 2b).

The calibration for formamide was constructed by plotting the maximum absorbance achieved for each concentration against the concentration of formamide (Fig. 2b). The LOD and LOQ were calculated to be 7.0 µM (0.31 mg/mL) and 21.4 µM (0.96 mg/mL), respectively. Thus, the AmiF – FDH cascade with the colorimetric detection of NADH can be used for the quantification of formamide as, e.g., a product of cyanide biodegradation. Having confirmed that this cascade is feasible, we combined it with the fCN hydration by CyH to develop the targeted fCN assay (see below).

Determination of cyanide concentration

To develop the cyanide assay, we replaced formamide standards in the above-mentioned cascade with samples from the hydration of KCN by CynH. Other conditions were the same as in the above assay. The absorbance at 460 nm increased for 40−50 min (Fig. 3a) and the reaction rate was thus slightly lower than in the reaction of formamide standard, probably because of pH change (pH 8.2 in the cyanide assay vs. pH 7.6 in the formamide assay). The response was almost linear for KCN concentrations between approximately 0.01 mM and 0.077 mM (Fig. 3b).

The LOD and LOQ (Table 1) for fCN were calculated to be 9.1 µM (0.24 mg/L) and 27.6 µM (0.72 mg/L), respectively. The LOD is near the United States Environmental Protection Agency (EPA) limit for cyanide in drinking water, i.e. 0.2 mg/L [29].

Table 1

Limit of detection (LOD) and limit of quantification (LOQ) for the determination of formamide (Fig. 2b) and free cyanide supplemented as KCN (Fig. 3b).
Compound		STEYX function	SLOPE function	LOD (µM)	LOQ (µM)
Formamide		0.058	27.16	7.0	21.4
KCN		0.026	9.43	9.1	27.6

When comparing the proposed assays with other cyanide assays, the results should be seen in a broader context, taking into account the sensitivity but also the possible use of harmful chemicals or harsh reaction conditions, and intereferences. In comparison with the picric acid method, a routine fCN assay detecting ≥ 1 mg fCN/L, the enzymatic assay is over four times more sensitive.

In addition, the hazardous chemical and harsh conditions are the disadvantages of the picric acid method. In contrast, the Spectroquant kit allows to determine very low levels of cyanide such as 0.01 mg/L in a 1 cm cuvette. However, the kit is not designed for microplate readers, and thus requires large amounts of reagents.

Another disadvantage of the kit is its sensitivity to certain interfering ions (e.g., Br⁻, NO₂⁻,

SCN⁻, Ag⁺, Cu²⁺, Hg²⁺, Ni²⁺) [8]. The enzymatic method is selective by principle, although formic acid is naturally an interfering compound. However, this disadvantage can be surpassed by performing a control reaction without formamidase. Certain compounds present in the assayed samples could inhibit the used enzymes. 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 sulfides or thiocyanates did not significantly inhibit the enzyme [25], which is encouraging. Sulfides and sulfites (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]. Sulfides and thiocyanates are usual components of some cyanide-containing wastewaters, especially coke plant wastewater, and occur in them in up to g/L concentrations [25].

We report a proof of concept for a new spectrophotometric fCN assay based on NADH as a readily detectable product. The amount of NADH produced is proportional to fCN and is determined with a sensitive method with color formazan as the final product. Formamide can be determined analogously using a part of the cascade reaction. The assay is in a high-throughput format, and the consumption of enzymes and chemicals is minimal. As far as we know, the CynH and AmiF enzymes have not yet been commercialized unlike FDH but their preparation is straightforward. The method can be still improved, e.g. by varying the assay conditions, in order to achieve even lower detection limits.

Competing interests

The authors declare no competing interests.

Ethical Approval

This research contains no human or animal studies.

Funding

This research was funded by the Czech Science Foundation (grant GA22-06785S), by the Ministry of Education, Youth and Sports of the Czech Republic grant Talking microbes - understanding microbial interactions within One Health framework (CZ.02.01.01/00/22_008/0004597), and by the Czech Academy of Sciences (grant RVO61388971).

Availability of data and materials

Data and materials are available on request.

Author Contribution

Katarína Šťastná: methodology, investigation, validation, and writing (review, and editing). Ludmila Martínková: conceptualization, methodology, validation, resources, supervision, project administration, funding acquisition, and writing (original draft, review, and editing). Lenka Rucká: methodology, investigation, and writing (review and editing). Barbora Křístková: investigation, and writing (review and editing). Pavla Bojarová: conceptualization, validation, supervision, funding acquisition, and writing (review, and editing). Miroslav Pátek: conceptualization, funding acquisition, and writing (review, and editing).

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No competing interests reported.

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

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