Development of an enzyme membrane reactor for treatment of cyanide-containing wastewaters from the food industry

Cyanidase, an immobilized enzyme preparation for hydrolyzing cyanide to ammonia and formate, was applied for the treatment of cyanide-containing waste waters from the food industry. Apricot seed extract was chosen as a model effluent. The enzymatic hydrolysis of pure amygdalin, the main cyanogenic glycoside in the extract, and the degradation of the cyanide formed was investigated and compared with the behavior of the real extract in a batch slurry reactor. A diffusional-type, flat-membrane reactor with immobilized cyanidase was developed, where the enzyme is effectively protected from adverse effects of high molecular components contained in the extract. For monitoring continuous-membrane reactor operation, a new unsegmented ammonia measurement system was developed and applied. In continuous operation the cyanidase retained its original activity for more than 400 hours on steam. (c) 1993 John Wiley & Sons, Inc.     

Effects of the Gut microbiota on Amygdalin and its use as an anti-cancer therapy: Substantial review on the key components involved in altering dose efficacy and toxicity

Conventional and Alternative Medicine (CAM) is popularly used due to side-effects and failure of approved methods, for diseases like Epilepsy and Cancer. Amygdalin, a cyanogenic diglycoside is commonly administered for cancer with other CAM therapies like vitamins and seeds of fruits like apricots and bitter almonds, due to its ability to hydrolyse to hydrogen cyanide (HCN), benzaldehyde and glucose. Over the years, several cases of cyanide toxicity on ingestion have been documented. In-vitro and in-vivo studies using various doses and modes of administration, like IV administration studies that showed no HCN formation, point to the role played by the gut microbiota for the commonly seen poisoning on consumption. The anaerobic Bacteriodetes phylum found in the gut has a high β-glucosidase activity needed for amygdalin hydrolysis to HCN. However, there are certain conditions under which these HCN levels rise to cause toxicity. Case studies have shown toxicity on ingestion of variable doses of amygdalin and no HCN side-effects on consumption of high doses. This review shows how factors like probiotic and prebiotic consumption, other CAM therapies, obesity, diet, age and the like, that alter gut consortium, are responsible for the varying conditions under which toxicity occurs and can be further studied to set-up conditions for safe oral doses. It also indicates ways to delay or quickly treat cyanide toxicity due to oral administration and, reviews conflicts on amygdalin's anti-cancer abilities, dose levels, mode of administration and pharmacokinetics that have hindered its official acceptance at a therapeutic level.     

Excreted thiocyanate detects live reef fishes illegally collected using cyanide--a non-invasive and non-destructive testing approach

Cyanide fishing is a method employed to capture marine fish alive on coral reefs. They are shipped to markets for human consumption in Southeast Asia, as well as to supply the marine aquarium trade worldwide. Although several techniques can be used to detect cyanide in reef fish, there is still no testing method that can be used to survey the whole supply chain. Most methods for cyanide detection are time-consuming and require the sacrifice of the sampled fish. Thiocyanate anion (SCN(-)) is a metabolite produced by the main metabolic pathway for cyanide anion (CN(-)) detoxification. Our study employed an optical fiber (OF) methodology (analytical time <6 min) to detect SCN(-) in a non-invasive and non-destructive manner. Our OF methodology is able to detect trace levels (>3.16 μg L(-1)) of SCN(-) in seawater. Given that marine fish exposed to cyanide excrete SCN(-) in the urine, elevated levels of SCN(-) present in the seawater holding live reef fish indicate that the surveyed specimens were likely exposed to cyanide. In our study, captive-bred clownfish (Amphiprion clarkii) pulse exposed for 60 s to either 12.5 or 25 mg L(-1) of CN(-) excreted up to 6.96±0.03 and 9.84±0.03 μg L(-1) of SCN(-), respectively, during the 28 days following exposure. No detectable levels of SCN(-) were recorded in the water holding control organisms not exposed to CN(-), or in synthetic seawater lacking fish. While further research is necessary, our methodology can allow a rapid detection of SCN(-) in the holding water and can be used as a screening tool to indicate if live reef fish were collected with cyanide.     

The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode beta-cyano-L-alanine hydratase/nitrilase

Nitrilases (nitrile aminohydrolases, EC ) are enzymes that catalyze the hydrolysis of nitriles to the corresponding carbon acids. Among the four known nitrilases of Arabidopsis thaliana, the isoform NIT4 is the most divergent one, and homologs of NIT4 are also known from species not belonging to the Brassicaceae like Nicotiana tabacum and Oryza sativa. We expressed A. thaliana NIT4 as hexahistidine tag fusion protein in Escherichia coli. The purified enzyme showed a strong substrate specificity for beta-cyano-l-alanine (Ala(CN)), an intermediate product of cyanide detoxification in higher plants. Interestingly, not only aspartic acid but also asparagine were identified as products of NIT4-catalyzed Ala(CN) hydrolysis. Asn itself was no substrate for NIT4, indicating that it is not an intermediate but one of two reaction products. NIT4 therefore has both nitrilase and nitrile hydratase activity. Several lines of evidence indicate that the catalytic center for both reactions is the same. The NIT4 homologs of N. tabacum were found to catalyze the same reactions and protein extracts of A. thaliana, N. tabacum and Lupinus angustifolius also converted Ala(CN) to Asp and Asn in vitro. NIT4 may play a role in cyanide detoxification during ethylene biosynthesis because extracts from senescent leaves of A. thaliana showed higher Ala(CN) hydratase/nitrilase activities than extracts from nonsenescent tissue.     

Synthesis and evaluation of diverse analogs of amygdalin as potential peptidomimetics of peptide T

Peptide T (ASTTTNYT) is a promising molecule to prevent the neuropsychometric symptoms of patients suffering AIDS and for the treatment of psoriasis. In order to fully prove its therapeutic benefits, efforts were put forward to design peptidomimetics of the peptide. In this direction, in a recent computational study the natural product amygdalin was identified as a prospective peptidomimetic of the peptide and later proved to exhibit a similar chemotactic profile to the peptide. However, the cyanide moiety of amygdalin provides to the molecule a toxic profile. The present study reports the synthesis of a set of amygdalin analogs lacking the cyanide group with improved chemotactic profiles.     

Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida

Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of [14C]aspartate or [3H]alanine, respectively, following acid hydrolysis of the labeled protein. When labeling with cyanide was conducted under denaturing conditions, 4 mol of [14C]cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits. In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of [14C]cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites. Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present. Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation. A labeled tryptic peptide was isolated from enzyme inactivated with [14C]cyanide. Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue. Such a finding indicates the dehydroalanine group is distinct from the active site cysteine. Treatment of crude extracts with [14C]cyanide and purification of the inactive enzyme yielded labeled protein that release [14C]aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Species comparison of methemoglobin reductase

Methemoglobin (MHb) formation is effective in treating cyanide (CN) poisoning. Endogenous activity of the enzyme MHb reductase (MR) reflects the capacity to reduce MHb and thus represents a key factor for evaluating anti-CN efficacy of MHb formers. MR activity was measured in whole blood of nine animal species and was compared with human MR activity. The animals in this comparative study included seven nonhuman primate (NHP) species, the beagle dog, and the ferret. Although exhibiting higher MR activity than in humans, the rhesus and aotus NHPs' average MR activity was the closest to humans', with raw data from each NHP showing overlap with human raw data. The beagle dog, used extensively to study anti-CN characteristics of MHb formers, was the sole species that displayed MR activity lower than in humans, with no data overlap. Based on MR activity, the rhesus and aotus NHPs may each represent a more accurate model for predicting human responses to MHb formers. The data from this study provides a unique interspecies enzyme comparison, which should facilitate future rational development of anti-CN MHb formers.     

氰化物污染的植物修复可行性研究

氰化物是目前世界范围内最常使用的提取黄金和白银等贵重金属的沥滤剂,其对自然生态环境的污染和破坏以及对人畜和其它生物的毒性作用是众所周知的.本试验用一自行设计的生物反应器来观察黄豆(Glycine max(L)Merr.)和玉米(Zea mays L.)对氰化物污染土壤的原位修复的可能性.室温条件下(23.0～26.0℃),低浓度的氰化物污染液对(≤45.5 CN mg·L^-1)二种测试植物的生长没有产生任何毒性作用;而在高浓度的氰化物试验组(≥91.0 CNmg·L^-1),二种测试植物的生长都出现了明显的滞长现象(生长率下降大于10%),但没有观察到其它毒性反应.同时二种测试植物的叶片细胞用来测定植物细胞线粒体中的氰丙氨酸合成酶(β-cyanoalanine synthase)转化氰化物的潜力.实验是在一封闭的玻璃器皿(100mL)中进行的(100mL的氰化钾溶液中加入1.5g(鲜重)植物的叶片,氰化钾溶液的浓度大约1.0 CNmg·L^-1).在为期28 h的时间内,水溶液中超过90%的氰化物被植物的叶片去除;黄豆和玉米的的叶片细胞对氰化物去除率分别测定为4.43mg CN·kg^-1(鲜重)·h^-1和3.42mg CN·kg^-1(鲜重)·h^-1.本实验结果表明,植物对氰化物污染的土壤原位修复方法是一种可行的和有效的选择.     

Quantitative determination of amygdalin epimers from armeniacae semen by liquid chromatography

D-amygdalin and its conversion product, neoamygdalin, were quantitatively analyzed on reverse-phase, high-performance liquid chromatography with an optimized eluent of 10 mM sodium phosphate buffer (pH 3.1) containing 8.5% acetonitrile. Linearity between concentrations and detector responses was obtained in the range from 0.05 to 0.5 mM. The detection limits for D-amygdalin and neoamygdalin were approximately 5 microM per injected amount. Armeniacae semen contains not only amygdalin but also emlusin, which is an enzyme that hydrolyzes amygdalin. When extracting amygdalin from a whole piece of armeniacae semen in boiling water, there was almost no influence of emulsin; which increased the extraction efficiency. However, conversion of d-amygdalin into neoamygdalin at high temperature was found. In this report, we solved this problem by using 4% citric acid as an extractant. This solution also prevented the extraction process from being affected by emulsin. In addition, the extraction efficiency remained the same as that when methanol was used as an extractant, regardless of the cutting size.     

Wild Bearded Capuchin Monkeys (Sapajus libidinosus) Strategically Place Nuts in a Stable Position during Nut-Cracking

Humans can use hand tools smoothly and effectively in varying circumstances; in other words, skillfully. A few other species of primates crack encased foods using hammer tools and anvils. Are they skilled? Positioning the food on the anvil so that it does not fall off when struck is a component of skilled cracking. We discovered that bearded capuchin monkeys deliberately place palm nuts in a relatively stable position on the anvil before striking them. In the first experiment, we marked the meridians of palm nuts where they stopped when rolled on a flat surface (“Stop meridian”). We videotaped monkeys as they cracked these nuts on an anvil. In playback we coded the position of the Stop meridian prior to each strike. Monkeys typically knocked the nuts on the anvil a few times before releasing them in a pit. They positioned the nuts so that the Stop meridian was within 30 degrees of vertical with respect to gravity more often than expected, and the nuts rarely moved after the monkeys released them. In the second experiment, 14 blindfolded people (7 men) asked to position marked nuts on an anvil as if to crack them reliably placed them with the Stop meridian in the same position as the monkeys did. In the third experiment, two people judged that palm nuts are most bilaterally symmetric along a meridian on, or close to, the Stop meridian. Thus the monkeys reliably placed the more symmetrical side of the nuts against the side of the pit, and the nuts reliably remained stationary when released. Monkeys apparently used information gained from knocking the nut to achieve this position. Thus, monkeys place the nuts skillfully, strategically managing the fit between the variable nuts and pits in the anvil, and skilled placement depends upon information generated by manual action.     

Evaluation of Cyanogenic Potentials of Local Cassava Species and Residual Cyanide Contents of Their Locally Processed Food Products in Southeast Nigeria

The cyanogenic potentials and residual cyanide contents of local cassava parenchyma and their locally processed food products in southeastern Nigeria were studied. Seven species of cassava locally grown and four main food products from them were analyzed colorimetrically for their cyanide contents. Results of the analyses indicated that five of the species contain cyanide potentials between 50 and 100 mg HCN/kg fresh weight while only one contains cyanogens level greater than 100 mg HCN/kg fresh weight. Of the cassava products analyzed, two contained cyanide above the level recommended by the WHO/FAO (10 mg HCN/kg). The result raises concern as these cassava products constitute about 80–90% of the diet of the local people and the facts known about cyanide poisoning from intake of high cyanide containing food.     

Biological cyanide degradation in aerobic fluidized bed reactors: treatment of almond seed wastewater

The continuous aerobic transformation of synthetic cyanide waste-water, amygdalin solutions and almond seed extract containing cyanide was investigated in several fluidized bed reactors. Various inocula consisting of activated sludge or soil slurry were used. Successful inoculation was achieved with simple soil slurry. No significant influence was found between the performance of the systems inoculated with a cyanide contaminated soil and a garden soil. The performance and stability of the reactors with respect to degradation rate were tested for a range of cyanide loading conditions, with feed containing only cyanide, and with different additional carbon sources, as well as various CN ratios at a hydraulic retention time of 24 h. No growth with cyanide as the sole source of carbon and nitrogen was observed. The system with lactate as the organic C-source was capable of operating at cyanide concentrations of 160 ppm cyanide with a conversion rate of 0.125 kg cyanide/m³ d. Ammonia was the end product and the effluent concentration was 0.5 ppm CN^–. The systems with ethanol as the organic C-source could degrade only 0.05 kg cyanide/m³ d, whose feed concentration was 60 ppm cyanide. Amygdalin, an organic cyanide-containing compound present in stone fruit seeds, was fed as a model substrate. Degradation rates up to 1.2 kg COD/m³ d could be measured with no free or organically bound cyanide in the effluent. These rates were limited by oxygen transfer, owing to the large amount of degradable COD. The further investigations with almond seed extracts, confirmed the applicability of the aerobic process to treat food-processing waste streams having low concentrations of cyanide with high COD content.The authors with to thank Dr. Ö.M. Kurt for useful discussions.     

Rapid quantitation of cyanide in whole blood by automated headspace gas chromatography

Cyanide (CN), a chemical asphyxiant, is a rapidly acting and powerful poison. We have developed a sensitive, rapid, simple, and fully automated method for measuring CN in whole blood. The assay is based on the use of gas chromatography (GC) with nitrogen-phosphorus detection and acetonitrile as an internal reference. Following the automated addition of phosphoric acid to the blood sample, the released hydrogen cyanide is analyzed using a fully automated headspace GC system. The assay, validated on human blood samples spiked with potassium cyanide and on clinical samples from fire victims who had smoke inhalation injury, can detect CN at a wide range of concentrations (30-6000 microg/l) in about 17 min (including incubation and GC run time, and <2 min for manual sample preparation). This automated, high-throughput, simple, and sensitive method is suitable for the rapid diagnosis of CN in clinical and forensic specimens.     

Cyanide enhances hydrogen peroxide toxicity by recruiting endogenous iron to trigger catastrophic chromosomal fragmentation

Hydrogen peroxide (HP) or cyanide (CN) are bacteriostatic at low‐millimolar concentrations for growing Escherichia coli, whereas CN + HP mixture is strongly bactericidal. We show that this synergistic toxicity is associated with catastrophic chromosomal fragmentation. Since CN alone does not kill at any concentration, while HP alone kills at 20 mM, CN must potentiate HP poisoning. The CN + HP killing is blocked by iron chelators, suggesting Fenton's reaction. Indeed, we show that CN enhances plasmid DNA relaxation due to Fenton's reaction in vitro. However, mutants with elevated iron or HP pools are not acutely sensitive to HP‐alone treatment, suggesting that, in addition, in vivo CN recruits iron from intracellular depots. We found that part of the CN‐recruited iron pool is managed by ferritin and Dps: ferritin releases iron on cue from CN, while Dps sequesters it, quelling Fenton's reaction. We propose that disrupting intracellular iron trafficking is a common strategy employed by the immune system to kill microbes.     

Inorganic Pyrophosphatase from E. coli as a Label for the Detection of Plant Viruses by ELISA

Inorganic pyrophosphatase (PPase) from E. coli was used as label in enzyme immunoassay for the detection of different plant viruses. The use of PPase-conjugated antibodies and tetrasodium pyrophosphate as a substrate in ELISA provides some advantages in detection of plant viruses in purified preparations and extracts from plant specimens: high sensitivity, negligible level of background reactions in control samples, bright blue-greenish colour which allows to detect the reaction visually and high stability of PPase-conjugated antibodies.     

Quantitative immunoassay of human deoxycytidine kinase in malignant cells.

Deoxycytidine kinase (dCK) is necessary for the activity of several nucleosides used for the chemotherapy of cancer and AIDS. However, the measurement of dCK catalytic activity in crude cell extracts may be imprecise, due to the presence of phosphatases and nucleotidases that degrade the enzyme products. We describe a simple immunoassay for dCK that can measure accurately as little as 5 ng enzyme protein in crude tissue extracts. The assay enabled us to show (i) that mutant cells deficient in dCK activity lack immunoreactive dCK protein, (ii) that dCK catalytic activity and immunoreactivity correlate closely in human tumors, and (iii) that immunoreactive dCK is particularly high in lymphocytes and lymphoid malignancies, although certain solid tumors may also contain the enzyme. The immunoassay of dCK could prove useful in the selection and monitoring of patients who are being treated with nucleosides that are activated by this enzyme.     

Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by Pseudomonas fluorescens NCIMB 11764

Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min⁻¹ ml of culture fluid⁻¹) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by ¹³C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO₂ (and NH₃) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.     

Mercaptopyruvate sulfurtransferase as a defense against cyanide toxication: molecular properties and mode of detoxification.

In cyanide poisoning, metalloproteins and carbonyl groups containing proteins are the main target molecules of nucleophilic attack by cyanide. To defend against this attack, cyanide is metabolized to less toxic thiocyanate via transsulfuration. This reaction is catalyzed by rhodanese and mercaptopyruvate sulfurtransferase (MST). Rhodanese is a well characterized mitochondrial enzyme. On the other hand, little was known about MST because it was unstable and difficult to purify. We first purified MST to homogeneity and cloned MST cDNA from rat liver to characterize MST. We also found that MST was an evolutionarily related enzyme of rhodanese. MST and rhodanese are widely distributed in rat tissues, and the kidney and liver prominently contain these enzymes. Immunohistochemical study revealed that MST is mainly distributed in proximal tubular epithelial cells in the kidney, pericentral hepatocytes in the liver, the perinuclear area of myocardial cells in the heart, and glial cells in the brain, and immunoelectron microscopical study concluded that MST was distributed in both cytoplasm and mitochondria, so that MST first detoxifies cyanide in cytoplasm and the cyanide which escapes from catalysis due to MST enters mitochondria. MST then detoxifies cyanide again in cooperation with rhodanese in mitochondria. Tissues other than the liver and kidney are more susceptible to cyanide toxicity because they contain less MST and rhodanese. Even in the same tissue, sensitivity to cyanide toxicity may differ according to the kind of cell. It is determined by a balance between the amount of proteins to be attacked and that of enzymes to defend.     

A Novel ELISA Format for the Rapid and Sensitive Detection of Staphylococcal Enterotoxin A

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.