Biodegradation of Cyanide under Alkaline Conditions by a Strain of Pseudomonas Putida Isolated from Gold Mine Soil and Optimization of Process Variables through Response Surface Methodology (RSM)

In regard to highly poisonous effects of cyanide ion, concerns have been focused recently on treatment of such compounds in different ways. Four bacterial strains (C1-C4) capable of using cyanide as nitrogen source were isolated from contaminated gold mine soil samples under alkaline conditions at 30 °C, pH 9.5-10.5, and agitation speed 150 rpm. The gram-negative bacterium C3 (identified as Pseudomonas parafulva NBRC 16636(T) by 16S rRNA gene sequencing) was able to tolerate cyanide up to 500 ppm besides removing 93.5% of 200 ppm cyanide in 13 days which was confirmed by microorganisms growth. The addition of basal salts enhanced the removal efficiency of C3 by 16%. Cyanide removal efficiency of co-culture was 30% less than C3. Optimization of three significant parameters including temperature, pH, and glucose concentration for cyanide biodegradation was studied using response surface methodology (RSM). The optimum conditions for maximizing cyanide biodegradation were temperature (32.23 °C), pH (9.95), and glucose concentration (0.73 g/l)

Efficient bioremediation of toxic waste by new nitrilases

Wastewater from certain industrial processes (gold and silver mining, electroplating with Cu, Ag and other metals, coal coking) contains high concentrations of free cyanide (hydrogen cyanide and cyanide ions; fCN) and can pose a threat to environmental safety. Modern approaches to wastewater treatment are primarily based on (physico)chemical methods and a final biological treatment with mixed microbial cultures. Enzymatic removal of fCN represents an environmentally promising approach. This work focuses on the remediation potential of enzymes from the nitrilase superfamily. In fungi, hypothetical nitrilases and cyanide hydratases were found through database searches. Selected enzymes were overproduced in Escherichia coli, purified and characterized, and their remediation potential was evaluated based on these results. Two of the enzymes belonged to the NIT4-type nitrilases (EC 3.5.5.4) according to their amino acid sequences: the NitTv1 enzyme from Trametes versicolor (protein XP_008032838.1) and the NitAb enzyme from Agaricus bisporus (protein XP_006462086.1). NIT4-type nitrilases are widespread plant enzymes that transform -cyano-L-alanine (AlaCN), an intermediate of fCN scavenging. AlaCN is converted to utilizable amino acids by NIT4. In this work, these enzymes were described for the first time in fungi. The fungal enzymes NitTv1 and NitAb exhibited high specific activities with AlaCN − approximately 132 and 40 U/mg of protein, respectively. In addition, the substrate specificity of these enzymes went beyond plant NIT4, as they could also transform other nitriles (phenylpropionitrile, cinnamonitrile, fumaronitrile). The stability properties were better for NitTv1 than NitAb. Presumably, these nitrilases play a role in plant-fungus interactions, allowing fungi to detoxify plant nitriles. Cyanide hydratases (CynHs) (EC 4.2.1.66) convert fCN into the much less toxic formamide and have been therefore considered for the decontamination of cyanide effluents. The purified CynH from Exidia glandulosa (protein KZV92691.1, enzyme NitEg) showed 5 high activity towards fCN with 784 U/mg of protein, kcat (turnover number) of 927/s, and kcat/KM (catalytic efficiency) of 42 l/s/mmol. pH and temperature optimum were 6–9 and 40–45 °C, respectively. Thus, the catalytic properties of NitEg surpassed those of the second CynH studied, which was from the fungus Stereum hirsutum (protein XP_007307917.1, enzyme NitSh). High concentrations of silver and copper (1 mM) reduced the activity of NitEg by 30–40%, whereas phenol, thiocyanate, sulfide, and ammonia, at levels typical for industrial effluents, did not lead to a significant reduction in fCN conversion. The enzyme was also functional at high fCN concentrations (100 mM), which is relevant to the remediation of wastewater from metal electroplating. NitEg is thus a promising biocatalyst for direct fCN detoxification, and NIT4 was shown to likely play an important role in fCN detoxification by fungi. Both enzymes are thus prospective for the detection and determination of fCN. Nevertheless, the potential of the enzymes is just emerging and requires further intensive research to improve and scale up their production and application.Odpadní vody z některých průmyslových procesů (těžba zlata a stříbra, galvanické pokovování Cu, Ag a dalšími kovy, koksování uhlí) obsahují vysoké koncentrace volného kyanidu (kyanovodík a kyanidové ionty) a mohou představovat hrozbu pro bezpečnost životního prostředí. Moderní přístupy k čištění odpadních vod jsou založeny především na (fyzikálně)-chemických metodách a konečném biologickém čištění pomocí směsných mikrobiálních kultur. Enzymatické odstraňování volného kyanidu představuje přístup šetrný k životnímu prostředí. Tato práce se zaměřuje na remediační potenciál enzymů z nadrodiny nitrilas. U hub byly pomocí vyhledávání v databázích identifikovány hypotetické nitrilasy a kyanidhydratasy. Vybrané enzymy byly nadprodukovány v heterologním hostiteli (Escherichia coli), purifikovány a charakterizovány a na základě těchto výsledků byl vyhodnocen jejich remediační potenciál. Dva z enzymů patřily podle aminokyselinových sekvencí k nitrilasám typu NIT4 (EC 3.5.5.4): enzym NitTv1 z houby Trametes versicolor (protein XP_008032838.1) a enzym NitAb z houby Agaricus bisporus (protein XP_006462086.1). Nitrilasy typu NIT4 jsou široce rozšířené rostlinné enzymy, které přeměňují β-kyano-L-alanin (AlaCN), meziprodukt detoxifikace a utilizace volného kyanidu. AlaCN je pomocí nitrilasy NIT4 přeměňován na využitelné aminokyseliny. V této práci byly tyto enzymy poprvé popsány u hub. Fungální enzymy NitTv1 a NitAb vykazovaly vysoké specifické aktivity pro AlaCN - přibližně 132, resp. 40 U/mg proteinu. Substrátová specifita těchto enzymů byla navíc širší ve srovnání s rostlinnou nitrilasou NIT4. Fungální enzymy přeměňovaly i jiné nitrily (fenylpropionitril, nitril kyseliny skořicové, fumaronitril). Stabilita byla lepší u NitTv1 než u NitAb. Pravděpodobně tyto nitrilasy hrají roli v interakcích mezi rostlinami a houbami a umožňují houbám detoxikovat rostlinné nitrily. 7 Kyanidhydratasy (EC 4.2.1.66) přeměňují volný kyanid na mnohem méně toxický formamid, a proto se o nich uvažuje jako o enzymu pro dekontaminaci kyanidových odpadních vod. Purifikovaná CynH z houby Exidia glandulosa (protein KZV92691.1, enzym NitEg) vykazovala vysokou aktivitu pro volný kyanid (asi 784 U/mg proteinu), kcat (číslo přeměny) 927/s a kcat/KM (katalytická účinnost) 42 l/s/mmol. pH a teplotní optimum byly 6-9 a 40-45 °C. Katalytické vlastnosti NitEg tak předčily vlastnosti druhého studovaného enzyme, CynH, který pocházel z houby Stereum hirsutum (protein XP_007307917.1, enzym NitSh). Vysoké koncentrace stříbra a mědi (1 mM) snížily aktivitu NitEg o 30-40 %, zatímco fenol, thiokyanát, sulfid a amoniak v koncentracích typických pro průmyslové odpadní vody nevedly k významnému snížení konverze fCN. Enzym byl funkční i při vysokých koncentracích fCN (100 mM), což je důležité pro sanaci odpadních vod z galvanického pokovování. NitEg je tedy slibným biokatalyzátorem pro přímou detoxikaci fCN a ukázalo se, že NIT4 pravděpodobně hraje důležitou roli při detoxikaci fCN houbami. Oba enzymy jsou také perspektivní pro detekci a stanovení fCN. Nicméně potenciál těchto enzymů se teprve objevuje a vyžaduje další intenzivní výzkum ke zlepšení a rozšíření jejich výroby a použití

Directed Evolution of Cyanide Degrading Enzymes

Cyanide is acutely toxic to the environment. However, this simple nitrile is used in several industrial applications especially the mining industry. Due to its high affinity to metals, cyanide has been used for years to extract gold and other precious metals from the ore. Cyanide nitrilases are considered for the detoxification of the industrial wastewaters contaminated with cyanide. Their application in cyanide remediation promises cheaper and safer processes compared to chemical detoxification. However, application of these enzymes in industry requires improving their characteristics. The goal of this dissertation is to better understand cyanide nitrilases, in particular the cyanide dihydratase from of Bacillus pumilus and Pseudomonas stutzeri and to improve their activity and stability. The lack of any high resolution structure of these enzymes calls for isolating or screening for mutants showing enhancement in enzyme properties. Described first is a simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification. This method is useful for generating large pools of randomly mutagenized clones after error-prone PCR mutagenesis. Several parameters were investigated to optimize this technique; length of homology region, vector treatment, induction time and ratio of fragment to vector. Using error-prone PCR for random mutagenesis, several CynDpum mutants were isolated for higher catalysis at pH 7.7. Three point mutations, K93R, D172N and E327K increased the enzyme’s thermostability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 suggesting an effect on the active site. However, the A202T mutation located in the dimerization or the A surface rendered the enzyme inactive by destabilizing it. No significant effect on activity at alkaline pH was observed for any of the purified mutants. Lastly, an important region for CynDstut activity was identified in the C-terminus. This same region increased the stability of CynDpum compared to the wild-type enzyme. Also, CynDpum-stut hybrid was found to be highly more stable than CynDpum. This same hybrid exhibited 100% activity at pH9, a pH where the parent enzyme is inactive, and retained 40% of its activity at pH 9.5 making it a true pH tolerant mutant

Cinética en la biodegradación de cianuro por Bacillus sp. en condiciones alcalinas

El cianuro es un compuesto altamente tóxico que se utiliza en diversas actividades industriales, que representa un riesgo ambiental difícil de controlar. Este estudio tuvo como objetivo aislar cepas de bacterias nativas provenientes de un pasivo ambiental minero con capacidad de tolerar cianuro y determinar la cinética de biodegradación de cianuro de la cepa más eficiente utilizando un biorreactor tipo batch en condiciones alcalinas. Los resultados permitieron aislar siete cepas bacterianas que tuvieron la capacidad de tolerar hasta 800 ppm de cianuro libre y una eficiencia de biodegradación entre 50% y 96%. La cepa 2 de Bacillus sp. presentó una eficiencia de degradación del 96,8 % en 36 horas. El análisis de la biodegradación siguió una cinética de primer orden (k1 = 0,06649 mg/(mg·h), R2 = 0,97), lo cual indica que la cepa presenta gran potencial para su aplicación en estudios de biorremediación de áreas contaminadas con cianuro

Rhizobacteria isolated from saline soil induce systemic tolerance in wheat (triticum aestivum l.) against salinity stress

Halotolerant plant growthpromoting rhizobacteria (PGPR) have the inherent potential to cope up with salinity. Thus, they can be used as an effective strategy in enhancing the productivity of saline agrosystems. In this study, a total of 50 isolates were screened from the rhizospheric soil of plants growing in the salt range of Pakistan. Out of these, four isolates were selected based on their salinity tolerance and plant growth promotion characters. These isolates (SR1. SR2, SR3, and SR4) were identified as Bacillus sp. (KF719179), Azospirillum brasilense (KJ194586), Azospirillum lipoferum (KJ434039), and Pseudomonas stutzeri (KJ685889) by 16S rDNA gene sequence analysis. In vitro, these strains, in alone and in a consortium, showed better production of compatible solute and phytohormones, including indole acetic acid (IAA), gibberellic acid (GA), cytokinin (CK), and abscisic acid (ABA), in culture conditions under salt stress. When tested for inoculation, the consortium of all four strains showed the best results in terms of improved plant biomass and relative water content. Consortiuminoculated wheat plants showed tolerance by reduced electrolyte leakage and increased production of chlorophyll a, b, and total chlorophyll, and osmolytes, including soluble sugar, proline, amino acids, and antioxidant enzymes (superoxide dismutase, catalase, peroxidase), upon exposure to salinity stress (150 mM NaCl). In conclusion, plant growthpromoting bacteria, isolated from saltaffected regions, have strong potential to mitigate the deleterious effects of salt stress in wheat crop, when inoculated. Therefore, this consortium can be used as potent inoculants for wheat crop under prevailing stress conditions

Regulation and Function of Versatile Aerobic and Anaerobic Respiratory Metabolism in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitously distributed opportunistic pathogen that inhabits soil and water as well as animal-, human-, and plant-host-associated environments. The ubiquity would be attributed to its very versatile energy metabolism. P. aeruginosa has a highly branched respiratory chain terminated by multiple terminal oxidases and denitrification enzymes. Five terminal oxidases for aerobic respiration have been identified in the P. aeruginosa cells. Three of them, the cbb3-1 oxidase, the cbb3-2 oxidase, and the aa3 oxidase, are cytochrome c oxidases and the other two, the bo3 oxidase and the cyanide-insensitive oxidase, are quinol oxidases. Each oxidase has a specific affinity for oxygen, efficiency of energy coupling, and tolerance to various stresses such as cyanide and reactive nitrogen species. These terminal oxidases are used differentially according to the environmental conditions. P. aeruginosa also has a complete set of the denitrification enzymes that reduce nitrate to molecular nitrogen via nitrite, nitric oxide (NO), and nitrous oxide. These nitrogen oxides function as alternative electron acceptors and enable P. aeruginosa to grow under anaerobic conditions. One of the denitrification enzymes, NO reductase, is also expected to function for detoxification of NO produced by the host immune defense system. The control of the expression of these aerobic and anaerobic respiratory enzymes would contribute to the adaptation of P. aeruginosa to a wide range of environmental conditions including in the infected hosts. Characteristics of these respiratory enzymes and the regulatory system that controls the expression of the respiratory genes in the P. aeruginosa cells are overviewed in this article

Regions Involved in the Oligomerization and Activity of the Spiral Forming Nitrilase Cyanide Dihydratase

Nitrilases offer an economic and environmentally friendly solution for the production of valuable chemicals, and the degradation of toxic industrial nitrile wastes. A lack of fine structural data has impeded rational engineering of these enzymes to expand their utility. The nitrilases form large spiral shaped oligomers, seen by electron microscopy. These spiral structures are seen in all active nitrilases, and mutations altering this structure influence activity, stability, and substrate specificity. Currently no spiral forming nitrilase has been amenable to crystallization, leaving a significant gap in our understanding of these enzymes. Crystal structures from the larger nitrilase-superfamily reveal a conserved dimer structure and active site. Three regions in nitrilases are hypothesized to associate these dimer building blocks into the spiral oligomer. Two stand out as insertions relative to the crystallized homologs. These regions are hypothesized to associate the basic nitrilase dimer to form the large spiral structure. I investigate this hypothesis in the nitrilase CynD_(pum) by constructing mutations throughout these insertion regions and ascertaining their effect on enzyme activity as well as on oligomerization. A third region that stands out from the nitrilase sequences in comparison to the crystallized superfamily members is the extension of the C-terminus. Mutations to the C-terminus in various nitrilases can alter the proteins stability, activation, and substrate specificity, but how it interacts with other regions of the protein is not understood. In CynD, exchanging the C-terminus between the Bacillus pumilus enzyme and that of Pseudomonas stutzeri (CynD_(stut)) shows a one-way compatibility. When CynD_(pum) is paired with the P. stutzeri C-terminus (CynD_(pum-stut)), the hybrid enzyme is active and more stable than either parent enzyme. Conversely, the reverse hybrid, where the P. stutzeri CynD is paired with the B. pumilus C-terminus (CynD_(stut-pum)) is not active. In this study I exploit the stabilizing effect of the C-terminus from B. pumilus to test whether it participates in spiral formation. Additionally, I identify regions interacting with the C-terminus by looking for suppressing substitutions in CynD_(stut-pum) that restore activity

Cyanide detoxification by soil microorganisms.

Cyanides enter the environment through both natural and man-made sources. Natural sources include cyanogenesis by bacteria, fungi and plants. A number of cyanide catabolising microorganisms have also been reported in literature. This is the first reported instance of cyanide catabolism in Trichoderma harzianum. Four strains of T. harzianum, one of T. pseudokoningii were evaluated. An investigation was made into the occurrence and distribution of the cyanide catabolising enzymes. Three enzymes, cyanide hydratase, beta-cyanoalanine synthase and rhodanese, were studied. All the strains showed a high capacity to degrade cyanide via both the cyanide hydratase and rhodanese pathways, beta-cyanoalanine synthase activity, however, was not detected in any of the selected strains. In the studies on the kinetic characterization of the rhodanese enzyme, a broad pH optimum of 8.5 - 10.5 was obtained for all the strains and a broad temperature optimum of 35 - 55 °C was also observed. The KmCN and Vmax values ranged from 7-16 mM and from 0.069 - 0.093 betamoles. Min-1. mg protein-1, respectively, between the selected strains of Trichoderma. Strong evidence of cyanide biodegradation and co-metabolism emerged from studies with flask cultures where glucose was provided as a co-substrate. The rate of degradation of 2000 ppm CIST was enhanced almost three times in the presence of glucose. Plant microcosm studies carried out using pea and wheat seeds too gave further corroboration of the cyanide degrading and plant growth promotion capabilities of Trichoderma. Microcosms set-up with cyanide at 50 or 100 ppm CN, in the presence of Trichoderma, showed germination of both pea and wheat seeds. There was no seed germination in any of the controls in the absence of Trichoderma inoculation