Browsing by Subject "Bioremediation"

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    Engineering pH-tolerant mutants of a cyanide dihydratase
    (Springer Verlag, 2012) Wang, Lan; Watermeyer, Jean M; Mulelu, Andani E; Sewell, Trevor B; Benedik, Michael J
    Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynDpum) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynDpum mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.
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    Genome mining of cyanide-degrading nitrilases from filamentous fungi
    (Springer, 2008) Basile, Lacy J; Wilson, Richard C; Sewell, B. Trevor; Benedik, Michael J
    A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. In this paper, we report on two previously unknown and uncharacterized cyanide hydratases from Neurospora crassa and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, Gibberella zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli, and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to remediate cyanide contaminated waste water from silver and copper electroplating baths. Although all four were similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range of >50% activity. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both heavy metals. The CHT of A. nidulans has the highest reaction rate of the four fungal nitrilases evaluated in this work. These data will help determine optimization procedures for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, both N. crassa and A. nidulans were induced to express CHT by growth in the presence of KCN.
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    Genome mining of cyanide-degrading nitrilases from filamentous fungi.
    (Springer Verlag, 2008) Basile, Lacy J; Willson, Richard C; Sewell, Trevor B; Benedik, Michael J
    A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. In this paper, we report on two previously unknown and uncharacterized cyanide hydratases from Neurospora crassa and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, Gibberella zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli, and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to remediate cyanide contaminated waste water from silver and copper electroplating baths. Although all four were similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range of >50% activity. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both heavy metals. The CHT of A. nidulans has the highest reaction rate of the four fungal nitrilases evaluated in this work. These data will help determine optimization procedures for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, both N. crassa and A. nidulans were induced to express CHT by growth in the presence of KCN.
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    A kinetic study on anaerobic reduction of sulphate, Part I: Effect of sulphate concentration
    (Elsevier, 2002) Moosa, S; Nemati, M; Harrison, S T L
    The kinetics of anaerobic reduction of sulphate was studied in continuous bioreactors. The effects of initial sulphate concentration and its volumetric loading on the kinetics of reaction and activity of sulphate-reducing bacteria were investigated. The increase in initial concentration of sulphate in the range 1.0–Full-size image (<1 K) enhanced the reaction rate from 0.007–Full-size image (<1 K). For a given initial sulphate concentration increasing the volumetric loading rate of sulphate led to a linear increase in volumetric reduction rate. The initial concentration of sulphate did not have a significant effect on maximum specific growth rate (μm), decay coefficient (kd) on bacterial yields (Yx/sulphate and Yx/acetate), with the values of these coefficients being Full-size image (<1 K) bacteria/g sulphate and Full-size image (<1 K) bacteria/g acetate, respectively. The saturation constant (Ks) was an increasing linear function of initial sulphate concentration, with the lowest and highest values being 0.027 and Full-size image (<1 K), respectively. Using the experimental data a kinetic model, incorporating terms for the effects of initial and residual concentrations of sulphate and biomass, was developed.
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    Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein
    (Springer Verlag, 2015) Crum, Mary Abou-Nader; Park, Jason M; Mulelu, Andani E; Sewell, Trevor B; Benedik, Michael J
    The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-termini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previous studies and investigated how the C-terminus affects the activity and stability of three nitrilases, the cyanide dihydratases from B. pumilus (CynDpum) and P. stutzeri (CynDstut) and the cyanide hydratase from Neurospora crassa. Enzymes in which the C-terminal residues were deleted decreased in both activity and thermostability with increasing deletion lengths. However, CynDstut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynDstut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynDstut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interactions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimerization interface A-surface region 195–206 (A2) from CynDpum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynDstutΔ302 or enhance the activity of full-length CynDstut and therefore does not act as a general stability motif.