This article describes an efficient method, combining chemical oxidation and acetone extraction, to produce carbonaceous nanomaterials from dairy manure biochar. The optical and mechanical properties are similar to methods previously reported carbonaceous nanomaterials from biomass. Our novel process cuts the processing time in half and drastically reduces the energy input required. The acetone extraction produced 10 fractions with dairy manure biochar-derived carbonaceous nanomaterials (DMB-CNs). The fraction with the carbonaceous nanomaterials, DMB-CN-E1, with highest fluorescence was selected for in-depth characterisation and for initial testing across a range of applications. DMB-CN-E1 was characterised using atomic force microscope, electrophoresis, and spectrophotometric methods. DMB-CN-E1 exhibited a lateral dimension between 11 and 28 nm, a negative charge, and excitation/emission maxima at 337/410 nm, respectively. The bioimaging potential of DMB-CN-E1 evidenced different locations and different interactions with the cellular models evaluated. DMB-CN-E1 was quenched by several heavy metal ions showing a future application of these materials in heavy metal ion detection and/or removal. The demonstrated capabilities in bioimaging and environmental sensing create the opportunity for generating added-value nanomaterials (NanoRefinery) from dairy manure biochar gasification and, thus, increasing the economic viability of gasification plants.

This article focuses on implementing multivariate analysis to evaluate biochar-derived carbonaceous nanomaterials (BCN) from three different feedstocks for the detection and differentiation of heavy metal ions in aqueous systems. The BCN were produced from dairy manure, rice straw and sorghum straw biochar using our NanoRefinery process. The NanoRefinery process transforms biochar into advanced nanomaterials using depolymerisation/chemical oxidation and purification of nanomaterials using solvent extraction. Dairy manure biochar-derived carbonaceous nanomaterials (DMB-CN), rice straw biochar-derived carbonaceous nanomaterials (RSB-CN) and sorghum straw biochar-derived carbonaceous nanomaterials (SSB-CN) were utilised as probes for the evaluation of their fluorescent properties and the detection of heavy metal ions. The BCN fluorescence quenching and fluorescence recovery was tested with lead (Pb2+), nickel (Ni2+), copper (Cu2+) and mercury (Hg2+). Principal component analysis (PCA) and discriminant analysis were used to differentiate among heavy metal ions in water samples. The BCN from different feedstocks had different characteristics and produced different interactions with heavy metal ions. DMB-CN had the highest quenching for Hg2+ and Ni2+ while SSB-CN and RSB-CN responded best to Cu2+ and Pb2+, respectively. The fluorescence quenching was modelled using linear and empirical functions. PCA and discriminant analysis used the quenching measurements to differentiate heavy metal ions in aqueous system. A key result was that the discriminant analysis had a 100% accuracy to detect Pb2+, 66% for Ni2+ and Cu2+, and 33% for Hg2+. This study has shown that biochar-derived carbonaceous nanomaterials could be used in heavy metal ions sensing applications. This is the first step in the development of a fast and accurate method for the detection of heavy metal ions in waters using environmentally friendly BCN.

This research seeks a coupled solution for managing the large amounts of biochar produced by microalgae biofuel production, and the necessity for novel, economic and accurate heavy metal sensing methods. Therefore, this study evaluated the transformation of microalgae biochar (MAB) into carbon dots (Cdots) and their subsequent application as heavy metal ion sensors in aqueous systems. The experimental phase included the transformation of MAB into microalgae biochar-derived carbon dots (MAB-Cdots), MAB-Cdot characterisation and the evaluation of the MAB-Cdots as transducers for the detection of four heavy metal ions (Pb-2 (broken vertical bar), Cu-2 (broken vertical bar), Cd-2 (broken vertical bar), and Ni-2 (broken vertical bar)). MAB-Cdot fluorescence was stable over a wide range of pH and resistant to photo-bleaching, making them suitable as fluorescence probes. The MAB-Cdot fluorescence was quenched by all of the metal ions and displayed different quenching levels. Depending upon the ions involved, MAB-Cdots were used to detect the presence of heavy metal ions from concentrations of 0.012 mu M up to 2mM by measuring the reduction in fluorescence intensity. Neutral and slightly alkaline pHs were optimal for Cu2+ Ni2+ and Pb2+ heavy metal quenching. To quantify the concentration of the heavy metal ions, linear and logarithmic functions were used to model the MAB-Cdot fluorescence quenching. The sensing mechanism was determined to be reversible and purely collisional with some fluorophores less accessible than the others. This work demonstrated the ability to produce Cdots from microalgae biochar, examined their application as a transducer for detecting heavy metal ions in aqueous systems and paves the way for novel sensing systems using MAB-Cdots. (C) 2018 Elsevier B.V. All rights reserved.

Three types of biochar (microalgae, rice straw and sorghum straw) from biomass thermal conversion production were tested for producing biochar-derived carbonaceous nanomaterials (BCN). BCN were obtained after using chemical depolymerisation and solvent extraction, NanoRefinery process. Microalgae biochar-derived carbonaceous nanomaterials (MAB-CN), rice straw biochar-derived carbonaceous nanomaterials (RSB-CN) and sorghum straw biochar-derived carbonaceous nanomaterials (SSB-CN) were characterised using spectroscopic and microscopic techniques. This characterisation evidenced significant differences among the three BCN with MAB-CN exhibiting greater structural differences compared to RSB-CN and SSB-CN. Biocompatibility, cellular uptake, and cellular localisation were evaluated using three yeast species, Saccharomyces cerevisiae, Candida albicans, and Yarrowia lipolytica. While all BCN were biocompatible, the degree of biocompatibility for each species was dependent on pH, BCN concentration and BCN type. Additionally, BCN were evaluated as transducers for the detection of 12 heavy metal ions. MAB-CN, RSB-CN, and SSB-CN had different responses to the 12 heavy metal ions. The SSB-CN/Cu (II) and the MAB-CN/Zn (II) combinations evidenced selectivity over the other metal ions with these combinations having limits of detection of 0.0125 mu M and 9 mu M, respectively. The results from this research pave the way for BCN novel applications for bioimaging and heavy metal ions sensing probes.

ABSTRACT The binding and cytochrome P45051 (CYP51) inhibition properties of a novel antifungal compound, VT-1161, against purified recombinant Candida albicans CYP51 ( ERG11 ) and Homo sapiens CYP51 were compared with those of clotrimazole, fluconazole, itraconazole, and voriconazole. VT-1161 produced a type II binding spectrum with Candida albicans CYP51, characteristic of heme iron coordination. The binding affinity of VT-1161 for Candida albicans CYP51 was high (dissociation constant [ K d ], ≤39 nM) and similar to that of the pharmaceutical azole antifungals ( K d , ≤50 nM). In stark contrast, VT-1161 at concentrations up to 86 μM did not perturb the spectrum of recombinant human CYP51, whereas all the pharmaceutical azoles bound to human CYP51. In reconstitution assays, VT-1161 inhibited Candida albicans CYP51 activity in a tight-binding fashion with a potency similar to that of the pharmaceutical azoles but failed to inhibit the human enzyme at the highest concentration tested (50 μM). In addition, VT-1161 (MIC = 0.002 μg ml −1 ) had a more pronounced fungal sterol disruption profile (increased levels of methylated sterols and decreased levels of ergosterol) than the known CYP51 inhibitor voriconazole (MIC = 0.004 μg ml −1 ). Furthermore, VT-1161 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. In summary, VT-1161 potently inhibited Candida albicans CYP51 and culture growth but did not inhibit human CYP51, demonstrating a >2,000-fold selectivity. This degree of potency and selectivity strongly supports the potential utility of VT-1161 in the treatment of Candida infections.

This study demonstrates use of recombinant yeast to simultaneously saccharify and ferment grass juice (GJ) to bioethanol. A modified Bacillus subtilis levanase gene (sacC) in which the native bacterial signal sequence was replaced with a yeast α-factor domain, was synthesised with yeast codon preferences and transformed into Saccharomyces cerevisiae (strain AH22) using the expression vector pMA91. AH22:psacC transformants secreted sacCp as an active, hyper-glycosylated (>180 kDa) protein allowing them to utilise inulin (β[2-1] linked fructose) and levan (β[2-6] linkages) as growth substrates. The control (AH22:pMA91) strain, transformed with empty plasmid DNA was not able to utilise inulin or levan. When cultured on untreated GJ levels of growth and bioethanol production were significantly higher in experiments with AH22:psacC than with AH22:pMA91. Bioethanol yields from AH22:psacC grown on GJ (32.7[±4] mg mL(-1)) compared closely to those recently achieved (Martel et al., 2010) using enzymatically pre-hydrolysed GJ (36.8[±4] mg mL(-1)).

ABSTRACT The recent decrease in the sensitivity of the Western European population of the wheat pathogen Mycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in the CYP51 gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutated M. graminicola CYP51 (MgCYP51) variants in a Saccharomyces cerevisiae mutant carrying a doxycycline-regulatable tetO 7 - CYC promoter controlling native CYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in S. cerevisiae . Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460, commonly identified in modern M. graminicola populations, have no effect on the capacity of the M. graminicola protein to function in S. cerevisiae . We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity of MgCYP51 to complement the S. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the M. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western European M. graminicola population emerged.

Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei beta-fructosidase was overexpressed as truncated cytosolic protein ((t)fosEp) in Escherichia coli. Purified (t)fosEp was thermostable (10-50 degrees C) with a pH optimum of 5; it showed highest affinity for bacterial levan (beta[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (beta[2-1] linkages) and sucrose (K(m) values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with (t)fosEp resulted in the release of >13 mg/ml more bioavailable fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewer's yeast and GJ+(t)fosEp were >25% higher than those achieved using untreated GJ feedstock (36.5[+/-4.3] and 28.2[+/-2.7]mg ethanol/ml, respectively). This constitutes the first specific study of the potential to ferment ethanol from grass juice and the utility of a novel core domain of beta-fructosidase from L. paracasei.

ABSTRACT The complete DNA sequence of Candida albicans DIT2 , encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli , where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N -formyl tyrosine into N , N ′-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min −1 and a k s of 26 μM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56 Δ mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.

ABSTRACT Inhibition of sterol-14α-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of Saccharomyces cerevisiae that was humanized with respect to the amino acids encoded at the CYP51 ( ERG11 ) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to >1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14α-methyl-ergosta-8, 24(28)-dien-3β-6α-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.

Conference Abstract| February 01 2001 Heterologous CYP51 expression to probe altered interactions with azole inhibitors T. Marczylo; T. Marczylo 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA. Search for other works by this author on: This Site PubMed Google Scholar D. C. Lamb; D. C. Lamb 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA. Search for other works by this author on: This Site PubMed Google Scholar D. E. Kelly; D. E. Kelly 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA. Search for other works by this author on: This Site PubMed Google Scholar S. L. Kelly S. L. Kelly 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2001) 29 (1): A43. https://doi.org/10.1042/bst029a043 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation T. Marczylo, D. C. Lamb, D. E. Kelly, S. L. Kelly; Heterologous CYP51 expression to probe altered interactions with azole inhibitors. Biochem Soc Trans 1 February 2001; 29 (1): A43. doi: https://doi.org/10.1042/bst029a043 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2001 Biochemical Society2001 Article PDF first page preview Close Modal You do not currently have access to this content.

Conference Abstract| February 01 2001 Yeast cytochrome P450 reductase; expression of a soluble and full-length form, kinetic mechanisms and crystallisation D. C. Lamb; D. C. Lamb 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA. Search for other works by this author on: This Site PubMed Google Scholar A. Warrilow; A. Warrilow 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA. Search for other works by this author on: This Site PubMed Google Scholar K. Venkateswarlu; K. Venkateswarlu 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA. Search for other works by this author on: This Site PubMed Google Scholar D. E. Kelly; D. E. Kelly 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA. Search for other works by this author on: This Site PubMed Google Scholar S. L. Kelly S. L. Kelly 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2001) 29 (1): A44. https://doi.org/10.1042/bst029a044 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation D. C. Lamb, A. Warrilow, K. Venkateswarlu, D. E. Kelly, S. L. Kelly; Yeast cytochrome P450 reductase; expression of a soluble and full-length form, kinetic mechanisms and crystallisation. Biochem Soc Trans 1 February 2001; 29 (1): A44. doi: https://doi.org/10.1042/bst029a044 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2001 Biochemical Society2001 Article PDF first page preview Close Modal You do not currently have access to this content.

Conference Abstract| February 01 2001 Manipulation of P450 in Acinetobacter and application to pollutant degradation S. L. Kelly; S. L. Kelly 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA Search for other works by this author on: This Site PubMed Google Scholar D. E. Kelly; D. E. Kelly 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA Search for other works by this author on: This Site PubMed Google Scholar G. Jones; G. Jones *Foundation for Water Research, Marlow, Bucks. Search for other works by this author on: This Site PubMed Google Scholar D. C. Lamb D. C. Lamb 1Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, UK, SY23 3DA Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2001) 29 (1): A43. https://doi.org/10.1042/bst029a043b Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation S. L. Kelly, D. E. Kelly, G. Jones, D. C. Lamb; Manipulation of P450 in Acinetobacter and application to pollutant degradation. Biochem Soc Trans 1 February 2001; 29 (1): A43. doi: https://doi.org/10.1042/bst029a043b Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2001 Biochemical Society2001 Article PDF first page preview Close Modal You do not currently have access to this content.

Fluconazole selectively inhibits fungal sterol 14α-demethylase, a cytochrome P450 enzyme found in plants, animals, fungi, and Mycobacteria. The mutation G464S, observed in the heme-binding domain of sterol 14α-demethylase in clinical strains of fluconazole-resistant Candida albicans, is shown here to cause resistance through substantially reducing the inhibitory effect of fluconazole and is associated with perturbation of the heme environment as indicated by spectral data. The protein exhibits 42% of the maximal enzymatic rate of the wild-type protein allowing continued production of the end product of fungal sterol biosynthesis, ergosterol, in resistant strains. This mutation may cause these phenotypes through altering the heme location, thus changing the ability of residues above the heme to bind the drug effectively. This perturbation would also account for the observation of reduced sterol demethylase catalytic activity by changing the location of the 14α-methyl group in relation to oxygen-bound heme during the catalytic cycle.

Human and Candida albicans CYP51 were purified to homogeneity after GAL10-based heterologous expression in yeast in order to resolve the basis for the selective inhibition of the fungal enzyme over the human orthologue by the azole drugs ketoconazole and itraconazole, used in the treatment of systemic fungal infection. The purified proteins have similar spectral characteristics, both giving a maximum at 448 nm in reduced carbon monoxide difference spectra. Substrate affinity constants of 20.8 and 29.4 microM and Vmax of 0. 15 and 0.47 nmol/min/nmol were observed for C. albicans and human enzymes, respectively, in reconstituted enzymatic assays, using an intermediate of the demethylation reaction [32-3H]-3beta-hydroxylanost-7-en-32-ol as the substrate. Both enzymes gave similar type II spectra on titration with drugs, but a reduced affinity was observed for human CYP51 using the ability of carbon monoxide to displace the drug as a ligand and by calculation of IC50. However, although the results indicate higher affinity of the drugs for their target CYP51 in the major fungal pathogen C. albicans, when compared directly to CYP51 from humans, the difference was less than 10-fold. This difference is an order of magnitude lower than previously reported data based on measurements using unpurified human CYP51 enzyme preparations. Consequently, increased azole doses to combat resistant candidaemia may well inhibit endogenous human CYP51 and the potential consequences are discussed.

The genome sequence of Mycobacterium tuberculosis (and also M. leprae) revealed a significant number of homologies to Saccharomyces cerevisiae sterol biosynthetic enzymes. We addressed the hypothesis of a potential sterol biosynthetic pathway existing in Mycobacterium using cultures of Mycobacterum smegmatis. Non-saponifiable lipid extracts subjected to analysis by gas chromatography-mass spectrometry (GC-MS) showed cholesterol was present. Sterol synthesis by M. smegmatis was confirmed using 14C-radiolabelled mevalonic acid and incorporation into C4-desmethyl sterol co-migrating with authentic cholesterol on TLC. The sterol biosynthetic pathway has provided a rich source of targets for commercially important bioactive molecules and such agents represent new opportunities for Mycobacteria chemotherapy.

Glyphosate (Roundup) is an herbicide used extensively worldwide which acts as an inhibitor of 5'enolpyruvylshikimate-3-phosphate synthase and for which transgenic herbicide resistant plants have been developed, Here we report for the first time that glyphosate is an inhibitor of cytochrome P450 using a functional expression system for Thlaspi arvensae CYP71B1 in Escherichia coli. CYP71B1 was fused to the soluble domain of a plant cytochrome P450 reductase (CPR) from Catharanthus roseus. CYP71B1 could obtain reducing equivalents in this fusion construct and metabolised the polycyclic aromatic hydrocarbon, benzo(a)pyrene, The fusion protein retained normal spectral characteristics having a Soret peak at 448 nm in the reduced carbon monoxide difference spectrum, Addition of the herbicide resulted in a Type II spectrum indicative of binding via the nitrogen group to haem as a sixth ligand, A K-s of 60 mu M was observed and an IC50 Of 12 mu M was observed for glyphosate inhibition of CYP71B1 activity. The implications of these results are discussed. (C) 1998 Academic Press.