Ryszard Brzezinski

Treatment of air polluted with high concentrations of toluene and xylene in a pilot-scale biofilter

Air biofiltration is now under active consideration for the removal of the volatile organic compounds from air polluted streams. In order to investigate the performance of this newly developed technology, a biofiltration pilot unit was operated for a continuous period of 8 months. The biofilter column was packed with commercially conditioned peat. At start-up, the filter bed was inoculated with four species of microorganisms. The resulting biofilter was fed with air contaminated with toluene, xylene or a mixture of toluene and xylene. The maximum elimination capacities attained were 165 g m−3 h−1 for toluene, 66 g m−3 h−1 for xylene and 115 g m−3 h−1 for the mixture of toluene and xylene. These specific performances exceed the values published in the technical and commercial literature for similar processes. Xylene isomers were degraded in decreasing order of reactivity, m-xylene, p-xylene, o-xylene. In the case of air polluted with a toluene and xylene mixture, it was noticed that the metabolism of toluene biodegradation was inhibited by the presence of xylene. Characterization of the biofilm microbial populations after several weeks of operation showed that the dominant strains among the isolated culturable strains from the biofilm, even if different from the initially inoculated strains, had at least one physiological property favoring degradation of aromatic organic rings. The performance of the biofilter was found to be dependent on the temperature of the filter media and the pressure drop through the bed. Finally, a steady state mathematical model was tested in order to theoretically describe the experimental results. This model is used to illustrate the operating diffusion and reaction regimes at steady state for the case of each pollutant.

X-ray structure of an anti-fungal chitosanase from streptomyces N174.

We report the 2.4 Å X-ray crystal structure of a protein with chitosan endo-hydrolase activity isolated from Streptomyces N174. The structure was solved using phases acquired by SIRAS from a two-site methyl mercury derivative combined with solvent flattening and non-crystallographic two-fold symmetry averaging, and refined to an R-factor of 18.5%. The mostly α-helical fold reveals a structural core shared with several classes of lysozyme and barley endochitinase, in spite of a lack of shared sequence. Based on this structural similarity we postulate a putative active site, mechanism of action and mode of substrate recognition. It appears that Glu 22 acts as an acid and Asp 40 serves as a general base to activate a water molecule for an SN2 attack on the glycosidic bond. A series of amino-acid side chains and backbone carbonyl groups may bind the polycationic chitosan substrate in a deep electronegative binding cleft.

Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-β-d-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or Δ4,5-anhydrogalaturonic acid (Δ4,5-GalA). Δ4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.

Biofiltration of air contaminated with toluene on a compost-based bed

Many studies have focused on problems created by emissions to the atmosphere of gaseous effluents containing volatile organic compounds (VOCs). Over the more recent decades, such studies have led to the development of various bioreactors such as the bioscrubber, the biotrickling filter and the biofilter. This paper presents the results of a study on the biofiltration of airborne toluene, the biofilter employed being operated at the laboratory-scale for a continuous period of 3 months. The focus of this particular study has been the development of a new compost-based filter-bed material, which consists of an association between matured compost and a proprietary organic binder that is intended to prolong the period of the beds efficient operations. No inoculum was added to the filter-bed material. During the experimental program, the performance of two different bed irrigation solutions was examined, the most effective nutrient supply solution then being used, along with toluene input levels varying from 0.6–2.6 g/m3, and toluene polluted air flow rates ranging from 0.4–1 m3/h, equivalent to empty bed residence times of 65–165 s. The results of this program have demonstrated removal efficiencies approaching 95%, while maximum elimination capacities of 55 g/m3 h, for an inlet load of 65 g/m3 h, have been achieved, supporting the view that the compost-based filter material tested in this work functions as a promising biofilter medium in this application. Finally, in order to present the biofilter performance observed under the best operating conditions, a simplified representation based on Ottengrafs model has been developed from the experimental results and is included here.

Purification and characterization of a chitosanase from Streptomyces N174

A highly efficient chitosanase producer, the actinomycete N174, identified by chemotaxonomic methods as belonging to the genus Streptomyces was isolated from soil. Chitosanase production by N174 was inducible by chitosan or d-glucosamine. In culture filtrates the chitosanase accounted for 50–60% of total extracellular proteins. The chitosanase was purified by polyacrylic acid precipitation, CM-Sepharose and gel permeation chromatography. The maximum velocity of chitosan degradation was obtained at 65° C when the pH was maintained at 5.5. The enzyme degraded chitosans with a range of acetylation degrees from 1 to 60% but not chitin or CM-cellulose. The enzyme showed an endo-splitting type of activity and the end-product of chitosan degradation contained a mixture of dimers and trimers of d-glucosamine.

Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial σ factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for σF, σC, and σK, and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of σ factor genes. Our results revealed that many σ factors are expressed from autoregulated promoters.

Posttranslational Regulation of Mycobacterium tuberculosis Extracytoplasmic-Function Sigma Factor σL and Roles in Virulence and in Global Regulation of Gene Expression

ABSTRACT In this report, we demonstrate that SigL is posttranslationally regulated by a specific anti-sigma factor, RslA, and contributes to the expression of at least 28 genes. Several of these genes could mediate important cell envelope-related processes. Importantly, a sigL-rslA mutant strain was significantly attenuated in a mouse model of infection.

Novel Mycobacterium tuberculosis anti-σ factor antagonists control σf activity by distinct mechanisms

The aetiological agent of tuberculosis, Mycobacterium tuberculosis, encodes 13 σ factors, as well as several putative anti‐, and anti‐anti‐ σ factors. Here we show that a σ factor that has been previously shown to be involved in virulence and persistence processes, σF, can be specifically inhibited by the anti‐σ factor UsfX. Importantly, the inhibitory activity of UsfX, in turn, can be negatively regulated by two novel anti‐anti‐σ factors. The first anti‐anti‐σ factor seems to be regulated by redox potential, and the second may be regulated by phosphorylation as it is rendered non‐functional by the introduction of a mutation that is believed to mimic phosphorylation of the anti‐anti‐σ factor. These results suggest that σF activity might be post‐translationally modulated by at least two distinct pathways in response to different possible physiological cues, the outcome being consistent with the bacterias ability to adapt to diverse host environments during disease progression, latency and reactivation.

A versatile shuttle cosmid vector for use in Escherichia coli and actinomycetes

A shuttle cosmid vector has been constructed for Escherichia coli and actinomycetes. This vector, pFD666, utilizes the origin of replication (ori) of the broad-host-range plasmid, pJV1, from Streptomyces phaeochromogenes, for replication in actinomycetes and is compatible with vectors derived from pIJ101. The pFD666 vector employs the neomycin phosphotransferase-encoding gene (neo) from transposon Tn5 as the selective marker. To achieve this, the native promoter of neo was replaced by one optimized for expression in both hosts. The polylinker used for cloning has nine unique sites flanked by the promoters for T7 and SP6 RNA polymerase for the production of specific RNA probes. Terminators on both sides of the polylinker protect the vector from transcription originating from cloned inserts. An M13 ori allows the production of single-stranded DNA.

Two exo-β-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases

A GlcNase (exo-beta-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with K(m) and kcat values of 0.16 mg/ml and 2832 min(-1). On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative beta-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable beta-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with beta-galactosidase, beta-glucuronidase or beta-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.