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The current article emphasized on cultural conditions for Alcaligenes sp. NCIM 5085 to synthesize Polyhydroxybutyrate (PHB) from sugar industry waste during batch fermentation. Alcaligenes sp. NCIM 5085 was found to grow best in conditions that included 40 g l -1 of cane molasses, 1 g l -1 of ammonium sulphate, 10% inoculum with neutral pH and 48 h of incubation time. Sudan Black B staining was employed to verify the PHB synthesis initially, and further TEM analysis was performed to confirm it. The structural analysis of recovered PHB was carried out by using GC-MS, FTIR, 1 H NMR and 13 C NMR analysis. The absorption peak at 1724.56 cm -1 revealed the presence of C=O (carbonyl) group by FTIR, which is an indicator of PHB presence. Furthermore, results of NMR and GC-MS analysis confirmed the recovered polymer was PHB. The thermal properties of recovered polymer were analyzed by TGA, DTG and DSC and showed thermal stability of PHB. The observed glass transient temperature (T g ) -2.8°C was within the normal PHB range of T g . However, melting temperature of recovered PHB was 161.7°C, where the degree of crystallinity was lower than standard PHB that widens the application possibilities.

Ulcerative colitis (UC) is a global disease, characterized by periods of relapse that seriously affects the quality of life of patients. Oligosaccharides are considered to be a prospective strategy to alleviate the symptoms of UC. The present study aimed to evaluate the effect of weilan gum oligosaccharide (WLGO) on a mouse UC model induced by dextran sulfate sodium (DSS). WLGO structural physical properties were characterized by electrospray mass spectrometry and fourier tansform infrared spectroscopy. MTT assays were performed to evaluate the non‑toxic concentration of WLGO. RT‑qPCR and ELISAs were conducted to determine the levels of inflammatory factors. The clinical symptoms and mucosal integrity of the DSS‑induced UC model were assessed by DAI and histological assessment. LPS‑induced Caco‑2 cells and DSS‑induced UC mice were used to explore the effects of WLGO on UC. Treatment of the mice with 4.48 g/kg/day WLGO via gavage for 7 days significantly relieved the symptoms of DSS‑induced UC model mice, whereas significant effects were not observed for all symptoms of DSS‑induced UC in the WLGO‑low group. The disease activity index score was decreased and the loss of body weight was reduced in DSS‑induced UC model mice treated with WLGO. Moreover, colonic damage and abnormally short colon length shortenings were relieved following WLGO treatment. WLGO treatment also reduced the concentration and mRNA expression levels of proinflammatory cytokines, including interleukin‑1β, interleukin‑6 and tumor necrosis factor α, in DSS‑induced UC model mice and lipopolysaccharide‑treated Caco‑2 cells. These results indicated that WLGO may be an effective strategy for UC treatment.

Cu-containing nitrite reductases that convert NO 2 - to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4-domain CuNiR that has both cytochrome and cupredoxin domains fused at the N terminus and the other, a classical 2-domain CuNiR (Br 2D NiR). We report the first enzymatic studies of a novel 4-domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3- and 2-domain variants, and Br 2D NiR revealing up to ~ 500-fold difference in catalytic efficiency in comparison with classical 2-domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self-contained donor-acceptor system, we demonstrate that 4-domain BrNiR utilizes N-terminal tethering for downregulating enzymatic activity instead. Both Br 2D NiR and an engineered 2-domain variant of BrNiR (Δ(Cytc-Cup) BrNiR) have 3 to 5% NiR activity compared to the well-characterized 2-domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc-Cup) BrNiR and Br 2D NiR with classical 2-domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally occurring 4-domain CuNiR and its engineered 3- and 2-domain variants. The reverse protein engineering approach utilized here has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes. ENZYME: Copper-containing nitrite reductase (CuNiR) (EC 1.7.2.1). DATABASE: The atomic coordinate and structure factor of Δ(Cytc-Cup) BrNiR and Br 2D NiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6THE and 6THF, respectively., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Glucan (from Alcaligenes faecalis ) is a polymer composed of β-1,3-linked glucose residues, and it has been addressed in different medical fields, namely in nanotechnology, as a vaccine or a drug delivery system. However, due to their small size, nanomaterials may present new risks and uncertainties. Thus, this work aims to describe the production of glucan nanoparticles (NPs) with two different sizes, and to evaluate the influence of the NPs size on immunotoxicity. Results showed that, immediately after production, glucan NPs presented average sizes of 129.7 ± 2.5 and 355.4 ± 41.0 nm. Glucan NPs of 130 nm presented greater ability to decrease human peripheral blood mononuclear cells and macrophage viability and to induce reactive oxygen species production than glucan NPs of 355 nm. Both NP sizes caused hemolysis and induced a higher metabolic activity in lymphocytes, although the concentration required to observe such effect was lower for the 130 nm glucan NPs. Regarding pro-inflammatory cytokines, only the larger glucan NPs (355 nm) were able to induce the secretion of IL-6 and TNF-α, probably due to their recognition by dectin-1. This higher immunomodulatory effect of the larger NPs was also observed in its ability to stimulate the production of nitric oxide (NO) and IL-1β. On the contrary, a small amount of Glu 130 NPs inhibited NO production. In conclusion, on the safe-by-design of glucan NPs, the size of the particles should be an important critical quality attribute to guarantee the safety and effectiveness of the nanomedicine.

Protein oligomers have gained interest, owing to their increased knowledge in cells and promising utilization for future materials. Various proteins have been shown to 3D domain swap, but there has been no domain swapping report on a blue copper protein. Here, we found that azurin from Alcaligenes xylosoxidans oligomerizes by the procedure of 2,2,2-trifluoroethanol addition to Cu(i)-azurin at pH 5.0, lyophilization, and dissolution at pH 7.0, whereas it slightly oligomerizes when using Cu(ii)-azurin. The amount of high order oligomers increased with the addition of Cu(ii) ions to the dissolution process of a similar procedure for apoazurin, indicating that Cu(ii) ions enhance azurin oligomerization. The ratio of the absorbance at 460 nm to that at ∼620 nm of the azurin dimer (Abs460/Abs618 = 0.113) was higher than that of the monomer (Abs460/Abs622 = 0.067) and the EPR A‖ value of the dimer (5.85 mT) was slightly smaller than that of the monomer (5.95 mT), indicating a slightly more rhombic copper coordination for the dimer. The redox potential of the azurin dimer was 342 ± 5 mV vs. NHE, which was 50 mV higher than that of the monomer. According to X-ray crystal analysis, the azurin dimer exhibited a domain-swapped structure, where the N-terminal region containing three β-strands was exchanged between protomers. The copper coordination structure was tetrahedrally distorted in the azurin dimer, similar to that in the monomer; however, the Cu-O(Gly45) bond length was longer for the dimer (monomer, 2.46-2.59 Å; dimer, 2.98-3.25 Å). These results open the door for designing oligomers of blue copper proteins by domain swapping.

This study evaluated the immunostimulatory activity of curdlan oligosaccharides (GOS) in cyclophosphamide (CTX)-induced immunosuppressed mice and in RAW264.7 cells. GOS was able to stimulate the release of nitric oxide (NO), cytokines (IL-1β, IL-6 and TNF-α) and improve the phagocytic rate of peritoneal macrophages and RAW264.7 cells. It further enhanced immunoglobulins (Ig) release (IgG by 50.6%-74.7%, IgA by 31.3%-34.9%, IgM by 28.3%-66.7%), splenic lymphocyte proliferation (by 74.8%-91.3%), nature killer cells cytotoxicity (by 32.0%-49.6%), immunophenotypes of splenic lymphocytes (from 1.7 to 2.4, 2.2 and 2.7) in immunosuppressed mice. Compared with curdlan, higher immunostimulatory activity of GOS was found in CTX-treated mice. Moreover, GOS could activate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways through toll-like receptor 2 (TLR2) and complement receptor 3 (CR3). These results indicated that GOS may be a favorable candidate of functional food in regulating immune responses., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cell-surface functional groups (amino, carboxyl, hydroxyl, as well as phosphate) were chemically modified in various ways to enhance the demulsification capability of the demulsifying bacteria Alcaligenes sp. S-XJ-1. Results demonstrated that the demulsifying activity was significantly inhibited by amino enrichment with cetyl trimethyl ammonium bromide, amino methylation, hydroxyl acetylation, and phosphate esterification, but was gradually promoted by carboxyl blocking with increasing the extents of esterification. Compared with the raw biomass, an optimal esterification of carboxyl moieties enhanced the demulsification ratio by 26.5% and shortened the emulsion half-life from 24 to 8.8 h. The demulsification boost was found to be dominated by strengthened hydrophobicity (from 53° to 74°) and weakened electronegativity (from -34.6 to -4.3 mV at pH 7.0) of the cell surface, allowing the rapid dispersion and adsorption of cells onto the oil-water interface. The chemical modification of the functional groups on the biomass surface is a promising tool for the creation of a high-performance bacterial demulsifier.

Damage to normal tissues is a consequence of both therapeutic and accidental exposures to ionizing radiation. A water-soluble heteropolysaccharide called AXEPS, composed of glucose, galactose, rhamnose and glucouronic acid in a molar ratio of nearly 1.0:1.6:0.4:2.3, respectively, was isolated from culture medium of strain Alcaligenes xylosoxidans MSA3 by ethanol precipitation followed by freeze-drying. Chemical analysis, Fourier-transform infrared (FTIR) and chromatographic studies revealed that the molecular weight was 1.6 × 10(4) g mol(-1). This study was designed to investigate the radioprotective and biological effects of AXEPS in alleviating the toxicity of ionizing radiation in female albino rats. A total of 32 female albino rats were divided into four groups. In the control group, rats were administered vehicle by tube for four weeks. The second group was administered AXEPS (100 mg/kg) orally by gavage for four weeks. Animals in the third group were exposed to whole-body γ-rays (5 Gy) and remained for 2 weeks without treatment. The fourth group received AXEPS (100 mg/kg) orally by gavage for two weeks before being exposed to whole-body γ-rays (5 Gy), then 24 h post γ-rays, they received AXEPS (100 mg/kg) in a treatment continuing till the end of the experiment (15 days after the whole-body γ-irradiation). Oral administration of AXEPS (100 mg/kg) significantly reversed the oxidative stress effects of radiation, as evidenced by the decrease in DNA damage in the bone marrow. Assessment of apoptosis and cell proliferation markers revealed that caspase-3 significantly increased in the irradiated group. Moreover, a significant decrease in the hematological constituents of peripheral blood, the chemotactic index and CD8+ T cells were observed in animals in the irradiation-only group, whereas an increase in the lymphocyte index was observed in animals in that group. In contrast, AXEPS treatment prevented these alterations. From our results, we conclude that AXEPS is a potent antioxidant and treatment agent for protection from γ-rays., (© The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Five-coordinate heme nitrosyl complexes (5cNO) underpin biological heme-NO signal transduction. Bacterial cytochromes c' are some of the few structurally characterized 5cNO proteins, exhibiting a distal to proximal 5cNO transition of relevance to NO sensing. Establishing how 5cNO coordination (distal vs proximal) depends on the heme environment is important for understanding this process. Recent 5cNO crystal structures of Alcaligenes xylosoxidans cytochrome c' (AXCP) and Shewanella frigidimarina cytochrome c' (SFCP) show a basic residue (Arg124 and Lys126, respectively) near the proximal NO binding sites. Using resonance Raman (RR) spectroscopy, we show that structurally characterized 5cNO complexes of AXCP variants and SFCP exhibit a range of ν(NO) (1651-1671 cm(-1)) and ν(FeNO) (519-536 cm(-1)) vibrational frequencies, depending on the nature of the proximal heme pocket and the sample temperature. While the AXCP Arg124 residue appears to have little impact on 5cNO vibrations, the ν(NO) and ν(FeNO) frequencies of the R124K variant are consistent with (electrostatically) enhanced Fe(II) → (NO)π* backbonding. Notably, RR frequencies for SFCP and R124A AXCP are significantly displaced from the backbonding trendline, which in light of recent crystallographic data and density functional theory modeling may reflect changes in the Fe-N-O angle and/or extent of σ-donation from the NO(π*) to the Fe(II) (dz(2)) orbital. For R124A AXCP, correlation of vibrational and crystallographic data is complicated by distal and proximal 5cNO populations. Overall, this study highlights the complex structure-vibrational relationships of 5cNO proteins that allow RR spectra to distinguish 5cNO coordination in certain electrostatic and steric environments.

There has been growing interest in aminopolysaccharide synthesis over the last two decades due to the critical natural functions of aminopolysaccharides, and their potential in biomedical applications. Regioselective introduction of amino groups into polysaccharide backbones is a challenge. Natural curdlan is a linear β-(1→3)-glucan that is of interest both for its physical properties and its biomedical applications. Aminated curdlan derivatives were synthesized in three steps. First, curdlan was regioselectively brominated at the C-6 position in lithium bromide-N,N-dimethylacetamide (DMAc/LiBr). Second, the bromide of the product 6-bromo-6-deoxycurdlan was displaced by nucleophilic substitution with sodium azide (NaN3) in dimethyl sulfoxide (DMSO). Third, O-acylated 6-amido-6-deoxycurdlan was produced by a one-pot method. 6-Azido-6-deoxycurdlan was subjected to Staudinger reduction, followed by reaction in situ with excess carboxylic anhydride, without isolation of the 6-amino-6-deoxycurdlan intermediate. Regioselectivity and degree of substitution (DS) of these derivatives were confirmed by (1)H and (13)C NMR spectroscopy, FTIR spectroscopy, and elemental analysis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochrome c' from Alcaligenes xylosoxidans. This allowed the fingerprinting and validation of different redox and ligand states, identification of vibrational modes and identification of intermediates together with monitoring of radiation-induced changes. This combined approach provides a powerful tool to obtain complementary data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.

Thermodynamic studies on ligand-protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4-7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH(0)/ΔG(0). The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design., (Copyright © 2014 John Wiley & Sons, Ltd.)

Alcaligenes sp. MTCC 10675 has been isolated from soil sample using enrichment method and has nitrilase catalytic system which is highly specific for the hydrolysis of arylaliphatic nitriles. Optimization of culture conditions using response surface methodology and inducer-mediated approach enhanced arylacetonitrilase production significantly (2.4-fold). Isobutyronitrile acted as an effective inducer for the induction of arylacetonitrilase, and it is highly specific for arylacetonitriles (phenyl acetonitrile and mandelonitrile). Arylacetonitrilase has no effect on its relative velocity (V r) up to 20 mM substrate (mandelonitrile) concentration and at 30 mM mandelonitrile, 23.4 % degree of inhibition (I d) was recorded. Half life of arylacetonitrilase of Alcaligenes sp. MTCC 10675 was 27.5 h at 25 °C. Hg(2+), Ag(+), Pb(3+), and Co(2+) were strong inhibitor of arylacetonitrilase activity which resulted into 100 %, 91 %, 84 %, and 83 % inhibition, respectively. Polar protic solvent (dichloromethane, dimethylsulphooxide, and n-butanol) reduce arylacetonitrilase activity up to 80-94 % at 10 % concentration. Alcaligenes sp. MTCC 10675 has higher biocatalytic activity, i.e., 3.9 gg(-1) dcw, which is highest in comparison to till reported organism. Arylacetonitrilase-mediated hydrolysis of racemic mandelonitrile resulted into R-(-) mandelic acid with 99.0 % enantiomeric excess (e.e.).

The main goal of this work was to analyze the effect of surface substances on demulsifying capability of the demulsifying strain Alcaligenes sp. S-XJ-1. The demulsifying substances were successfully separated from the cell surface with dichloromethane-alkali treatment, and exhibited 67.5% of the demulsification ratio for water-in-kerosene emulsions at a dosage of 356mg/L. FT-IR, TLC and ESI-MS analysis confirmed the presence of a carbohydrate-protein-lipid complex in the demulsifying substances with the major molecular ions from mass-to-charge ratio (m/z) 165 to 814. After the substances separated, the cell morphology changed from aggregated to dispersed, and the concentration of cell surface functional groups decreased. Cell surface hydrophobicity and the ability of cell adhesion to hydrophobic surface of the treated cells was also reduced compared with original cell. It was proved that the demulsifying substances had a significant effect on cell surface properties and accordingly with demulsifying capability of Alcaligenes sp. S-XJ-1., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Extraction and identification of surface active substance of Alcaligenes sp. S-XJ-1, as well as description of its emulsion breaking process were conducted to reveal the demulsifying characteristics of this demulsifying strain. Alkali solvent was adopted in the extraction process with conditions optimized as 35 degrees C, 0.08 mol x L(-1) of alkali concentration, 12 g x L(-1) of sample to solution ratio, and 4 h of extraction time by launching both single-factor and orthogonal tests. Under this optimal condition, the extracted surface active substance (the extraction ratio was 36.1%) achieved 77% emulsion breaking ratio for 500 mg x L(-1) within 48 h. FT-IR showed the existence of glycolipids, lipids and proteins in the surface active substance, the molecular weight of which mainly scattered between 55 and 61 256. Saccharides, lipids and proteins were identified as the three chief components in surface active substance with the content of 22.2%, 7.5% and 13.4%, respectively. The proteins were further proved to take the most responsibility for the emulsion breaking ability. Moreover, obvious difference in the emulsion breaking process was demonstrated between the original demulsifying strain S-XJ-1 and the extracted surface active substance by real time observation of Turbiscan Lab Expert. The results suggested that the demulsifying efficiency of the strain was jointly contributed by its surface active substance and demulsifying cell morphology, and the former possessed higher functional priority than the latter.

Rheological properties of welan gum and xanthan gum solutions have been characterized systematically at various concentrations, temperatures and salinities. It is found that the viscoelasticity of welan gum is higher than that of xanthan gum at the same condition though the molecular weight of welan gum is lower. In view of this, welan gum will make a good performance in enhanced oil recovery, especially in high temperature and high salinity reservoirs. Network structure can be formed in solutions of welan gum and xanthan gum for the dynamic modulus has exponential relationship with the concentration. Moreover, the molecular aggregates of welan gum adopt a different arrangement with that of xanthan gum, adjacent double helices of welan gum arrange in parallel as the zipper model. The structure formed by zipper model is still stable in high temperature and high salinity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The chemical acylation of natural antioxidants may improve their oxidative and thermal stability, as well as modify their hydrophile-lipophile balance (HLB). These processes are generally carried out under harsh conditions using strongly corrosive acids. In contrast, lipase-catalyzed acylation is characterized by mild reaction conditions, low energy requirements, and a minimization of side reactions. We report the one-step enzymatic acylation of a phenolic antioxidant (α-tocopherol) and a polyphenol (resveratrol) by lipase-catalyzed transesterification. In particular, the regioselectivity of resveratrol acylation can be controlled by an adequate selection of the biocatalyst.

Considering that the surface properties of demulsifying cells correlate with their demulsification efficiency, the demulsifying bacteria Alcaligenes sp. S-XJ-1 with various surface properties were obtained using different vegetable oils as carbon sources. The results show that better performance was achieved with demulsifying bacteria S-XJ-1 possessing a relatively high cell surface hydrophobicity (CSH) and total unsaturated degree for the cell-wall bound fatty acids. There also appeared to be a correlation between the specific cell-wall bound fatty acid components of the bacteria, in terms of carbon chain length or degree of unsaturation, and either CSH or demulsification efficiency. The fatty acids attached to the cell wall were mainly composed of palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). C18:1 and C18:2 had a positive effect on the formation of CSH, while C18:0 and C18:3 had the opposite effect., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The long-term destabilization process of a water-in-oil emulsion was investigated with two different biodemulsifiers produced under different culture conditions by Alcaligenes sp. S-XJ-1. Biodemulsifier I was obtained by using paraffin as substrate at initial culture pH of 10 and biodemulsifier II was produced with waste frying oils at pH of 7. The former exhibited higher demulsifying ability and interfacial activity than the latter. Bottle test, microscopy and Turbiscan Lab(®) Expert were used to investigate the biological demulsification process. It was found that biodemulsifiers' ability to decrease the interfacial tension played a more important role in demulsification than their ability to decrease the surface tension. Owing to their amphiphilic nature, demulsification process began with the adsorption of the biodemulsifiers onto the water-oil interface. Then the biodemulsifiers reacted with the emulsifiers because of their interfacial activity. As a result, thin liquid film was removed from the surface of dispersed droplets and coalescence occurred. This led to the settling of the dispersed droplets and the clarification of the continuous phase. Turbiscan Lab(®) Expert can be used to evaluate the demulsification efficiency and to analyze the destabilization process of different biodemulsifiers. It is a rapid and accurate method to screen high-efficiency demulsifiers from other bioproducts., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Soluble microbial products (SMP) in the wastewater treatment process not only cause fouling to the membrane, but also generate disinfection by-products (DBP) in the effluent, thus get increasing attention. In this study, SMP produced by activated sludge and isolates under different stressful conditions, i.e. starvation, salinity, heavy metals, low pH and high temperature, were characterized to investigate the effects of these conditions on the amount of SMP and their compositions. The analysis results using size exclusion chromatography (SEC), high pressure liquid chromatography (HPLC) and fluorescence excitation emission matrix (FEEM) showed that activated sludge and isolates suffered with the same stressful condition contained almost the same concentration and composition of SMP, indicating that the stressful condition instead of the microbial species played the crucial role in the production of SMP. Among of stressful conditions tested, high temperature had stimulated the production of polysaccharides and polycarboxylate-type humic acid with high hydrophilicity, which is in positive proportion to the foulants formation potential, thus should be avoided in membrane bioreactors. Low pH had promoted the generation of hydrophobic humic acid-like or protein-like organics, which had been proved as the main disinfection byproduct (DBP) precursor, thus should be avoided in the biological treatment. Starvation had less effect on SMP production as the seeding microbes had no substrates., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

4-Chlorobenzoate:CoA ligase (CBL) belongs to the adenylate-forming family of enzymes that catalyze a two-step reaction to first activate a carboxylate substrate as an adenylate and then transfer the carboxylate to the pantetheine group of either coenzyme A or an acyl-carrier protein. The active site is located at the interface of a large N-terminal domain and a smaller C-terminal domain. Crystallographic structures have been determined at multiple steps along the reaction pathway and form the basis for a proposal that the C-terminal domain rotates by approximately 140 degrees between the two states that catalyze the adenylation and thioester-forming half-reactions. The domain rotation is accompanied by a change in the main chain torsional angles of Asp402, a conserved residue located at the interdomain hinge position. We have mutated the Asp402 residue to Pro in order to test the impact of reduced main chain flexibility at the putative hinge position. The crystal structure of the D402P mutant shows that the enzyme adopts the proposed adenylate-forming conformation with very little change to the overall structure. To examine the impact of this mutation on the ability of the enzyme to catalyze the complete reaction, single turnover kinetic experiments were performed. Whereas the ability of this mutant to catalyze the adenylate-forming half-reaction is reduced by approximately 3-fold, catalysis of the second half-reaction is reduced by 4 orders of magnitude. The impact of the alanine replacement of Asp402 on the thioester-forming reaction is significant, although not as dramatic as the proline mutation, and provides evidence that the Asp402 carboxylate group, through ion pair formation with N-terminal domain residue Arg400, assists in the transition to the thioester-forming conformer. Together these results support the domain alternation hypothesis.

Metal-substituted blue copper proteins (cupredoxins) have been successfully used to study the effect of metal-ion identity on their active-site properties, specifically the coordination geometry and metal-ligand bond strengths. In this work, low-temperature (77 K) resonance Raman (RR) spectra of the blue copper protein Alcaligenes xylosoxidans azurin I and its Ni(II) derivative are reported. A detailed analysis of all observed bands is presented and responsiveness to metal substitution is discussed in terms of structural and bonding changes. The native cupric site exhibits a RR spectrum characteristic of a primarily trigonal planar (type 1) coordination geometry, identified by the nu(Cu-S)(Cys) markers at 373, 399, 409, and 430 cm(-1). Replacement of Cu(II) with Ni(II) results in optical and RR spectra that reveal (1) a large hypsochromic shift in the main (Cys)S --> M(II) charge-transfer absorption from 622 to 440 nm, (2) greatly reduced metal-thiolate bonding interaction, indicated by substantially lower nu(Ni-S)(Cys) stretching frequencies, (3) elevation of the cysteine nu(C( beta )-S) stretching, amide III, and rho (s)(C( beta )H(2)) scissors vibrational modes, and (4) primarily four-coordinated, trigonally distorted tetrahedral geometry of the Ni(II) site that is marked by characteristic nu(Ni-S)(Cys) stretching RR bands at 347, 364, and 391 cm(-1). Comparisons of the electronic and vibrational properties between A. xylosoxidans azurin I and its closely structurally related azurin from Pseudomonas aeruginosa are made and discussed. For cupric azurins, the intensity-weighted average M(II)-S(Cys) stretching frequencies are calculated to be nu(Cu-S)(iwa) = 406.3 and 407.6 cm(-1), respectively. These values decreased to nu(Ni-S)(iwa) = 359.3 and 365.5 cm(-1), respectively, after Ni(II) --> Cu(II) exchange, suggesting that the metal-thiolate interactions are similar in the two native proteins but are much less alike in their Ni(II)-substituted forms.

In the present study, we used microwave energy instead of conventional heating to transform poly-3-hydroxybutyrate (PHB) into methyl 3-hydroxybutyrate (Me-3HB) in acidified methanol (H2SO4, 10%, v/v) mixture in less than 4 min at 10% microwave power. The microwave assisted method was then applied to analyze PHB produced by Alcaligenes latus. The PHB content in the biomass determined using microwave heating was comparable to the amount found by conventional heating. Moreover, the new esterification method was at least 50 times faster than the conventional method, affording a significant saving of time and energy.

Biopolymers such as poly(hydroxyalkanoates) (PHAs) have received much attention due to their physico-chemical properties, biodegradability, and biocompatibility that make them good candidates for industrial and medical applications. Produced by some microorganisms PHAs accumulate within the cells of these organisms. The optimization of microbial processes to produce PHAs at a lower cost requires rapid and accurate techniques for quantification of the biopolymer in biomass. The present study describes a method based on solid-phase microextraction (SPME) coupled to gas chromatography (GC) for the determination of poly(3-hydroxybutyrate) (PHB) in Alcaligenes latus cells. First PHB was depolymerized by either methanolic or hydrolytic digestion into methyl 3-hydroxybutyrate (Me-3-HB) or crotonic acid (CA), respectively. The resulting analytes were then subjected to analysis by headspace SPME/GC with flame ionization detection (FID). The two depolymerization/SPME/GC-FID methods were optimized and applied to the analysis of PHB in bacterial biomass harvested from a fermentation process that uses A. latus. Results were compared with those obtained using GC-FID analysis of MeOH/CHCl(3) digested samples. Excellent agreement was found between the three methods but the two SPME-based methods were environmentally friendly and easier to perform.

Electrochemical studies of three c-type cytochromes (cyt c from horse heart, cyt c(2) from Rhodospirillum rubrum, and cyt c(553) from Alcaligenes xylosoxidans GIFU 1051) were performed by using the optically active Co(III) complex-modified Au electrode. Three cytochromes gave different redox behaviors reflected from the respective structural information. From relationship between their redox behaviors and their structural characteristics, we evaluated the solution structure around heme center of cyt c(553).

Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.

We address some physical features associated with long-range interfacial electron transfer (ET) of metalloproteins in both electrochemical and electrochemical scanning tunneling microscopy (ECSTM) configurations, which offer a brief foundation for understanding of the ET mechanisms. These features are illustrated experimentally by new developments of two systems with the blue copper protein azurin and enzyme nitrite reductase as model metalloproteins. Azurin and nitrite reductase were assembled on Au(111) surfaces by molecular wiring to establish effective electronic coupling between the redox centers in the proteins and the electrode surface for ET and biological electrocatalysis. With such assemblies, interfacial ET proceeds through chemically defined and well oriented sites and parallels biological ET. In the case of azurin, the ET properties can be characterized comprehensively and even down to the single-molecule level with direct observation of redox-gated electron tunnelling resonance. Molecular wiring using a pi-conjugated thiol is suitable for assembling monolayers of the enzyme with catalytic activity well-retained. The catalytic mechanism involves multiple-ET steps including both intramolecular and interfacial processes. Interestingly, ET appears to exhibit a substrate-gated pattern observed preliminarily in both voltammetry and ECSTM.

Soluble guanylate cyclase (sGC), the mammalian receptor for nitric oxide (NO), is a heme protein with a histidine as the proximal ligand. Formation of a five-coordinate heme-NO complex with the associated Fe-His bond cleavage is believed to trigger a conformational change that activates the enzyme and transduces the NO signal. Cytochrome c' (cyt c') is a protobacteria heme protein that has several similarities with sGC, including the ability to form a five-coordinate NO adduct and the fact that it does not bind oxygen. Recent crystallographic characterization of cyt c' from Alcaligenes xylosoxidans (AXCP) has yielded the discovery that exogenous ligands are able to bind to the Fe center from either side of the porphyrin plane. In this paper, we explore the molecular basis of the NO interaction with AXCP using hybrid quantum-classical simulation techniques. Our results suggest that Fe-His bond breaking depends not only on the iron-histidine bond strength but also on the existence of a local minimum conformation of the protein with the histidine away from the iron. We also show that AXCP is a useful paradigm for NO interaction with heme proteins, particularly regarding the activation/deactivation mechanism of sGC. The results presented here fully support a recently proposed model of sGC activation in which NO is not only the iron ligand but also catalyzes the activation step.

The reduction thermodynamics (Delta H degrees '(rc) and Delta S degrees '(rc)) for native Paracoccus versutus amicyanin, for Alcaligenes faecalis S-6 pseudoazurin, and for the G45P, M64E, and K27C variants of Pseudomonas aeruginosa azurin were measured electrochemically. Comparison with the data available for other native and mutated blue copper proteins indicates that the features of metal coordination and the electrostatic potential due to the protein matrix and the solvent control the reduction enthalpy in a straightforward way. However, the effects on the reduction potential are rather unpredictable owing to the entropic contribution to E degrees ', which is mainly determined by solvent reorganization effects. Analysis of all the Delta H degrees '(rc) and Delta S degrees '(rc) values available for this protein class indicates that enthalpy-entropy compensation occurs in the reduction thermodynamics of wt cupredoxins from different sources, as well as for mutants of the same species. The findings indicate that the reduction enthalpies and entropies for these species are strongly affected by reduction-induced reorganization of solvent molecules within the solvation sphere of the protein. The absence of a perfect enthalpy-entropy compensation is due to the fact that while the differences between reduction entropies are dominated by solvent reorganization effects, those between reduction enthalpies are significantly controlled by intrinsic molecular factors related to the selective stabilization of the reduced form by coordination features of the copper site and electrostatic effects at the interface with the protein matrix.

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecular mass of chitinase was estimated to be 45 kDa and 44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 degrees C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l-1. Cu2+ and Na+ at 5 mM inhibited chitinase activity to 25% while Ca2+, Mg2+ and Ba2+ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.

Loop-contraction mutagenesis has been applied to the cupredoxin pseudoazurin to introduce the active-site loop of amicyanin. The mutation has a limited effect on the spectroscopic properties, and therefore structure, of the cupric protein. The loop contraction results in the increase of the pKa for the detachable His ligand of pseudoazurin by two pH units, similar to the value as found in amicyanin.

Pseudoazurin is an electron transfer copper protein, a member of the cupredoxin family. The protein is frequently found in denitrifying bacteria, where it is the electron donor of nitrite reductase. The copper at the active site is coordinated to His40, Cys78, His81 and Met86 in a distorted tetragonal geometry. We have recorded and assigned the (1)H NMR spectra of Co(II)-substituted pseudoazurin from Achromobacter cycloclastes. The (1)H NMR spectrum of Co(II)-pseudoazurin closely resembles that of Co(II)-rusticyanin, reflecting an altered conformation for the Met-Co(II)-Cys moiety in both proteins, compared to Co(II)-azurin, amicyanin and stellacyanin. The electron spin density onto the Sgamma(Cys) is larger in Co(II)-pseudoazurin compared to Co(II)-rusticyanin. Instead, the Co(II)-Met interaction is similar in both derivatives. Hence, the different metal-ligand interactions might be independently modulated by the protein structure. The present work also shows that the electron spin density onto the Co(II)-S(cys) bond is sensibly smaller than the Cu(II)-S(cys). Notwithstanding, NMR data on Co(II)-substituted blue copper proteins can be safely extrapolated to native Cu(II) proteins.

Semi-purified lipases from Candida rugosa, Pseudomonas cepacia and Alcaligenes sp. were chemically modified with a wide range of hydrophobic groups such as benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, t-butoxycarbonyl, lauroyl and acetyl moieties. The Candida rugosa lipase MY modified with the benzyloxycarbonyl group (modification ratio = 84%) brought about a 15-fold increase in enantioselectivity (E value) towards the hydrolysis of racemic butyl 2-(4-ethylphenoxy)propionate in an aqueous buffer solution, although the enzymatic activity was decreased. The origin of the enantioselectivity enhancement by chemical modification of the lipase is attributed to a significant deceleration in the initial reaction rate for the incorrectly binding enantiomer.

This paper focuses on the development of MALDI sample preparation protocols for the analysis of a bioactive beta-(1 --> 3) polysaccharide, i.e. Curdlan. The crude Curdlan sample was first separated into a low molecular weight water-soluble portion and a high molecular weight water-insoluble portion. The water-soluble portion was analyzed using a standard MALDI sample preparation method developed for dextran analysis. Two low-mass (<4000 Da) polysaccharide distributions differing by 16 Da were observed. For the analysis of the water-insoluble portion, several sample preparation protocols were evaluated using GPC-fractionated samples. A sample preparation method based on the deposition of the analyte solution with a mixture of 2,5-dihydroxybenzoic acid (DHB) and 3-aminoquinoline (3AQ) matrices in dimethyl sulfoxide (DMSO) at elevated temperature of 70 degrees C was found to reliably produce good MALDI spectra. MALDI analysis of the water-insoluble Curdlan portion gave number-average (Mn) and weight-average (Mw) molecular weights and polydispersity of 8000 Da, 8700 Da, and 1.10, respectively., (Copyright 2003 John Wiley & Sons, Ltd.)

The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyanin from Thiobacillus ferrooxidans, have been investigated by (1)H NMR at a magnetic field of 18.8 T. Hyperfine shifts of the protons belonging to the coordinated ligands have been identified by exchange spectroscopy, including the indirect detection for those resonances that cannot be directly observed (the beta-CH(2) of the Cys ligand, and the NH amide hydrogen bonded to the S(gamma)(Cys) atom). These data reveal that the Cu(II)-Cys interaction in pseudoazurin and rusticyanin is weakened compared to that in classic blue sites (plastocyanin and azurin). This weakening is not induced by a stronger interaction with the axial ligand, as found in stellacyanin, but might be determined by the protein folding around the metal site. The average chemical shift of the beta-CH(2) Cys ligand in all BCPs can be correlated to geometric factors of the metal site (the Cu-S(gamma)(Cys) distance and the angle between the CuN(His)N(His) plane and the Cu-S(gamma)(Cys) vector). It is concluded that the degree of tetragonal distortion is not necessarily related to the strength of the Cu(II)-S(gamma)(Cys) bond. The copper-His interaction is similar in all BCPs, even for the solvent-exposed His ligand. It is proposed that the copper xy magnetic axes in blue sites are determined by subtle geometrical differences, particularly the orientation of the His ligands. Finally, the observed chemical shifts for beta-CH(2) Cys and Ser NH protons in rusticyanin suggest that a less negative charge at the sulfur atom could contribute to the high redox potential (680 mV) of this protein.

Chemically fully sulfated polysaccharides including xylan (-->4Xylbeta-(1-->4)Xylbeta1-->), amylose (-->4Glcalpha-(1-->4)Glcalpha1-->), cellulose (-->4Glcbeta-(1-->4)Glcbeta1-->), curdlan (-->3Glcbeta-(1-->3)Glcbeta1-->) and galactan (-->3Galbeta-(1-->3)Galbeta1-->), which have been isolated from Korean clam, were prepared, and their anticoagulant activity was investigated. The results strongly suggest that the activity might not be depending on anomeric configuration (alpha or beta) or monosaccharide species but on the glycosidic linkage, either (1-->3) or (1-->4). 1H NMR studies of these modified polysaccharides show that the neighboring sulfate groups at the C-2 and C-3 positions might have caused the conformational changes of each monosaccharide from 4C(1) to 1C(4). Furthermore, the effect of 6-sulfate residues on the anticoagulant activity was investigated using a specific desulfated reaction for the chemically fully sulfated polysaccharides. The 6-sulfate group is very important in determining anticoagulant activity of (1-->3)-linked polysaccharides, whereas the activity is not affected by presence or absence of the 6-sulfate group in (1-->4)-linked polysaccharides.

The paramagnetic (1)H NMR spectrum of Cu(II) pseudoazurin [PACu(II)] contains eight directly observed hyperfine-shifted resonances which we have assigned using saturation transfer experiments on a 1:1 mixture of PACu(I) and PACu(II). The spectrum exhibits a number of similarities to those of other cupredoxins, but differences are found concerning the Cu-S(Met) interaction. The spectrum is dependent on pH* in the range 8.5-4.5 (pK(a)* 6.4), and a conformational change involving movement of the copper ion away from the Met toward the equatorial ligands, as a consequence of protonation of the surface His6 residue, is identified. Corresponding changes are also seen in the UV/vis spectrum. The protonation/deprotonation equilibrium of His6 influences the reduction potential of the protein in the same pH range. The self-exchange rate constant of PACu at pH* 6.0 (25 degrees C) is considerably smaller (1.1 x 10(3) M(-1) s(-1)) than the value obtained at pH* 7.6 (3.7 x 10(3) M(-1) s(-1)). The effect on the self-exchange reactivity is mainly due to an alteration in the reorganization energy of the copper site brought about by the structural change resulting from His6 protonation.

Background: Arsenite oxidase from Alcaligenes faecalis NCIB 8687 is a molybdenum/iron protein involved in the detoxification of arsenic. It is induced by the presence of AsO(2-) (arsenite) and functions to oxidize As(III)O(2-), which binds to essential sulfhydryl groups of proteins and dithiols, to the relatively less toxic As(V)O(4)(3-) (arsenate) prior to methylation., Results: Using a combination of multiple isomorphous replacement with anomalous scattering (MIRAS) and multiple-wavelength anomalous dispersion (MAD) methods, the crystal structure of arsenite oxidase was determined to 2.03 A in a P2(1) crystal form with two molecules in the asymmetric unit and to 1.64 A in a P1 crystal form with four molecules in the asymmetric unit. Arsenite oxidase consists of a large subunit of 825 residues and a small subunit of approximately 134 residues. The large subunit contains a Mo site, consisting of a Mo atom bound to two pterin cofactors, and a [3Fe-4S] cluster. The small subunit contains a Rieske-type [2Fe-2S] site., Conclusions: The large subunit of arsenite oxidase is similar to other members of the dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes, particularly the dissimilatory periplasmic nitrate reductase from Desulfovibrio desulfuricans, but is unique in having no covalent bond between the polypeptide and the Mo atom. The small subunit has no counterpart among known Mo protein structures but is homologous to the Rieske [2Fe-2S] protein domain of the cytochrome bc(1) and cytochrome b(6)f complexes and to the Rieske domain of naphthalene 1,2-dioxygenase.

NosL, one of the accessory proteins of the nos (nitrous oxide reductase) gene cluster, has been heterologously expressed, purified, and characterized. NosL is a monomeric protein of 18,540 MW that specifically and stoichiometrically binds Cu(I). The copper ion in NosL is ligated by a Cys residue, and one Met and one His are thought to serve as the other ligands. While it is possible to oxidize Cu(I)-NosL with ferricyanide, the Cu(II) ion thus formed appears to dissociate from the protein. The function of Cu(I)NosL is not yet known, but the data indicate that NosL does not act as an electron transfer partner to nitrous oxide reductase. NosL is encoded on the same transcript as three other gene products (NosD, NosF, and NosY). These have been shown to be required for assembly of the active site in nitrous oxide reductase, which is thought to be a copper cluster. Accordingly, it is possible that NosL is a copper chaperone involved in metallocenter assembly.

The mutant replacing the Met86 ligand of Achromobacter cycloclastes pseudoazurin (Ac-pAz) with Gln has been prepared and spectroscopically and electrochemically characterized. Ac-pAz has four ligands (2His, Cys, and Met) and donates one electron to its cognate Cu-containing nitrite reductase (Ac-NIR). The mutant ([Met86Gln]pAz) shows the electronic absorption and CD spectra considerably similar to those of zucchini mavicyanin (Mv) and lacquer and cucumber stellacyanins (St) having 2His, Cys, and Gln. The EPR signal of the mutant has an axial character, although those of Mv and St show rhombic signals. The findings indicate that the Cu site having Gln might be a distorted trigonal geometry. The half-wave potentials (E(1/2)) of [Met86Gln]pAz and the intermolecular electron-transfer rate constant (kET) from the mutant to Ac-NIR were determined by cyclic voltammetry at pH 7.0 and 25 degrees C. The E(1/2) is +134 mV (versus NHE) and the coordination of Gln instead of Met negatively shifts the E(1/2) of Ac-pAz (+260 mV (versus NHE)). The kET of [Met86Gln]pAz (1.2x10(6) M(-1) s(-1)) is larger than that of the recombinant Ac-pAz (7.5x10(5) M(-1) s(-1)).

The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited by p-chloromercuribenzoic acid, EDTA, o-phenanthroline, HgCl(2), and CuSO(4). The EDTA-inactivated enzyme was completely restored by the addition of Zn(2+) or Co(2+). In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme.

The dielectric permeability of the degradable biopolymer polyhydroxybutyrate synthesized by hydrogen-oxidizing bacteria Alcaligenes eutrophus was investigated by the resonance method using original highly sensitive microstrip sensors. For the first time, a linear growth of dielectric permeability (delta epsilon/delta T = 7 x 10(-4) degree-1) due to the flexibility of the polymer chain in the temperature range from 10 to 70 degrees C was revealed. The energy of a bend of the nearest fragments was evaluated (E = 392 K), and its correspondence to the energies of bends of the alcyl groups of low-molecular substances like liquid crystals was established. It was shown that at low values of dielectric permeability in the high-frequency range (epsilon' = 2.25 +/- 0.02), which are stable, in a wide range of frequencies of the electromagnetic field (1 MHz - 1 Hz), polyoxybutyrate can be used in the microwave equipment.

Crystallographic structures of oxidized and reduced forms of azurin II are reported at 1.75 A resolution. Data were collected using one crystal in each case and by translating the crystal after each oscillation range to minimize photoreduction. Very small differences are observed at the Cu site upon reduction and these cannot be determined with confidence at current resolution. A comparison with the three-dimensional EXAFS reveals a good correspondence for all the ligand distances except for Cu-His46, where a larger deviation of approximately 0.12-0.18 A is observed, indicating that this ligand is more tightly restrained in the crystallographic refinement at the current resolution.

A knee-joint exudate culture yielded on two occasions a gram-negative bacterium. Regular methods for speciation did not provide an identification. The infection was successfully treated with ciprofloxacin. The unknown isolate, CCUG 36768, was subjected to further investigation, including 16S rDNA sequencing, protein profiling, cellular fatty acid analysis, and various biochemical tests, in order to produce a species identification. The 1469 bp-long 16S rDNA sequence did not reveal identity with any known species sequence. CCUG 36768 clustered in a group of species, including Alcaligenes defragrans, Denitrobacter permanens, Taylorella equigenitalis, Alcaligenes faecalis, and four strains of Alcaligenes species without a specific species name. Bordetella species also showed a high degree of similarity with CCUG 36768. Protein profiling, cellular fatty acid analysis and computer-assisted analysis of biochemical profiles indicated similarity with Bordetella-Alcaligenes species, often close to B. holmesii and B. avium. API 20 NE indicated the profile of Moraxella species of poor identity. It is concluded that CCUG 36768 represents a new bacterial species of pathogenic potential in humans. It is related to the Bordetella-Alcaligenes group. Powerful new methods for speciation are available and it is recommended that unknown isolates from normally sterile sites be submitted for further analysis. Several isolates are required for the definition of new species.

A lysozyme-osmotic shock method is described for fractionation of Alcaligenes faecalis which uses glucose to adjust osmotic strength and multiple osmotic shocks. During phenylethylamine-dependent growth, aromatic amine dehydrogenase, azurin, and a single cytochrome c were localized in the periplasm. Their induction patterns are different from those for the related quinoprotein methylamine dehydrogenase and its associated redox proteins.

Recently it was shown that motility of Vibrio alginolyticus facilitated cell attachment to glass surfaces. In the present study the same relationship between motility and cell attachment was confirmed for Alcaligenes and Alteromonas spp. These findings clearly answer a long-standing question: does motility facilitate attachment? However, they are contradictory to a general view on cell attachment that the energy barrier due to electrostatic repulsion between negatively charged bacterial cells and a glass surface is much greater than both the thermal kinetic energy of the bacterial cell and the bacterial swimming energy. It is shown that the energy barrier becomes far less than that usually estimated when bacterial cells are rich in polymers at their surfaces. This finding reasonably explains the dependence of bacterial attachment rate on cell motility and demands reconsideration of the mechanism of bacterial attachment.

The crystal structures of oxidized and reduced pseudoazurins from a denitrifying bacterium, Achromobacter cycloclastes IAM1013, have been determined at 1.35- and 1.6-A resolutions, respectively. The copper site in the oxidized state exhibits a distorted tetrahedral structure like those of other pseudoazurins. However, not only a small change of the copper geometry, but concerted peptide bond flips are identified. The imidazole ring of remote His6 has a hydrogen bonding distance of 2.73 A between N-delta1(His6) and O-gamma1(Thr36) in the oxidized protein. When the protein is reduced at pH 6.0, the imidazole ring rotates by 30.3 degrees and moves 1.00 A away from the position of the oxidized state. A new hydrogen bond between N-epsilon2(His6) and O-epsilon1(Glu4) is formed with a distance of 3.03 A, while the hydrogen bond between N-delta1(His6)-O-gamma1(Thr36) is maintained with an interatomic distance of 2.81 A. A concomitant peptide bond flip of main chain between Ile34 and Thr36 occurs.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is involved in photosynthesis where it catalyzes the initial step in the fixation of carbon dioxide. The enzyme also catalyzes a competing oxygenation reaction leading to loss of fixed carbon dioxide, thus reducing the net efficiency of photosynthesis significantly. Rubisco has therefore been studied extensively, and a challenging goal is the engineering of a more photosynthetically efficient enzyme. Hexadecameric rubiscos fall in two distinct groups, "green-like" and "red-like". The ability to discriminate between CO2 and O2 as substrates varies significantly, and some algae have red-like rubisco with even higher specificity for CO2 than the plant enzyme. The structure of unactivated rubisco from Alcaligenes eutrophus has been determined to 2.7 A resolution by molecular replacement and refined to R and Rfree values of 26.6 and 32.2 %, respectively. The overall fold of the protein is very similar to the rubisco structures solved previously for green-like hexadecameric enzymes, except for the extended C-terminal domains of the small subunits which together form an eight-stranded beta-barrel which sits as a plug in the entrance to the central solvent channel in the molecule. The present structure is the first which has been solved for a red-like rubisco and is likely to represent a fold which is common for this group. The small subunits in general are believed to have a stabilizing effect, and the new quaternary structure in the oligomer of the present structure is likely to contribute even more to this stabilization of the assembled rubisco protein., (Copyright 1999 Academic Press.)