Purification of two isofunctional hydrolases (EC 3.7.1.8) in the degradative pathway for dibenzofuran inSphingomonas sp. strain RW1

Sphingomonas sp. strain RW1, when grown in salicylate-salts medium, synthesized the enzymes for the degradation of dibenzofuran. The reaction subsequent tometa cleavage of the first benzene ring was found to be catalyzed by two isofunctional hydrolases, H1 and H2, which were purified by chromatography on anion exchange, hydrophobic interaction and gel filtration media. Each enzyme was able to hydrolze 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate to produce salicylate and benzoate, respectively. SDS/PAGE of each purified enzyme showed a single band ofM _r 31 000 (H1) or 29 000 (H2). The N-terminal amino acid sequences of the two proteins showed 50% homology.Abbreviations DHB 2,3-dihydroxybiphenyl - DSM German Culture Collection (Braunschweig) - FPLC protein liquid chromatograph(y) - HOHPDA 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate - HOPDA 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate - THB 2,2,3-trihydroxybiphenyl     

A Thermostable Xylanase from a Thermophilic Acidophilic Bacillus sp.

Bacillus sp. 11-IS, a strain of thermophilic acidophilic bacteria, produced an extracellular xylanase during growth on xylan. The enzyme purified from the culture supernatant solution was homogeneous on disc-gel electrophoresis. The molecular weight was calculated to be 56,000 by SDS-gel electrophoresis. The enzyme had a pH optimum for activity at 4.0, and its stability range was pH 2.0 ~ 6.0. The temperature optimum was 80°C (10-min assay); however, the enzyme retained full activity after incubation at 70°C for 15 min. The enzyme acted on carboxymethyl cellulose (CMC) and cellulose, as well as on xylan. The Michaelis constants for larchwood xylan and CMC were calculated to be 1.68 mg xylose eq/ml and 0.465 mg glucose eq/ml, respectively. The predominant hydrolysis products from larchwood xylan were xylobiose, xylotriose, and xylose; the release of arabinose from rice-straw arabinoxylan was not detected. CMC was cleaved to cellobiose and larger oligosaccharides. Thus, the enzyme is considered to be an endoenzyme which degrades the β-1,4-glycosyl linkages in xylan and cellulose.     

Transformation of 2-hydroxydibenzofuran by laccases of the white rot fungi Trametes versicolor and Pycnoporus cinnabarinus and characterization of oligomerization products

Laccase, a ligninolytic enzyme, was secreted by each ofthe white rot fungi Trametes versicolor and Pycnoporus cinnabarinusduring growth in a nitrogen-rich medium under agitated conditions. Afteraddition of 2-hydroxydibenzofuran to cell-freesupernatants of the cultures, yellow precipitates wereformed. These precipitates were poorly soluble in waterand therefore readily separated from the supernatant. Theproducts formed were more hydrophobic than thesubstrate, as indicated by their longer retention times on areverse phase high-performance liquid chromatographycolumn. Mass spectrometric analysis of the purifiedproducts indicated the formation of oligomers. Analysis ofthe mixture of products by gas chromatography and massspectrometry after derivatization with diazomethanesuggested the formation of at least three dimeric and ninetrimeric products. Carbon-carbon and carbon-oxygenbonds were identified in the dimers and trimers,respectively. The nuclear magnetic resonance spectrum ofthe main dimer suggested coupling of the two monomersat the carbon one position.     

Polybrominated biphenyls as aryl hydrocarbon hydroxylase inducers: structure-activity correlations

The synthesis of all possible laterally-substituted polybrominated biphenyl (PBB) congeners containing two para bromines is described. Using enzymic, electrophoretic and ligand-binding assays that distinguish between phenobarbitone(PB)- and 3-methylcholanthrene(MC)-type inducers, the synthetic PBBs were evaluated as inducers of liver microsomal drug-metabolizing enzymes in the immature male Wistar rat. 4,4'-Dibromobiphenyl resembled PB in its mode of induction whereas all the meta-brominated derivatives of 4,4'-dibromobiphenyl, namely 3,4,4'-tri, 3,4,4',5-tetra-, 3,3', 4,4'-tetra-, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexabromobiphenyl, resembled MC in their mode of induction. The results obtained with 3,4,4'-tribromobiphenyl demonstrate that, in contrast to the polychlorinated biphenyls (PCBs), a single meta halogen substituent is sufficient to abolish the PB-type characteristics of 4,4'-dibromobiphenyl and convert it to a strictly MC-type inducer. PBBs which induce AHH activity must be substituted at both para positions and at one, two, three or four meta positions. Ortho-substitution of PBBs which contain only lateral bromine groups may also give compounds which are aryl hydrocarbon hydroxylase (AHH) inducers. One of the MC-type PBBs, namely 3,3',4,4'-tetrabromobiphenyl, which has been tentatively identified in the commercial PBB mixture, fireMaster BP-6, was at least 50 times more potent as an inducer of AHH activity than the commercial PBB mixture. The induction of AHH by 3,3',4,4'-tetrabromobiphenyl was accompanied by a dose-dependent decrease in both thymus and spleen weights. The thymus and/or spleen weights were decreased in rats treated with the other MC-type PBBs which further supports the correlation between the toxicity of the PBBs and their ability to induce AHH.     

O‐Methyltransferases involved in biphenyl and dibenzofuran biosynthesis

Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O‐methylation reactions. cDNAs encoding the O‐methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab‐causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5‐dihydroxybiphenyl, supplied by the first pathway‐specific enzyme, biphenyl synthase (BIS). 3,5‐Dihydroxybiphenyl underwent a single methylation reaction in the presence of S‐adenosyl‐l ‐methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5‐hydroxyferulic acid. Both substrates were only methylated at the meta‐positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor‐treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N‐ and C‐terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.     

混合菌群DBFC对二苯并呋喃的降解特性及其代谢途径

[目的] 二苯并呋喃（DBF）是研究多环芳烃降解过程的模式化合物,研究其代谢过程和代谢途径对于阐明多环芳烃的代谢机制有重要意义。[方法] 从辽河河口区石油污染土壤中筛选到1个高效降解DBF的混合菌群DBFC。提取总DNA对菌群的生物多样性进行分析,通过稀释涂布平板法对菌株进行分离纯化。通过测定OD₆₀₀的吸收值对混合菌群的最适生长条件进行研究。在最适生长条件下研究底物浓度、底物谱、营养物质及表面活性剂对菌群降解效率的影响。利用超高分辨质谱检测混合菌群降解DBF的中间代谢物质,并推测其代谢途径。[结果] 生物多样性分析表明该混合菌群的组成为类芽孢杆菌（84.06%）、无色杆菌（8.17%）、假单胞菌（0.77%）、其他菌株（2.13%）。分离得到苍白杆菌、无色杆菌、寡养单胞菌和细杆菌。生长测定结果显示苍白杆菌、无色杆菌、寡养单胞菌和细杆菌均不能在DBF培养基中生长。混合菌群DBFC的最适生长条件为30℃、pH 8.0。在该条件下,混合菌群DBFC能将1.0 g/L的DBF在8 d内完全降解。在DBF浓度1.0 g/L条件下,混合菌群DBFC的最大降解速率为0.031 mmol/（L·h）。在培养基中添加葡萄糖、酵母粉和蛋白胨能将菌群降解DBF的效率分别提高1.38倍、1.14倍和1.24倍。在培养基中添加十二烷基磺酸钠或Triton-X-100能够抑制混合菌群降解DBF的效率。利用超高分辨质谱检测到4种中间代谢物质（2,2'',3-三羟基联苯、2,4-已二烯酸、龙胆酸和水杨酸）,并推测了DBF代谢途径。[结论] 本研究分离到高效DBF降解菌群,该菌群能在碱性（pH 8.0）条件下完全降解DBF,为该类污染物的原位修复提供优良菌系;利用超高分辨质谱分析得到了DBF降解途径,为该类物质的混合菌群代谢研究提供了参考。     

Biodegradation of Dibenzo-p-dioxin, Dibenzofuran, and Chlorodibenzo-p-dioxins by Pseudomonas veronii PH-03

The dioxin-degrading strain Pseudomonas veronii PH-03 was isolated from contaminated soil by selective enrichment techniques. Strain PH-03 grew on dibenzo-p-dioxin and dibenzofuran as a sole carbon source. Further, 1-chlorodibenzo-p-dioxin, 2-chlorodibenzo-p-dioxin and other dioxin metabolites, salicylic acid, and catechol were also metabolized well. Resting cells of strain PH-03 transformed dibenzo-p-dioxin, dibenzofuran, 2,2',3-trihydroxybiphenyl, and some chlorodioxins to their corresponding metabolic intermediates such as catechol, salicylic acid, 2-hydroxy-(2-hydroxyphenoxy)-6-oxo-2,4-hexadienoic acid, and chlorocatechols. The formation of these metabolites was confirmed by comparison of gas chromatography-mass spectrometry (GC-MS) data with those of authentic compounds. Although we did observe the production of 3,4,5,6-tetrachlorocatechol (3,4,5,6-TECC) from 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) with resting cell suspensions of PH-03, growth of strain PH-03 in the presence of 1,2,3,4-TCDD was poor. This result suggests that strain PH-03 is unable to utilize 3,4,5,6-TECC, even at very low concentration (0.01 mM) due to its toxicity. In cell-free extracts of DF-grown cells, 2,2',3-trihydroxybiphenyl dioxygenase, 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase, and catechol-2,3-dioxygense activities were detected. Moreover, the activities of meta-pyrocatechase and 2,2',3-trihydroxybiphenyl dioxygenase from the crude cell-free extracts were inhibited by 3-chlorocatechol. However, no inhibition was observed in intact cells when 3-chlorocatechol was formed as intermediate.     

The bphC gene-encoded 2,3-dihydroxybiphenyl-1,2-dioxygenase is involved in complete degradation of dibenzofuran by the biphenyl-degrading bacterium Ralstonia sp. SBUG 290

AIMS: Biphenyl-degrading bacteria are able to metabolize dibenzofuran via lateral dioxygenation and meta-cleavage of the dihydroxylated dibenzofuran produced. This degradation was considered to be incomplete because accumulation of a yellow-orange ring-cleavage product was observed. In this study, we want to characterize the 1,2-dihydroxydibenzofuran cleaving enzyme which is involved in dibenzofuran degradation in the bacterium Ralstonia sp. SBUG 290. METHODS AND RESULTS: In this strain, complete degradation of dibenzofuran was observed after cultivation on biphenyl. The enzyme shows a wide substrate utilization spectrum, including 1,2-dihydroxydibenzofuran, 2,3-dihydroxybiphenyl, 1,2-dihydroxynaphthalene, 3- and 4-methylcatechol and catechol. MALDI-TOF analysis of the protein revealed a strong homology to the bphC gene products. We therefore cloned a 3.2 kb DNA fragment containing the bphC gene of Ralstonia sp. SBUG 290. The deduced amino acid sequence of bphC is identical to that of the corresponding gene in Pseudomonas sp. KKS102. The bphC gene was expressed in Escherichia coli and the meta-fission activity was detected using either 2,3-dihydroxybiphenyl or 1,2-dihydroxydibenzofuran as substrate. CONCLUSIONS: These results demonstrate that complete degradation of dibenzofuran by biphenyl degraders can occur after initial oxidation steps catalysed by gene products encoded by the bph-operon. The ring fission of 1,2-dihydroxydibenzofuran is catalysed by BphC. Differences found in the metabolism of the ring fission product of dibenzofuran among biphenyl degrading bacteria are assumed to be caused by different substrate specificities of BphD. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time that the gene products of the bph-operon are involved in the mineralization of dibenzofuran in biphenyl degrading bacteria.     

Membrane separations using molecularly imprinted polymers

This review presents an overview on the promising field of molecularly imprinted membranes (MIM). The focus is onto the separation of molecules in liquid mixtures via membrane transport selectivity. First, the status of synthetic membranes and membrane separation technology is briefly summarized, emphasizing the need for novel membranes with higher selectivities. Innovative principles for the preparation of membranes with improved or novel functionality include self-assembly or supramolecular aggregation as well as the use of templates. Based on a detailed analysis of the literature, the main established preparation methods for MIM are outlined: simultaneous membrane formation and imprinting, or preparation of imprinted composite membranes. Then, the separation capability of MIM is discussed for two different types, as a function of their barrier structure. Microporous MIM can continuously separate mixtures based on facilitated diffusion of the template, or they can change their permeability in the presence of the template ("gate effect"). Macroporous MIM can be developed towards molecule-specific membrane adsorbers. Emerging further combinations of molecularly imprinted polymers (MIPs), especially MIP nanoparticles or microgels, with membranes and membrane processes are briefly outlined as well. Finally, the application potential for advanced MIM separation technologies is summarized.