Identification of two porins in Pelobacter venetianus fermenting high-molecular-mass polyethylene glycols.

Porins were purified from cells of the anaerobic gram-negative bacterium Pelobacter venetianus grown with 20-kDa polyethylene glycol. After treatment of the cell envelope fraction with sodium dodecyl sulfate-containing solutions, the murein contained only two major peptidoglycan-associated proteins of 14 and 23 kDa. Both proteins were released from the peptidoglycan by the detergent Triton X-100. Genapol X-80 released only the 23-kDa protein. This protein was purified by chromatography on a hydroxyapatite column. It did not form sodium dodecyl sulfate-resistant oligomers. Reconstituted in lipid bilayer membranes, the 23-kDa protein formed cation-selective channels with a single-channel conductance of 230 pS in 1 M KCl. The channel is not a general-diffusion pore, since its conductance depends only moderately on the salt concentration. The channel conducted ammonium much better than potassium or rubidium ions, suggesting that it is probably involved in ammonium uptake. The outer membrane of P. venetianus contains a further, non-murein-associated pore with an unknown molecular mass. It is also cationically selective and has a single-channel conductance of 1.6 nS in 1 M KCl, which suggests that its effective diameter is similar to that of porins from enteric bacteria.     

Fermentative degradation of polyethylene glycol by a strictly anaerobic, gram-negative, nonsporeforming bacterium, Pelobacter venetianus sp. nov.

The synthetic polyether polyethylene glycol (PEG) with a molecular weight of 20,000 was anaerobically degraded in enrichment cultures inoculated with mud of limnic and marine origins. Three strains (Gra PEG 1, Gra PEG 2, and Ko PEG 2) of rod-shaped, gram-negative, nonsporeforming, strictly anaerobic bacteria were isolated in mineral medium with PEG as the sole source of carbon and energy. All strains degraded dimers, oligomers, and polymers of PEG up to a molecular weight of 20,000 completely by fermentation to nearly equal amounts of acetate and ethanol. The monomer ethylene glycol was not degraded. An ethylene glycol-fermenting anaerobe (strain Gra EG 12) isolated from the same enrichments was identified as Acetobacterium woodii. The PEG-fermenting strains did not excrete extracellular depolymerizing enzymes and were inhibited by ethylene glycol, probably owing to a blocking of the cellular uptake system. PEG, some PEG-containing nonionic detergents, 1,2-propanediol, 1,2-butanediol, glycerol, and acetoin were the only growth substrates utilized of a broad variety of sugars, organic acids, and alcohols. The isolates did not reduce sulfate, sulfur, thiosulfate, or nitrate and were independent of growth factors. In coculture with A. woodii or Methanospirillum hungatei, PEGs and ethanol were completely fermented to acetate (and methane). A marine isolate is described as the type strain of a new species, Pelobacter venetianus sp. nov. Its physiology and ecological significance, as well as the importance and possible mechanism of anaerobic polyether degradation, are discussed.     

Fermentation of 2-methoxyethanol by Acetobacterium malicum sp. nov. and Pelobacter venetianus

Anaerobic bacteria degrading 2-methoxyethanol were enriched from freshwater sediments, and three strains were isolated in pure culture. Two of them were Grampositive non-spore-forming rods and grew strictly anaerobically by acetogenic fermentation. Optimal growth occurred at 30°C, initial pH 7.5–8.0. 2-Methoxyethanol and 2-ethoxyethanol were fermented to acetate and corresponding alcohols. Hydrogen plus carbon dioxide, formate, acetoin, l-malate, lactate, pyruvate, fructose, and methoxyl groups of 3,4,5-trimethoxybenzoate and 3,4,5-trimethoxycinnamate were fermented to acetate. 1,2-Propanediol was fermented to acetate, propionate, and propanol. Strain MuME1 was described as a new species, Actetobacterium malicum. It had a DNA base composition of 44.1 mol% guanine plus cytosine. The third strain, which was identified as Pelobacter venetianus, fermented 2-methoxyethanol to methanol, ethanol, and acetate.     

Enzymes Involved in Anaerobic Polyethylene Glycol Degradation by Pelobacter venetianus and Bacteroides Strain PG1

In extracts of polyethylene glycol (PEG)-grown cells of the strictly anaerobically fermenting bacterium Pelobacter venetianus, two different enzyme activities were detected, a diol dehydratase and a PEG-degrading enzyme which was characterized as a PEG acetaldehyde lyase. Both enzymes were oxygen sensitive and depended on a reductant, such as titanium citrate or sulfhydryl compounds, for optimal activity. The diol dehydratase was inhibited by various corrinoids (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin) by up to 37% at a concentration of 100 μM. Changes in ionic strength and the K⁺ ion concentration had only limited effects on this enzyme activity; glycerol inhibited the enzyme by 95%. The PEG-degrading enzyme activity was stimulated by the same corrinoids by up to 80%, exhibited optimal activity in 0.75 M potassium phosphate buffer or in the presence of 4 M KCI, and was only slightly affected by glycerol. Both enzymes were located in the cytoplasmic space. Also, another PEG-degrading bacterium, Bacteroides strain PG1, contained a PEG acetaldehyde lyase activity analogous to the corresponding enzyme of P. venetianus but no diol dehydratase. Our results confirm that corrinoid-influenced PEG degradation analogous to a diol dehydratase reaction is a common strategy among several different strictly anaerobic PEG-degrading bacteria.     

Inducible or constitutive polyethylene glycol dehydrogenase involved in the aerobic metabolism of polyethylene glycol

2, 6-Dichlorophenolindophenol (DCIP)-dependent polyethylene glycol (PEG) dehydrogenase activity was found in the particulate fractions of cell-free extracts prepared from PEG-utilizing bacteria (Pseudomonas and Flavobacterium species). This result suggested that PEG dehydrogenase is linked to the respiratory chain of each bacterium and that the enzyme plays a major role in the aerobic metabolism of PEG. Enzyme activities were strongly inhibited by 1, 4-benzoquinone. No metal ion was indispensable for the enzyme activities. Enzyme activities of PEG-utilizing bacteria were induced by PEG except for the activity of PEG 4000-utilizing Flavobacterium sp. no. 203 which had a constitutive enzyme. Although PEG-utilizing bacteria had different growth substrate specificities toward PEGs 200–20,000, their PEG dehydrogenases oxidized the same molecular wt. range of PEGs (dimer-20,000). Cell-free extracts of PEG 400-, 1000- or 4000-utilizing bacteria oxidized PEG 6000 and 20,000 though these bigger PEGs could not be utilized as the sole carbon and energy sources by the bacteria. Methanol, ethylene glycol and glycerol were not or only barely dehydrogenated by all the enzyme preparations.     

Phosphorus contamination in polyethylene glycol

Concentrations of Fe, Mn, Cu, Zn, Ca, Mg, K, and P were examined in untreated and ion exchange resin-treated solutions of polyethylene glycol, molecular weight 3000 to 3700, polyethylene glycol (PEG 4000). Relatively high levels of P were found in untreated PEF-4000 solutions. The concentration of contaminating P in solutions prepared from untreated PEG 4000, even at high water potentials (−1 to −3 bars), was greater than what is usually found in soil solution. Occurrence of significant amounts of P in untreated PEG could introduce problems in experiments where ³²P and PEG are used together and where phosphate interactions may occur.     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Fermentative Degradation of Polyethylene Glycol by a Strictly Anaerobic, Gram-Negative, Nonsporeforming Bacterium, Pelobacter venetianus sp. nov

[This corrects the article on p. 1913 in vol. 45.].     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol.

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Radioprotective effect of polyethylene glycol

Polyethylene glycol of molecular weight 400 (PEG-400) had a radioprotective effect of about 20% against lethality when given ip 20 min prior to single or fractionated X-ray doses to the head and neck. Dose modification factors (DMF) based on LD50/15 values ranged from 1.14 to 1.24. A similar DMF of 1.12 based on LD50/30 values was obtained using single doses of whole-body X irradiation. Mice given head and neck irradiation had significantly reduced rectal temperatures (31.3 +/- 3.0 degrees C) 9 days post irradiation compared with unirradiated controls (35.4 +/- 0.6 degrees C). No such reduction was observed when PEG-400 was given with radiation (36.3 +/- 0.9 degrees C). PEG-400 also lessened, but not significantly, the frequency of shivering in irradiated animals. Histopathologic examination of the oral structures demonstrated only marginal protection by PEG-400. Estimation of the alpha/beta ratio from LD50 data on head and neck-irradiated mice yielded values of 4.4 +/- 1.9 (95% confidence limits) Gy without PEG-400 and 7.9 +/- 1.4 Gy with PEG-400. Since it is a non-thiol radioprotector, PEG-400 may be more useful when combined with more conventional thiol-containing radioprotectors.     

Water potential of aqueous polyethylene glycol

Water potential (Ψω) values were determined for aqueous colloids of four molecular sizes of polyethylene glycol (PEG) using freezing-point depression and vapor-pressure deficit methods. A significant third-order interaction exists between the method used to determine Ψω, PEG molecular size, and concentration. At low PEG concentrations, freezing-point depression measurements result in higher (less negative) values for Ψω than do vapor-pressure deficit measurements. The reverse is true at high concentrations. PEG in water does not behave according to van't Hoff's law. Ψω is related to molality for a given PEG but not linearly. Moreover, Ψω varies with the molecular size of the PEG. It is suggested that the Ψω of PEG in water may be controlled primarily by the matric forces of ethylene oxide subunits of the PEG polymer. The term matricum is proposed for PEG in soil-plant-water relation studies.     

Bacterial oxidation of polyethylene glycol.

The metabolism of polyethylene glycol (PEG) was investigated with a synergistic, mixed culture of Flavobacterium and Pseudomonas species, which are individually unable to utilize PEGs. The PEG dehydrogenase linked with 2,6-dichlorophenolindophenol was found in the particulate fraction of sonic extracts and catalyzed the formation of a 2,4-dinitrophenylhydrazine-positive compound, possibly an an aldehyde. The enzyme has a wide substrate specificity towards PEGs: from diethylene glycol to PEG 20,000 Km values for tetraethylene glycol (TEG), PEG 400, and PEG 6,000 were 11, 1.7, and 15 mM, respectively. The metabolic products formed from TEG by intact cells were isolated and identified by combined gas chromatography-mass spectrometry as triethylene glycol and TEG-monocarboxylic acid plus small amounts of TEG-dicarboxylic acid, diethylene glycol, and ethylene glycol. From these enzymatic and analytical data, the following metabolic pathway was proposed for PEG: HO(CH2CH2O)nCH2CH2OH leads to HO(CH2CH2O)nCH2CHO leads to HO(CH2CH2O)nCH2COOH leads to HO(CH2CH2O)n-1CH2CH2OH.     

Alterations in phospholipid polymorphism by polyethylene glycol

Summary The fusogen polyethylene glycol is shown to alter the polymorphism of dimyristoyl phosphatidylcholine, soybean phosphatidylethanolamine, bovine phosphatidylserine, egg phosphatidylcholine/cholesterol mixture, dilinoleoylphosphatidylethanolamine/palmitoyl-oleoylphosphatidylcholine mixture, and egg lysolecithin. Suspension of these lipids in 50% polyethylene glycol (mol wt=6000) reduces both the lamellar and the hexagonal II repeat spacings as measured by X-ray diffraction. An increase in the gel to liquid crystalline and bilayer to hexagonal transition temperatures are observed by freeze-fracture, X-ray diffraction, differential scanning calorimetry and³¹P NMR. Freeze-fracture electron micrographs revealed different bilayer defects depending on the physical states of the lipid. Lipidic particles in mixtures containing unsaturated phosphatidylethanolamine is eliminated. Some of the influences of polyethylene glycol on lipids may be explained by its dehydrating effect. However, other nonfusogenic dehydrating agents failed to produce similar results. These findings are consistent with the proposal that close bilayer contact and the formation of bilayer defects are associated with the fusogenic properties of polyethylene glycol.     

Fermentation of acetylene by an obligate anaerobe,Pelobacter acetylenicus sp. nov.

Four strains of strictly anaerobic Gram-negative rod-shaped non-sporeforming bacteria were enriched and isolated from marine and freshwater sediments with acetylene (ethine) as sole source of carbon and energy. Acetylene, acetoin, ethanolamine, choline, 1,2-propanediol, and glycerol were the only substrates utilized for growth, the latter two only in the presence of small amounts of acetate. Substrates were fermented by disproportionation to acetate and ethanol or the respective higher acids and alcohols. No cytochromes were detectable; the guanine plus cytosine content of the DNA was 57.1±0.2 mol%. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyltransferase, and acetate kinase were found in high activities in cell-free extracts of acetylene-grown cells indicating that acetylene was metabolized via hydration to acetaldehyde. Ethanol was oxidized to acetate in syntrophic coculture with hydrogen-scavenging anaerobes. The new isolates are described as a new species in the genusPelobacter, P. acetylenicus.Dedicated to Professor Dr. Norbert Pfennig on occasion of his 60th birthday     

Protein refolding is improved by adding nonionic polyethylene glycol monooleyl ethers with various polyethylene glycol lengths

Protein refolding from bacterial inclusion bodies is a crucial step for the production of recombinant proteins, but the refolding step often results in significantly lower yields due to aggregation. To prevent aggregation, chemical additives are often used. However, the ability of additives to effectively increase refolding yields are protein dependent, and therefore, it is important to understand the manner in which the substructures of additives confer suitable properties on protein refolding. We focused attention on nonionic detergents, the polyethylene glycol monooleyl ether (PGME) series, and systematically studied the influence of two to 90 polyethylene glycol (PEG) lengths of PGMEs on the refolding of pig muscle lactate dehydrogenase (LDH), hen egg white lysozyme, and yeast α‐glucosidase. PGMEs with longer PEG lengths such as PGME20, 50, and 90 suppressed aggregation, and increased refolding yields. Notably, PGME20 increased the LDH yield to 56.7% from 2.5% without additives. According to the refolding kinetic analysis of LDH, compared with PGME50 and 90, the refolding rate constant in PGME20 solutions remained relatively high at a broad range of concentrations because of its weaker steric hindrance of intramolecular interactions involved in folding, leading to a preference for refolding over aggregation. These findings should provide basic guidelines to identify appropriate PEG‐based nonionic detergents for protein refolding.     

Branched polyethylene glycol for protein precipitation

The use of linear PEGs for protein precipitation raises the issues of high viscosity and limited selectivity. This paper explores PEG branching as a way to alleviate the first problem, by using 3-arm star as the model branched structure. 3-arm star PEGs of 4,000 to 9,000 Da were synthesized and characterized. The effects of PEG branching were then elucidated by comparing the branched PEG precipitants to linear versions of equivalent molecular weights, in terms of IgG recovery from CHO cell culture supernatant, precipitation selectivity, solubility of different purified proteins, and precipitation kinetics. Two distinct effects were observed: PEG branching reduced dynamic viscosity; secondly, the branched PEGs precipitated less proteins and did so more slowly. Precipitation selectivity was largely unaffected. When the branched PEGs were used at concentrations higher than their linear counterparts to give similar precipitation yields, the dynamic viscosity of the branched PEGs were noticeably lower. Interestingly, the precipitation outcome was found to be a strong function of PEG hydrodynamic radius, regardless of PEG shape and molecular weight. These observations are consistent with steric mechanisms such as volume exclusion and attractive depletion.     

Lectin-binding assay by polyethylene glycol 8000

A quantitative lectin-binding assay using a precipitation technique and polyethylene glycol 8000 (PEG) as a precipitating agent has been described. Carcinoscorpin, a sialic acid-binding lectin isolated from the hemolymph of Indian horseshoe crab, Carcinoscorpius rotunda cauda, and iodinated fetuin, a sialoglycoprotein, were appropriately incubated as the components of the binding assay. The specific interaction between these two components developed the lectin-glycoprotein-bound complex. This was subsequently precipitated by the addition of PEG together with a coprecipitant gamma-globulin. Radioactivity of the precipitated bound complex was estimated to quantify the binding. The formation of the bound complex was effectively inhibited by a specific sialodisaccharide, O-(N-acetylneuraminyl)-(2----6)-2-acetamido-2-deoxygalactitol, implying the specific interaction for such precipitation. The probable effect of PEG was to stabilize the bound complex, precipitating it along with added gamma-globulin. This was further evident from the prevention of dissociation of the bound complex and increased binding of glycoprotein to the immobilized lectin in the presence of PEG. The assay was also applicable to other sialoglycoproteins such as alpha 1-acid glycoprotein and human chorionic gonadotropin. Moreover, the method yielded a saturation plateau with a characteristic hyperbolic binding curve. The assay was simple, quick, safe, economic, and highly sensitive.     

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