Ester-exchange catalyzed by lipase modified with polyethylene glycol

Lipoprotein lipase was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine; forty-six percent out of seven amino groups in the molecule were substituted. The modified lipase catalyzed ester-exchange reactions between an ester and an alcohol, between an ester and an acid, and between two esters. The modified enzyme catalyzed these reactions not only in organic solvents, but also in straight hydrophobic substrates. As the modified enzyme was extremely stable at elevated temperature, for example at 70 degrees C, this can find many practical applications.     

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.     

Polymerization of phenols catalyzed by peroxidase in nonaqueous media

Polymers produced by horseradish-peroxidase-catalyzed coupling of phenols have been explored as potential substitutes for phenol-formaldehyde resins. To overcome low substrate solubilities and product molecular weights in water, enzymatic polymerizations in aqueous-organic mixtures have been examined. Peroxidase vigorously polymerizes a number of phenols in mixtures of water with water-miscible solvents such as dioxane, acetone, di-methylformamide, and methyl formate with the solvent content up to 95%. As a result, various phenolic polymers with average molecular weights from 400 to 2.6 x 10(4) D were obtained depending on the reaction medium composition and the nature of the phenol. Peroxidase-catalyzed copolymerization of different phenols in 85% dioxane was demonstrated. Poly(p-phenylphenol) and poly(p-cresol) were enzymatically prepared on a gram scale. They had much higher melting points, and in addition, poly(p-phenylphenol) was found to have a much higher electrical conductivity than phenol-formaldehyde resins.     

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.     

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.     

Immobilization of nitrifying bacteria by polyethylene glycol prepolymer

The effects of immobilizing materials on the activity of nitrifying bacteria were investigated by using 11 kinds of prepolymers of polyethylene glycol. Relative respiratory activity of immobilized nitrifying bacteria with polyethylene glycol metacrylate prepolymer was higher than that of polyethylene glycol acrylate prepolymer, and there was a tendency for relative respiratory activity to be higher with a prepolymer of greater molecular weight. With the polyethylene glycol prepolymer, there was a drastic improvement over the conventional method of immobilization by acrylamide in the relative respiratory activity of the pellet. Inorganic synthetic wastewater was treated under a high loading rate of 1.14 kg-N/m³·d. Influent NH₄-N could be removed to 2 mg/l or less and the nitrogen removal was 90%.     

Electrophoresis–chemiluminescence detection of phenols catalyzed by hemin

Based on the catalytic activity of hemin, an efficient biocatalyst, an indirect capillary electrophoresis–chemiluminescence (CE‐CL) detection method for phenols using a hemin–luminol–hydrogen peroxide system was developed. Through a series of static injection experiments, hemin was found to perform best in a neutral solution rather than an acidic or alkaline medium. Although halide ions such as Br^? and F^? could further enhance the CL signal catalyzed by hemin, it is difficult to apply these conditions to this CE‐CL detection system because of the self‐polymerization of hemin, as it hinders the CE process. The addition of concentrated ammonium hydroxide to an aqueous/dimethyl sulfoxide solution of hemin–luminol afforded a stable CE‐CL baseline. The indirect CE‐CL detection of five phenols using this method gave the following limits of detections: 4.8 × 10^?8 mol/L (o‐sec‐butylphenol), 4.9 × 10^?8 mol/L (o‐cresol), 5.4 × 10^?8 mol/L (m‐cresol), 5.3 × 10^?8 mol/L (2,4‐dichlorophenol) and 7.1 × 10^?8 mol/L (phenol). Copyright © 2013 John Wiley & Sons, Ltd.     

Enhancement of enzymatic production of cyclodextrins by adding polyethylene glycol or polypropylene glycol

Enzymatic production of cyclodextrins (CDs) from soluble starch was studied using either Bacillus macerans or Bacillus ohbensis cyclomaltodextrin glucanotransferase (CGTase). The production yield of CDs was found to be increased up to 1.5–2 times by the addition of low molecular weight polyethylene glycol (PEG 400) or polypropylene glycol (PPG 425) to the reaction medium. Such results were interpreted as being due to a conformational change of the substrate as well as reduction of hydrolytic activity of the enzyme in the presence of these additives.     

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.     

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.     

Acceleration of nucleic acid hybridization rate by polyethylene glycol

The addition of polyethylene glycol to filter-bound nucleic acid hybridization greatly increases the hybridization rate. With single-stranded probes, the increase obtained with polyethylene glycol is significantly greater than that obtained with dextran sulfate. Additionally, polyethylene glycol is easier to manipulate and less expensive than dextran sulfate.     

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.     

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.     

A procedure for the fusion of cells in suspension by means of polyethylene glycol

The nuclei of chick and human fibroblasts are readily distinguishable in Leishman stained heterokaryons. The use of these two types of cell has allowed development of a method for fusion in suspension based on the use of polyethylene glycol (PEG) of MW 1000 in Dulbecco's medium containing 15% dimethylsulfoxide (DMSO).     

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.     

Cryoprotection of purified rat kidney transamidinase by polyethylene glycol.

Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme is frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol 4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 mM ethylenediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes.