Enhancement effect of polyethylene glycol on enzymatic synthesis of cephalexin

In an enzymatic synthesis of cephalexin (CEX) using an acylase from Xanthomonas citri, the effect of polyethylene glycol (PEG) on the synthetic reaction of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenyl-glycine methyl ester (PGM) to CEX was investigated. The addition of PEG (MW 300-20,000) increased the yield significantly. This yield enhancement effect tended to increase with the increasing molecular weight of PEG. Addition of PEG to the reaction system did not affect both the CEX and PGM hydrolytic reactions. The PEG added to the reaction medium used in these experiments did not depress the water activity significantly, and the product yield improvement could not be explained by the activity alone. The PEG stabilized the enzyme activity to some extent, but this stabilizing effect was only partially attributable to the yield enhancement of CEX. The enhancing effect of PEG on the synthetic yield increased with the increasing PEG molecular weight or the length of the poly(oxy-1,2-ethanediyl) chain, which increases the hydrophobicity of PEG. This finding consequently has led to the conclusion that the PEG structure renders the affinity between enzyme and 7-ADCA, which is a hydrophobic substrate. The microenvironmental hydrophobicity of PEG and its interaction with the hydrophobic substrate was found to be the main reason for the improvement of the CEX yield. In fact, the Michaelis-Menten kinetic constant for 7-ADCA, K(7-ADCA) in the presence of PEG was smaller than that in the control system (without PEG addition). (c) 1993 John Wiley & Sons, Inc.     

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.     

Molecular engineering of G-quadruplex ligands based on solvent effect of polyethylene glycol

Because various non-parallel G-quadruplexes of human telomeric sequences in K(+) solution can be converted to a parallel G-quadruplex by adding polyethylene glycol (PEG) as a co-solvent, we have taken advantage of this property of PEG to study the covalent attachment of a PEG unit to a G-quadruplex ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC). The hybrid ligand with the PEG unit, BMVC-8C3O or BMVC-6C2O by substituting either the tetraethylene glycol or the triethylene glycol terminated with a methyl-piperidinium cation in N-9 position of BMVC, not only induces structural change from different non-parallel G-quadruplexes to a parallel G-quadruplex but also increases the melting temperature of human telomeres in K(+) solution by more than 45°C. In addition, our ligand work provides further confidence that the local water structure plays the key to induce conformational change of human telomere.     

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.     

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.     

Differential effect of sorbitol and polyethylene glycol on antioxidant enzymes in rice leaves

Polyethylene glycol (PEG) and sorbitol (ST) have each been used inosmotically induced water stress studies in plants, however, these osmotica maynot have equivalent effects in plants. The present study was designed to examinewhether antioxidant enzyme responses in rice leaves are different for PEG and STof osmotic potential –1.5 MPa. As judged by relative watercontent, PEG treatment resulted in a higher degree of water stress in riceleaves than ST treatment. PEG treatment markedly increased lipid peroxidation,judged by malondialdehyde content, in rice leaves. However, ST treatment had noeffect on lipid peroxidation. An increase in peroxidase (POX), ascorbateperoxidase (APX) and glutathione reductase (GR) activities was observed in riceleaves treated with ST. PEG treatment had no effect on POX and APX activitiesand decreased GR activity in rice leaves. The decrease in superoxide dismutaseactivity induced by PEG was more pronounced than by ST. Cycloheximide blockedthe enhanced activities of POX, APX and GR by ST, indicating de novo synthesisof the enzymes. Results suggest that ST but not PEG treatment can up-regulateantioxidant system in rice leaves.     

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.     

Radioprotective effect of pyrazolone derivatives]

The intraperitoneal injection of analgin (1000 mg/kg), antipurine (400 mg/kg), amidopyrine (100 mg/kg) 3 hours before the irradiation of mice in a dose of 800 R led to survival of 30 to 45% of the animals (against 12.5% in control) and to increase in the average duration of life of the animals that perished. 80-95% of mice survived the period of "intestinal deaths" (the 7th day after the irradiation) after combined prophylactic use of purasolone derivatives and cystamine before the irradiation of these animals in a dose of 1050 R. The radioprotective effect of pyrasolone derivatives given in therapeutic doses was less pronounced.     

Arginine-specific modification of proteins with polyethylene glycol

In this study, the residue-selective modification of proteins with polymers at arginine residues is reported. The difficulty in modifying arginine residues lies in the fact that they are less reactive than lysine residues. Consequently, typical chemo-selective reactions which employ "kinetic" selectivity (active esters, Michael addition, etc.) cannot be used to target these residues. The chemistry exploited herein relies on "thermodynamic" selectivity to achieve selective modification of arginine residues. ω-Methoxy poly(ethylene glycol) bearing an α-oxo-aldehyde group was synthesized and used to demonstrate the selective modification of lysozyme at arginine residues. In addition, the optimization of reaction conditions for coupling as well as the stability of the formed adduct toward dilution, toward a nucleophilic buffer, and toward acidification are reported. It was concluded that this approach is a convenient, mild, selective, and catalyst-free method for protein modification.     

聚乙二醇对PEG化重组人促红细胞生成素热稳定性和生物学活性的影响

为了研究聚乙二醇对PEG化rhEPO热稳定性和生物学活性的影响,用酶学方法切除rhEPO和PEGrhEPO分子中的N连接糖基,研究酶切产物在37℃时287nm和350nm吸光度随时间的变化;用MTT比色法,网织红细胞法和HCT法比较体内和体外生物学活性的改变。结果显示聚乙二醇修饰能显著减少无N连接糖基的rhEPO分子间的聚集,降低rhEPO体外生物学活性,但增高体内生物学活性;无N连接糖基的PEG化rhEPO具有与rhEPO相当的体内生物学活性。因此聚乙二醇能提高rhEPO热稳定性;聚乙二醇可替代糖基,维持rhEPO的体内生物学活性。用PEG修饰原核细胞表达的rhEPO是开发rhEPO制品的新思路。     

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.     

Effect of ethylene glycol and polyethylene glycol on the acid-unfolded state of trypsinogen

Effect of increasing concentrations of two of the polyols, ethylene glycol (EG) and polyethylene glycol (PEG), was studied by near and far circular dichroism (CD), fluorescence emission spectroscopy, and binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show the transition of acid-unfolded trypsinogen from an unordered state to an intermediate state having ordered secondary structure. Interestingly, near-UV CD spectra show some amounts of stabilizing effect on the tertiary structure of the protein also. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on addition of EG and PEG. Maximum ANS binding occurs in presence of 80% EG and 90% PEG (v/v), suggesting the presence of an intermediate or molten globule-like state at high concentrations of the two polyols.