The permeability of the barrier of the gastrointestinal tract for the macromolecules of polyethylene glycol-4000: an assessment of the mechanism and its reproducibility]

Polyethylene glycol (PEG)-4000 is proposed as a tracer of intestinal macromolecular permeability. The reproducibility of permeability testing with PEG-4000 and the mechanism of its penetration through intestinal mucosa were studied in adult rats. Permeability measurement for PEG-4000 was reproducible when repeated twice for 2 days. This makes it possible to repeat PEG-4000 permeability testing before and after any experimental impact on the intestine. I.p. administration of colchicine to rats (125 micrograms/100 g b. w.) significantly inhibited intestinal absorption of PEG-4000 fed to the animals 3 hours later. Hence, PEG-4000 penetration through the intestinal mucosa is mediated by the system of enterocyte cytoplasmic microtubules. Mucosal permeability for PEG-4000 may be consequently considered as a valuable model of permeability for protein macromolecules.     

Sizing the pore of the volume-sensitive anion channel by differential polymer partitioning

Partitioning of ethylene glycol and its polymeric forms into the pore of the volume-sensitive outwardly rectifying (VSOR) anion channel was studied to assess the pore size. Polyethylene glycol (PEG) PEG 200-300 (Rh = 0.27-0.53 nm) effectively suppressed the single-channel currents, whereas PEG 400-4000 (Rh = 0.62-1.91 nm) had little or no effect. Since all the molecules tested effectively decreased electric conductivity of the bulk solution, the observed differential effects between PEG 200-300 and PEG 400-4000 on the VSOR single-channel current are due to their limited partitioning into the channel lumen. The cut-off radius of the VSOR channel pore was assessed to be 0.63 nm.     

Permeability of the peptidic GH secretagogues hexarelin and EP 51389, across rat jejunum.

The intestinal permeability of hexarelin and EP 51389, two growth hormone releasing hexa- and tri- peptide analogues, was assessed in vitro with side-by-side diffusion chambers in the apical-to-basolateral (AP-to-BL) and in the basolateral-to-apical (BL-to-AP) direction using excised rat jejunal segments. The effect of EP 51389 on P-glycoprotein (P-gp) was evaluated by rhodamine 123 accumulation on monolayers of CH(R)C5 cells with increasing concentrations of EP 51389. Hexarelin and EP 51389 permeability were found to be < 1%. Permeability coefficients (P(app)) were 18.87 +/- 2.86 (x10(-7) cm/s) and 5.87 +/- 0.45 (x10(-7) cm/s) for hexarelin and EP 51389, respectively. Bidirectional studies revealed that hexarelin transport was similar in both directions. EDTA did not influence hexarelin permeability. Permeability was predominantly secretory for EP 51389 as P(app) in the BL-to-AP direction [32.56 +/- 6.11 (x10(-7) cm/s)] was greater than AP-to-BL. Confirming involvement of a secretory transport system, chlorpromazine inhibited EP 51389 transport across the jejunum. EP 51389 inhibited P-gp in a dose dependent manner resulting in the intracellular accumulation of rhodamine in CH(R)C5 cells. These results suggest that: 1) the intestinal permeability of hexarelin and EP 51389 is poor; 2) the passage of hexarelin is mainly via a transcellular passive pathway since the contribution of paracellular permeability to the overall permeability is rather low; 3) P-gp may act as a potential barrier for the intestinal absorption of EP 51389.     

Permeability of polyethylene glycol in remnant small bowel after massive intestinal resection

Studies were designed to determine if permeability of adapted (remnant) small bowel mucosa to polyethylene glycol (PEG) was altered after major intestinal resection. Rats underwent 50% small bowel resection with preservation of duodenum and terminal ileum. Sham-operated animals served as controls. Two and four weeks later we cannulated the portal vein and measured mucosal permeability to luminal [3H]PEG and [14C]PEG in isotonic Ringer solution in remnant proximal or distal in situ closed intestinal loops. A lumen-to-portal blood gradient of at least 1000/1 persisted throughout the one-hour experimental period in both resected and sham-operated animals. Thus the adapted remnant intestinal mucosa was highly impermeable to luminal radiotracer PEG. In separate experiments 2 and 4 weeks after 70% small bowel resection or sham operation, in vivo segments of proximal and distal small intestinal were perfused through the lumen for one hour with hypertonic (800 mOsm) mannitol or NaCl solution containing [3H]PEG. There was equal and almost total recovery of [3H]PEG at the end of the experimental period in resected and control animals. The combined data of all experiments indicate that radiotracer PEG may be confidently used as a luminal water phase marker in transport studies of remnant bowel following intestinal resection.     

Effect of high temperature and water stress on in vitro germination and growth in isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin

In non-irrigated agricultural fields in tropical zones, high temperature and water stress prevail during the main cropping season. Natural epizootics of Beauveria bassiana on lepidopteran pests occur during winter. Application of B. bassiana during hot months when pest populations are at their climax may prove an effective management strategy. Therefore, 29 isolates of B. bassiana were tested for their ability to germinate and grow in temperature and water availability conditions prevailing during the pest season in these fields. The effect of temperature cycles with 8 h duration of high temperature fluctuating with 16 h duration of lower temperature (similar to field conditions); low water availability; and a combination of these two stress conditions was studied. Germination and growth assays were done at fluctuating temperature cycles of 32, 35, 38, and 42+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and in media with water stress created by 10, 20, 30, and 40% polyethylene glycol (PEG 6000). Assays set at a continuous temperature of 25+/-1 degrees C with no PEG in the medium served as controls. Stress was assessed as percentage germination or as growth relative to control. Isolates showing 90% growth relative to the control at temperature cycles including high temperatures of 35 and 38+/-1 degrees C were identified. One isolate (ARSEF 2860) had a thermal threshold above 43 degrees C. At 25 degrees C, all but one isolate of B. bassiana showed >90% growth relative to the control in 10% PEG (-0.45 MPa). Some isolates were found with >90% growth relative to control in medium having 30% PEG with water availability (1.33 MPa), nearly equivalent to that in soils which induce permanent wilting point of plants. When isolates that showed >90% growth relative to the control at both stress conditions, were stressed simultaneously, a decrease in growth was observed. Growth was reduced by approximately 20% at 35+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG and was affected to a greater degree in combinations of harsher stress conditions. The isolate ARSEF 2860 with a thermal threshold of >43 degrees C showed approximately 80% relative growth at a combined stress of 38+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG. These findings will aid the selection of isolates for use in field trials in hot or dry agricultural climates.     

Polyethylene glycol behaves like weak organic solvent

Effect of polyethylene glycol (PEG) on protein solubility has been primarily ascribed to its large hydrodynamic size and thereby molecular crowding effect. However, PEG also shows characteristics of organic solvents. Here, we have examined the solubility of glycine and aliphatic and aromatic amino acids in PEG solutions. PEG400, PEG4000, and PEG20000 decreased the solubility of glycine, though to a much smaller magnitude than the level achieved by typical organic solvents, including ethanol and dimethyl sulfoxide. PEG4000 showed varying degree of interactions with amino acid side chains. The free energy of aliphatic side chains marginally increased by the addition of PEG4000, indicating their weak unfavorable interactions. However, it significantly decreased the free energy of the aromatic side chains and hence stabilized them. Thus, it was concluded that PEG behaves like weak organic solvents; namely PEG destabilized (interacted unfavorably with) polar and charged groups and stabilized (interacted favorably with) aromatic groups. Interestingly, the interaction of PEG20000, but neither PEG400 nor PEG4000, with glycine resulted in phase separation under the saturated concentration of glycine.     

Soluble complement receptor type 1 ameliorates the local and remote organ injury after intestinal ischemia-reperfusion in the rat.

We examined the role of C activation in ischemia reperfusion injury by inhibiting C activation in a rat model of mesenteric arterial occlusion. In anesthetized rats, 60 min of mesenteric arterial occlusion was followed by 3 h of reperfusion. PBS alone or containing soluble C receptor 1 (3 or 6 mg) was administered i.v. Controls underwent laparotomy without ischemia. Relative serum C activities were assessed by hemolytic assay, neutrophil (polymorphonuclear leukocyte) sequestration by tissue content of myeloperoxidase (MPO) activity, intestinal mucosal injury by histologic grading, lung vascular permeability by the ratio of bronchoalveolar lavage to blood concentration of radiolabeled BSA, and endothelial cell injury was quantified by measurement of plasma factor VIII-related Ag. After reperfusion, PBS-treated animals had increased intestinal MPO (0.048 +/- 0.007 U/g) compared to sham (0.022 +/- 0.005 U/g (p less than 0.05)) and intestinal mucosal injury score (2.490 +/- 0.221) compared to sham (0.331 +/- 0.045 (p less than 0.05)). Treatment with 6 mg soluble C receptor 1 15 min before reperfusion reduced intestinal MPO (0.017 +/- 0.003 U/g (p less than 0.05)) and mucosal injury (1.733 +/- 0.168 (p less than 0.05)) compared to PBS control. PBS-treated animals also demonstrated increased lung MPO (0.314 +/- 0.025 U/g vs 0.085 +/- 0.018 in sham (p less than 0.05)) and increased lung permeability (bronchoalveolar lavage/blood cpm 11.32 +/- 1.35 x 10(-3) vs sham 2.22 +/- 0.19 x 10(-3) (p less than 0.05)). Treatment with 6 mg soluble C receptor 1 15 min before reperfusion or at reperfusion reduced the lung permeability (bronchoalveolar lavage/blood cpm 3.90 +/- 0.79 x 10(-3) and 5.08 +/- 0.75, respectively (both p less than 0.05)) compared to PBS control, but did not reduce lung MPO (0.342 +/- 0.031 U/g and 0.246 +/- 0.025), respectively. Treatment with sCR1 also reduced the release of factor VIII-related Ag, 5-day mortality, and C hemolytic activity. In this model, C is a major mediator of intestinal injury and extraintestinal injury.     

Purification and characterization of constitutive polyethylene glycol (PEG) dehydrogenase of a PEG 4000-utilizing Flavobacterium sp. no. 203

Polyethylene glycol (PEG) 4000-utilizing bacterium no. 203 was identified as a Flavobacterium species. 2, 6-Dichlorophenol-indophenol (DCIP)-dependent PEG dehydrogenase was constitutively formed in nutrient broth, glucose and PEG media. However, the enzyme formation was repressed in the presence of an excess amount (over 0.25%) of PEG 400 or 1000. PEG dehydrogenase was purified approximately 34 fold by precipitation with ammonium sulfate, solubilization with benzalkonium chloride, chromatography with DEAE-Toyopearl 650 M and hydroxylapatite and gel filtration on Toyopearl HW-55. The molecular weight of the purified PEG dehydrogenase was calculated to be approximately 2.20 × 10⁵, a value which seemed to consist of four subunits with the same molecular weight of 5.70 × 10⁴. The enzyme was stable below 40°C and in the pH range of 7.0 and 8.0. The optimum pH and temperature of the activity were around 8.0 and 40°C, respectively. The enzyme reduced DCIP and coenzyme Q₁ and Q₂. PEG dehydrogenase showed activity toward various PEG molecules (dimer-PEG 20,000). The apparent K_m values for PEG 400, 1000, 4000 and 6000 were about 1.0, 1.7, 2.8 and 5.9 mM, respectively. The enzyme oxidized primary aliphatic alcohols of C₃–C₁₂, the corresponding aldehydes of C₃–C₇, aromatic alcohols and aldehydes, diols, etc. The enzyme was inactive on ethylene glycol, glycerol, secondary alcohols and sugar alcohols. The enzyme activity was strongly inhibited by sulfhydryl agents or heavy metals and 1, 4-benzoquinone. The purified enzyme showed absorption apectrum similar to that of PEG 6000 dehydrogenase which has already been reported to be a quinoprotein. The prosthetic group of the enzyme was extracted with methanol and identified as PQQ from its prosthetic group capability for glucose dehydrogenase and the fluorescence spectrum.     

Release of regulatory gut peptides somatostatin, neurotensin and vasoactive intestinal peptide by acid and hyperosmolal solutions in the intestine in conscious rats

The impact of exposure of the intestinal mucosa to acid and hyperosmolal solutions on the release of the inhibitory gut peptides somatostatin (SOM), neurotensin (NT) and vasoactive intestinal peptide (VIP) was studied in conscious rats during pentagastrin-stimulated gastric acid secretion. The animals were equipped with a chronic gastric fistula to measure acid secretion and a jejunal Thiry-Vella loop for intestinal challenge with saline, hydrochloric acid (HCl, 200 mmol L(-1)) or hyperosmolal polyethylene glycol (PEG, 1200 mOsm kg(-1)). Gut peptide concentrations were measured in intestinal perfusates, and in plasma samples collected during stimulated acid secretion, and at the end of experiments with luminal challenge of the loops. After pentagastrin-stimulation acid secretion was dose-dependently inhibited by intravenous administration of the gastrin receptor antagonist gastrazole, as well as ranitidine and esomeprazole by maximally 73+/-10%; 95+/-3%; 90+/-10%, respectively. Acid perfusion of the Thiry-Vella loop caused a prominent release of SOM both to the lumen (from 7.2+/-5.0 to 1279+/-580 pmol L(-1)) and to the circulation (from 18+/-5.2 to 51+/-9.0 pmol L(-1)) simultaneously with an inhibition of gastric acid secretion. The release of NT and VIP was not affected to the same extent. PEG perfusion of the loop caused a release of SOM as well as NT and VIP, but less. Simultaneously acid secretion was slightly decreased. In conclusion, intestinal perfusion with acid or hyperosmolal solutions mainly releases SOM, which seems to exert a major inhibitory action in the gut, as shown by inhibition of acid secretion. The other peptides NT and VIP also participate in this action but to a much lesser degree. The operative pathways of these gut peptides hence involve both endocrine (SOM) and paracrine actions (SOM, NT, VIP) in order to exert inhibitory functions on the stomach. The inhibitory action of gastrazole, was in a similar range as that of SOM implying that physiological acid-induced inhibition of gastric acid may primarily be exerted through inhibition of gastrin endocrine secretion.     

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.     

Evaluation of Polyethylene Glycol as a Water-soluble Marker: Absorption and Excretion of 14C-labeled Polyethylene Glycol in Rats

The absorbability of polyethylene glycol (PEG), a water-soluble nutritional marker, from the gastrointestinal tract of rat was examined using the [¹⁴C]-labeled compound ([¹⁴C]PEG) having a molecular weight of 4000. Intravenously injected [¹⁴C]PEG was readily excreted and recovered almost completely in the urine and neither hepatic nor renal uptake of the PEG was observed. Intragastrically administered [¹⁴C]PEG was eliminated in the urine with an average recovery of only 0.43 ± 0.13% (Mean ± S.D., n= 10) of the dose over 24 hr. From the gel column chromatographic profile of the radioactivity excreted in the urine after an oral dose, [¹⁴C]PEG was suggested to be absorbed in two forms, as an original form and as a low molecular weight component. The latter component might be the degraded product of PEG in the gastrointestinal tract. From these results it was confirmed that PEG with a molecular weight of 4000 is a satisfactory marker because of its low absorbability.     

An ultrarapid filtration method adapted to the measurements of water and solute permeability of synthetic and biological vesicles.

An ultrarapid filtration method was adapted to the determination of water and solute permeability of membrane vesicles. This method consisted of measuring substance washout from vesicles first loaded with 3H2O or labeled solutes, placed on filters, and rinsed at high rates for short periods. The retention of the vesicles on the filters was analyzed and was found to be a function of the nature and porosity of the filters as well as of the vesicle origin. Washing buffer flow rate and washing duration did not affect vesicle retention. The diffusional water permeability of cholesterol-free liposomes was determined at 16 degrees C. Its value was reduced by a factor of 2.5 when the liposomes were prepared with 20% cholesterol and a threefold increase was noted when the liposomes were preincubated with gramicidin (6 mg/g lipid). Water permeability of liposomes was strongly temperature-dependent: Ea = 15.3 kcal/mol. Diffusional water permeability of pink ghosts was also measured: a value of (4.4 +/- 0.2) X 10(-3) cm/s (n = 3) was obtained at 13 degrees C. This permeability was reduced by 45.2% with 0.4 mM HgCl2. The urea permeability of intestinal and renal brush-border membrane vesicles was (1.15 +/- 0.18) X 10(-6) cm/s (n = 7) and (1.67 +/- 0.08) X 10(-6) cm/s (n = 9), respectively. The renal value was reduced by a factor of 4.4 by 100 mM thiourea. This ultrarapid filtration technique provides an accurate method of transport measurement in sealed membranes such as liposomes and plasma membrane vesicles.     

Optimization of fibrinolytic enzyme production by Bacillus subtilis DC-2 in aqueous two-phase system (poly-ethylene glycol 4000 and sodium sulfate)

The central composite rotable design (CCRD) was used to determine optimal conditions for fibrinolytic enzyme production by Bacillus subtilis DC-2 in poly-ethylene glycol 4000 (PEG 4000) and sodium sulfate (Na(2)SO(4)) aqueous two-phase system (ATPS). PEG 4000 and Na(2)SO(4) concentration, fermentation time and temperature, and pH were selected as variables to evaluate the fibrinolytic activity in PEG phase. Using response surface methodology (RSM), a second-order polynomial equation was obtained by multiple regression analysis. The predicted maximal fibrinolytic activity in PEG phase was 1241.02 IU/ml with 9.05% PEG 4000 concentration, 5.06% Na(2)SO(4) concentration, 118.77 h fermentation time, 37.57 degrees C fermentation temperature and pH 6.52. The validity of the response model was verified by a good agreement between predicted and experimental results. The fibrinolytic activity obtained from experimental results in PEG phase (1223.61 IU/ml) was higher than that produced in homogeneous fermentation (1165.58 IU/ml).     

Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.     

Development of an improved medium for germination of Cajanus cajan (L.) Millsp. pollen in vitro.

A simple, reliable medium for pollen germination of Cajanus cajan was developed by modifying Brewbaker and Kwack (BK) medium. Past attempts of C. cajan pollen germination in artificial media were not successful. A medium containing polyethylene glycol 4000 (PEG) showed more than 90% germination for C. cajan var. Pusa 33 only when the young buds (36 h before anthesis) were kept in pollen germination medium (PGM) for 36 h before pollen extraction. Supplementation of PGM with epsilon-amino caproic acid (EACA), an amino acid, showed improved pollen germination in Pusa 33 and also helped to avoid preconditioning of young buds before pollen extraction. It was also observed that there is a genotypic difference in the level of EACA required for in vitro pollen germination. Thus a complete medium for C. cajan genotypes consists of 37.5% sucrose+ 15% PEG 4000+250 mg l(-1) boric acid+300 mg l(-1) calcium nitrate+100 mg l(-1) potassium nitrate+ 200 mg l(-1) magnesium sulphate+1% agar+EACA (0, 100, 250, 500, 750 or 1000 mg l(-1)).     

Development of white spruce (Picea glauca (Moench.) Voss) somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage

The limit of permeability of white spruce (Picea glauca [Moench.]Voss) somatic embryo cell walls to molecules was in the orderof 30 . Polyethylene glycols (PEGs) and dextrans of molecularweights greater than 1000 and 6000, respectively, produced anonpermeating (non-plasmolysing) water stress which improvedembryo development. Somatic embryos converted to plantlets atfrequencies of 76–84% following slow drying and storageat –20 C for 1 year, which was similar to the 77% recordedfor control somatic embryos slowly dried then germinated withoutfreezing or storage. Culture for 7–8 weeks with mediumcontaining abscisic acid, 3% sucrose, and 7.5% PEG 4000 yieldedsomatic embryos with five times the embryo storage lipid contentrecorded for zygotic embryos. During culture with PEG the moisturecontent of the somatic embryos decreased from 96% for immaturesuspension-cultured somatic embryos, to 47% for mature embryos.Somatic embryos cultured for 7–8 weeks survived rapiddrying to 5% moisture content, and converted to plantlets atfrequencies of 60–70%, but no somatic embryos survivedrapid drying when cultured for only 4 weeks; however, slow dryingdid induce desiccation tolerance in 3-week cultured somaticembryos. Abscisic acid was important to maintain embryos ina developmental state, but ABA alone did not induce desiccationtolerance. In order to induce desiccation tolerance a waterstress treatment was required. Tolerance of rapid drying coincidedwith moisture contents below 55%, which occurred after 5 weeksof culture in the presence of PEG 4000 and abscisic acid. Key words: Dextran, molecular weight, polyethylene glycol, triacylglycerol, water stress     

Modification of cellulase and its application to extraction of diosgenin from Dioscorea zingiberensis C.H. Wright

In order to enhance the thermostability and efficiency of cellulase in the extraction of diosgenin from Dioscorea zingiberensis C.H. Wright, we applied polyethylene glycol (PEG) (400, 1000, 2000, and 4000) to modify cellulase. The modified cellulase, α-amylase and β-glycosidase were used to hydrolyze the material. The results show that the thermostability of modified cellulase is better than that of natural cellulase, the optimum pH value and temperature of modified cellulase are wider than that of natural cellulase, the activity of cellulase modified by activated PEG2000 is higher than that of cellulase modified by other modifiers, and its remaining activity is 58% of its initial value. With this technique, the purity of the product reaches 96%, the melting point is 201–204 °C, the yield rate and the extraction rate of the diosgenin reaches 2.80% and 96.6%, respectively. IR spectra and ¹H NMR spectroscopy were used to confirm the structure of the product.     

Optimization of conditions for acid protease partitioning and purification in aqueous two-phase systems using response surface methodology

Aspergillopepsin I, an acid protease, was purified using an aqueous two-phase system that comprised various combinations of polyethylene glycol (PEG), NaH₂PO₄ and NaCl. Partition of the enzyme depended upon the molecular mass of the PEG and the presence of NaCl. With PEG 1500, 4000 and 6000, the partition coefficients were increased by 1,500-, 1,800- and 560-fold compared to values without NaCl. The presence of NaCl (8.75%, w/w) increased purification by 3.8, 9.5 and 2.8 times into these respective PEGs. The optimal aqueous two-phase system for acid protease purification was developed using response surface methodology. This system contained 17.3% of PEG 4000 (w/w), 15% NaH₂PO₄ (w/w) and 8.75% NaCl (w/w) and provided the best partition coefficient (Ke > 1,100) and yield over 99% in the same phase. The optimal ATPS purification factor of acid protease was over 5.     

Aqueous micellar systems in membrane protein crystallization. Partial miscibility of a nonionic surfactant in the presence of salt or polyethylene glycol

The effects of NaCl and polyethylene glycol (PEG) 4000 on the lower consolution boundary (LCB) of a nonionic surfactant (C8E5) were studied and compared. Micellar systems were NaCl and PEG 4000 are present are often used in membrane protein crystallization. While sodium chloride shifts the surfactant LCB to lower temperatures without a significant change in the shape of the boundary, PEG produces a large solubility change strongly depending on the surfactant concentration. The salt effect is explained by a reduced interaction of the micellar oligooxyethylene chains with the water and the PEG effect by an unfavourable configurational interaction between the C8E5 micelles and PEG molecules.     

Lipase immobilization on magnetic microspheres via spacer arms: Effect of steric hindrance on the activity

Poly(styrene-acrylic acid) magnetic microspheres with an average diameter of 2 μm were successfully prepared and used as carriers to immobilize lipase. Lipase immobilized on microspheres with no spacer arm exhibited low activities, which were attributed to steric hindrance on the lipase conformation. To avoid steric effects, ethylenediamine and poly(ethylene glycol) (PEG) 400/800/4000 were utilized as spacer arms to bind the lipase to the microspheres. The immobilized lipase activities were improved using PEG 800/4000 as a spacer arm. Furthermore, the influence of enzyme loading on lipase activity was investigated, and the results indicated that enzyme overloading could exert steric effect on lipase activity. The degree of PEG modification was demonstrated to affect lipase activity because excess PEG on the surface of microspheres could interact with lipase due to its mobility, consequently reducing lipase activity.