Preparation of superabsorbent cellulosic hydrogels

In this work, milled softwood (SW) bleached kraft fibers were crosslinked by esterification with poly(vinyl methyl ether-co-maleic acid) (PVMEMA) and polyethylene glycol (PEG). The effects of fiber length, crosslinking reaction time, and dosage of PVMEMA on water absorption and retention value (WAARV) for the crosslinked fibers were determined. The results show that as the softwood fiber length is mechanically decreased from 2.41 to 0.50 mm and employing a weight ratio of fiber to polymers equivalent to 1.00:1.28 the WAARV increased from 86.50 to 189.20 g/g. Analysis of the crosslinked fibers by SEM and light microscope indicated that the polymers and fibers form a crosslinked fibrous matrix. FT-IR spectroscopy was employed to detect the ester linkage between PVMEMA and PEG/SW kraft pulp fibers. The results suggested that the ester crosslinked pulps exhibit excellent water absorbent properties and have the potential of utilizing milled bleached SW kraft fibers, such as refiner dust or pulp fines, for novel water absorbent applications.     

Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with gamma-cyclodextrin

Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction. To manipulate the product specificity of the Paenibacillus sp. A11 and Bacillus macerans cyclodextrin glycosyltransferases towards the preferential formation of gamma-cyclodextrin (CD(8)), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies. The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD(8) showed pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases. However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were higher than those of the native cyclodextrin glycosyltransferases. When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD(8) synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased. Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced CD(6) : CD(7) : CD(8) : > or = CD(9) ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40 degrees C with starch substrates. In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD(8) and > or = CD(9), and the overall cyclodextrin yield was increased by 12% compared to the native enzymes. The crosslinked imprinted cyclodextrin glycosyltransferases also showed higher stability in organic solvents, retaining 85% of their initial activity after five cycles of synthesis reactions.     

Esterification crosslinking structures of rayon fibers with 1,2,3,4-butanetetracarboxylic acid and their water-responsive properties

1,2,3,4-Butanetetracarboxylic acid (BTCA) was applied to crosslink amorphous regions of cotton-type rayon fibers via anhydride-mediated esterification for the purpose of enhanced mechanical properties. Crosslinking was conducted under a series of curing temperature with the presence of sodium hypophosphite (SHP) as the effective catalyst for anhydride formation. The conversion rate based on Fourier transform infrared (FTIR) spectra was in good agreement with that from the direct calculation based on titrated carboxyl groups and ester linkages. Crosslinking degree increased linearly with the curing temperature. The optimal tensile properties were derived from the curing temperature of 180 °C with an intermediate crosslinking degree. Moreover, the crosslinked structures of the rayon fibers had a great impact on absorption of liquid and gaseous water. The liquid water retention capacity diminished substantially with the crosslinking degree, while interaction between moisture and the fibers revealed that sorption–desorption cycles and concomitant hystereses were scarcely affected by the crosslinked fiber structures.     

Improved biochemical characteristics of crosslinked β-glucosidase on nanoporous silica foams

Large mesoporous cellular foam (LMCF) materials were synthesized using the microemulsion templating route. For the enzyme stabilization, β-glucosidase was immobilized onto mesocellular silica foams (MCFs) in a simple and effective way, a process achieved using enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of crosslinked enzyme aggregates (CLEAs) of nanometer scale. The structural and chemical properties of these prepared materials were characterized by TG, CPMAS NMR and nitrogen adsorption measurements. The crosslinked immobilizates retained activity over wider ranges of temperature and pH than those of the free enzyme. Kinetic parameter (K_m) of the immobilized β-glucosidase is lower than that of its free counterpart. The resulting CLEA was proved to be active and recyclable up to 10 cycles without much loss in activity. This demonstrates its prospects for commercial applications. The immobilizate exhibited enhanced storage stability characteristics than the native enzyme. In contrast to adsorbed GL and covalently bound glucosidase, the resulting crosslinked enzyme aggregates (CLEAs) showed an impressive stability with high enzyme loadings.     

Peptide Synthesis Catalyzed by Crosslinked α-Chymotrypsin in Water/Dimethylformamide Solvent System

-Chymotrypsin was crosslinked to give a water-insoluble polymer by treatment with the bifunctional reagent glutaraldehyde. The specific activity of the crosslinked enzyme in aqueous media was three orders of magnitude lower than for the native chymotrypsin. In a medium containing more than 50% (v/v) of dimethylformamide the specific activities of both enzymes were comparable. In addition, the insoluble polymer was more stable in the presence of 60% (v/v) dimethylformamide compared with the native enzyme. Therefore, in this medium enzymatic peptide synthesis could be successfully accomplished with the crosslinked enzyme, but not with the same amount of native chymotrypsin.     

Thermodynamic aspects of the gelatinization and swelling of crosslinked starch

Samples of epichlorohydrin crosslinked potato starch were prepared by using a high ratio of starch to water and alkali concentration below the gelatinization level. This yielded crosslinked samples that were partially crystalline, and where the number of crosslinks could be varied between 1 and 20 crosslinks per 100 anhydroglucose units. The degree of swelling varied regularly with degree of crosslinking, and the molecular weight between crosslinks M_c as derived from swelling data in a good swelling agent compared favorably with M_c derived from chemical analysis. From the heat of gelatinization of the crosslinked starches, as observed in a differential scanning calorimeter, for gelatinization in glycerol, water, and dimethylsulfoxide, a model for the gel state of the crosslinked starch is proposed. It is concluded that the entropy of melting is the determining factor in establishing the temperature of gelatinization.     

Crosslinked polyethylenimine: An enzyme carrier with spacers of various lengths introduced in crosslinking reaction

Polyethylenimine (PEI) reacted with epichlorhydrin to various degrees of crosslinking was further activated with thiophosgene or with succinic anhydride; the carboxyl derivatives obtained were converted to hydrazide derivatives. d-Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4), glucoamylase (exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) and cholinesterase (acetyl-, butyryl-) [acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7), butyryl cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8)] were bound in their native forms to the first two types of carriers, while in the case of the hydrazide derivative of crosslinked PEI this occurred just after periodate oxidation of a glycoenzyme. The PEI derivatives prepared from the crosslinked PEI with the lowest degree of crosslinking, i.e. from that having the longest mean length of the spacer (～ 11.1 Å), revealed the best binding properties towards low-molecular thiols, amino acids and proteins/enzymes. After enzyme coupling, the isothiocyanate derivatives of crosslinked PEI gave preparations with the highest residual activities.     

Comparison of hydrolytic activity in water and heptane for thirty-two commercial lipase preparations

The protein content and the rates of hydrolysis of p-nitrophenyl palmitate (pNPP) in water (soluble enzyme and emulsified substrate) and in heptane (soluble substrate and insoluble enzyme) were measured for thirty-two commercial lipase preparations. The protein content of the powders varied in a wide range as well as the activity on emulsified pNPP showing the high heterogeneity of the commercial samples. Activity in heptane also varied but to a lesser extent than that in water. There was no direct correlation between activities in water and in heptane as assayed with the same hydrolytic reaction. The ratio of activity in heptane to that in water, R(O/A) ratio, was introduced to characterize activity in organic media. Six lipases showed R(O/A) values higher than 1 demonstrating a higher activity in organic solvent than in water. A linear correlation of R(O/A) with activity in water (log plot) suggested the strong influence of diffusional limitations on activity of solid enzyme suspended in organic solvents.     

Adsorption of humic acid from aqueous solution onto irradiation-crosslinked carboxymethylchitosan

This paper presents the adsorption of humic acid from aqueous solution onto crosslinked chitosan derivative (carboxymethylchitosan), formed by additionless irradiation technique. The surface charge and swelling properties of crosslinked samples were investigated. The adsorption of humic acid onto crosslinked carboxymethylchitosan was carried out by the batch method at room temperature, and it was found to be strongly pH-dependent. Maximum amount of humic acid was adsorbed under acidic conditions at the optimum pH value of 3.5. Adsorption kinetic studies indicated the adsorption process was transport-limited at the same pH. The adsorption isotherm analysis data under various initial humic acid concentrations confirms that experimental data fitted well into the Langmuir equation. X-ray photoelectron spectroscopy (XPS) revealed that the amino groups of carboxymethylated chitosan were protonated, suggesting the formation of organic complex between the protonated amino groups and humic acid. From these preliminary evaluations, it was concluded that crosslinked carboxymethylated chitosan derivatives have a great potential in water treatment for the removal of humic acid and other polarized or electrically charged species.     

Biodegradable superabsorbent hydrogels derived from cellulose by esterification crosslinking with 1,2,3,4-butanetetracarboxylic dianhydride

Superabsorbent hydrogels were prepared from native celluloses dissolved in lithium chloride and N-methyl-2-pyrrolidinone (LiCl/NMP) by esterification crosslinking with 1,2,3,4-butanetetracarboxylic dianhydride (BTCA). Subsequent conversion of the unreacted carboxyl groups to sodium carboxylates by the addition of aqueous NaOH was performed to enhance the water affinity of the gels. The absorbency of the products was strongly dependent on the amount of BTCA that was esterified to cellulose, and the highest absorbency was observed for the hydrogel composed of approximately 0.25 molecules of BTCA per anhydroglucose unit (AGU) of cellulose. Furthermore, it was confirmed that the absorbency was enhanced as the average degree of polymerization (DP) of the starting cellulose increased. The use of cotton cellulose with a high DP of about 2400 produced a hydrogel with an absorbency of 720 times its dry weight, which exceeded the absorbency of commercial crosslinked sodium polyacrylate superabsorbent hydrogel (SPA). The hydrogels exhibited good biodegradability, with a maximum degradation of 95% within 7 days using cellulase.     

Direct immobilization of milk xanthine oxidase in milk protein with and without addition of gelatin

Summary Milk or a solution of gelatin and milk or commercial xanthine oxidase were lyophilized and the powdered freeze-dried materials crosslinked with glutaraldehyde. The resulting immobilized xanthine oxidase preparations have a good stability, are highly active and well suited for organic synthesis.     

Biological activity in human neutrophils of di-tripeptides, analogues of the chemotactic fMLP

for-Met-Ser(for-Met-Leu-Phe)-Phe-OMe 1 and for-Met-Lys(for-Met-Leu-Phe)-Phe-OMe 2 were synthesized in order to investigate biological activities on human neutrophils of crosslinked di-tripeptides. Our results seem to highlight that the tested di-tripeptides (i) do not step up chemotaxis, (ii) can elicit superoxide anion production which is dependent on the nature of the residue at position 2, chosen in the tripeptide that is crosslinked to the fMLP-OMe.     

NADPH-cytochrome P-450 reductase-cytochrome b5 interactions: crosslinking of the phospholipid vesicle-associated proteins by a water-soluble carbodiimide

Detergent-solubilized and purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome b5 were coreconstituted into phospholipid vesicles. When the proteoliposomes were incubated with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a new higher-molecular-weight band was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The band was purified by chromatography on DEAE-Sepharose CL-6B, 2'5'-ADP-Sepharose 4B, and Sephadex G-100. The heme absorption spectrum and fluorophotometric assay of flavin of the purified material demonstrate that this product is a 1:1 crosslinked complex containing one molecule each of the flavoprotein and cytochrome. Proteolysis of the crosslinked form indicates that the hydrophilic catalytic domains participate in the covalent attachment, and that the hydrophobic membrane-attachment peptide is necessary for the protein interaction. The purified crosslinked derivative showed no activities for reduction of either cytochrome c or ferricyanide. About half of the enzyme-associated flavin was reduced rapidly by NADPH, as was 20-30% of the crosslinked cytochrome, indicating that, in at least some of the complexes, the flavin-mediated pathway for reduction of cytochrome by pyridine nucleotide was intact. These data suggest that the output- rather than the input-electron transfer site(s) in the flavoprotein was (were) blocked by the covalently attached cytochrome.     

Crosslinking of cytochrome c and cytochrome b5 with a water-soluble carbodiimide. Reaction conditions, product analysis and critique of the technique

A water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), has been used to crosslink horse heart cytochrome c and trypsin-solubilized bovine liver microsomal cytochrome b5. The reaction was conducted under a variety of solution conditions, and the products were purified by a combination of gel filtration and ion-exchange chromatography. Under all conditions of pH, ionic strength, EDC/protein ratio and reaction time that were studied, multiple 1:1 crosslinked complexes were observed with no evidence of a single, dominant species. Acetate, which is often used as a quencher of such reactions, was found to increase the complexity of the reaction products, presumably through EDC-promoted coupling to cytochrome c. Hydroxylamine treatment of the crosslinked complexes, a procedure frequently used to reverse EDC modification of tyrosyl residues, did not reduce the number of crosslinked components observed. The cytochrome b5 heme group was readily extracted from each of the 1:1 crosslinked complexes by standard techniques, so the crosslinking of heme propionate 7 with Lys79 of cytochrome c that might have been anticipated on the basis of molecular graphics modeling [Salemme, F.R. (1976) J. Mol. Biol. 102, 563-568] was not evident from this analysis. Analysis of HPLC tryptic peptide maps produced from crosslinked complexes revealed reduced specificity of trypsin in hydrolysis of EDC-crosslinked protein-protein complexes and unsatisfactory resolution of crosslinked or branched peptides. Nevertheless, it was possible to demonstrate that residues 52-72 of cytochrome b5, a region predicted to be critical to interaction with cytochrome b5 [Salemme, F.R. (1976) J. Mol. Biol. 102, 563-568] was absent from all peptide maps of 1:1 cytochrome c.cytochrome b5 complexes. Based on these results and a review of the literature involving EDC crosslinking of electron transfer proteins, we conclude that the techniques available for specific protein hydrolysis and separation of crosslinked peptides are not adequate to permit routine unambiguous identification of crosslinking sites in carbodiimide-crosslinked complexes.     

Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.     

Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.     

The properties of proteoglycan prepared from human articular cartilage by using associative caesium chloride gradients of high and low starting densities.

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves' serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes' radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.     

Effects of Crosslinking Agent-DFDNB on Energy-transducing Reactions of Chloroplast Thylakoid Membrane Proteins

The effects of crosslinking agent-DFDNB (difluoro dinitro benzene) on functions of chloroplast thylakoid membrane proteins were investigated. DFDNB inhibited activities of PSP and membrane-bound ATPase in chloroplasts. It decreased proton uptake of light-inducted chloroplast thylakoids and the relative value of fluorescence quenching of 9-aminoacridine, and inhibited the rate of fast electrogenic phase of absorption change at 515 nm in chloroplasts. In addition, the isolated CF1-ATPase was crosslinked with DFDNB. The pattern of polymers of crosslinked CF1-ATPase was observed on SDS-PAGE.     

Development of a stable and efficient zein based nanopesticides through green and simple way for improving utilization efficiency

Nanopesticides represent one of the effective ways of reducing the use of pesticides and the environmental pollution. Natural zein as a potential carrier for nanoencapsulation of pesticides has the disadvantages of large amount of organic solvents and poor dispersibility. In this study, a water soluble of zein peptides was obtained by hydrolyzed under alkaline conditions with heated and stability of nanopesticides were prepared through crosslinked after self-assemble. As the results, the zein peptides spontaneously assembled into spherical particles in an aqueous solution, the nanopesticides (avermectin, AVM), with uniform size distribution and size can be adjusted by the glutaraldehyde, have a great stability, and redispersed in water after freeze-drying. Moreover, nanoencapsulation protects avermectin from UV light, with 24% of the encapsulated AVM remaining intact after exposure to UV for 60 min, compared to less than 6% for the bare AVM. In addition, the nanoformulation had similar insecticidal activity to commercial EC and the cross-linking of particles slowed down the release rate and the curves were consistent with Higuchi model different from the abnormal diffusion in the uncross linked state. In short, this study provides a simple method to obtain stabilizable nanopesticides based on natural polymer on a green way.     

Preparation and characterization of chitosan membranes by using a combined freeze gelation and mild crosslinking method

When gelification is performed by freezing–thawing repeated cycles, the resultant gel-like polymer systems are called cryogels. This work aims to assess the effect of the addition of glutaraldehyde and 18 Crown Ether-6 on surface properties and protein loading of dried chitosan cryogel films. Residual water content of treated chitosan membranes ranged between 11.93 and 13.86%, while their water activities vary from 0.5 to 0.7 (measured from 4 to 60 °C). Based on thermal data, water evaporation peak and degradation temperatures of chitosan membranes shifted to a higher temperature for crosslinked samples. X-ray diffractograms provide high values of crystallinity for all the samples (70.67–92.86%), the highest value being for the glutaraldehyde-treated membrane. Candida rugosa lipase can be immobilized successfully on chitosan membranes. Lipase immobilized on glutaraldehyde-crosslinked chitosan yielded the highest efficiency in terms of total coupled protein and protein loading efficiency.