Synthesis of water-soluble cationic cellulose derivatives with tertiary amino groups

Water-soluble cellulose derivatives with tertiary amino groups up to substitution degree 0.8 (4.2% of coupled nitrogen) were prepared in a controlled manner by the interaction of cellulose acetate with N,N-diethylepoxypropylamine. It was shown that two reactions take place simultaneously, i.e., hydrolysis of acetyl groups and aminoalkylation of free hydroxyl groups of cellulose. The amino groups coupled to the cellulose are of middle basicity with pK _α ~9.5. Solubility of the products was found to be determined by the chemical composition of the cellulose derivative.     

Man-tailored cellulose-based carriers for invertase immobilization

Cellulose-based carriers Granocel were specially prepared and optimised for covalent immobilization of enzymes. The effects of carrier characteristics such as pore size, chemistry of anchor groups and their density on invertase immobilization efficiency were evaluated. It was found that the preferential adsorption/binding of the enzyme to a carrier during coupling and its activity after immobilization depended on microenvironmental effects created by hydrophilic surface of the carrier, functional groups and their activators. The best preparations (activity approx. 300 U/mL, high storage stability) were obtained for NH₂-Granocel activated with glutaraldehyde. It is probably due to Granocel modification with pentaethylenehexamine that gave a 19-atom spacer arm. The enzyme concentration in coupling mixture was optimised as well. The kinetic parameters of sucrose hydrolysis for native and immobilized invertase were evaluated. Compared to the native invertase, K _m value of immobilized enzyme was only twice higher with about three times lower substrate inhibition. Reaction runs in a well mixed batch reactors with native and immobilized invertase showed slightly slower reaction rate in the case of the enzyme covalently bound to Granocel. Very good stability of cellulose-based carrier was proved experimentally by 20 successive reaction runs in a batch reactor.     

Application and properties of butyl acrylate/pentaerythrite triacrylate copolymers and cellulose-based Granocel as carriers for trypsin immobilization

The main point was the search for a proper carrier and the kind of carrier activation for trypsin (EC 3.4.21.4) immobilization. The acrylic and cellulose-based carriers were specially prepared in that they possessed the most often used anchor groups: -OH, -NH(2), DEAE and/or -COOH. The immobilization procedures were selected to apply mainly to protein amine groups and appropriate anchor groups on the carrier. As activity tests low (N-benzoyl-dl-arginine-p-nitroanilide, BAPNA) and high (casein) molecular weight substrates were used. It was found, as a rule, that trypsin bound to -COOH groups with the help of carbodiimide was less active and that the amount of bound protein and measured activity (BAPNA) are considerably higher when protein is immobilized via divinyl sulfone. Both rules were observed irrespective of the nature of the polymer matrix. Both types of carriers were found suitable for trypsin immobilization and they were far better than the corresponding Eupergit C-bound enzyme preparations. Taking into account storage stability and activity for both substrates, the divinylsulfone linkage formed between unmodified Granocel and trypsin was the most effective method for the enzyme immobilization. For this preparation, BAPNA and casein conversion, thermal stability at 60 degrees C and estimated kinetic parameters were compared with those obtained for the native enzyme. It was shown that mass transport limitations could be effectively eliminated by suitable conditions and immobilized trypsin was considerably more stable. The values k(cat)/K(m) indicated that the immobilized enzyme was even better as amidase activity was regarded and its potential for protein hydrolysis was only less than twice.     

Interaction of cellulose-based cationic polyelectrolytes with mucin

Mucoadhesivity of water-soluble polymers is an important factor, when testing their suitability for controlled drug delivery systems. For this purpose, the interaction of new cationic cellulose polyelectrolytes with lyophilized mucin was investigated by means of turbidimetric titration, microscopy and measurement of zeta potential and particle size changes in the system. Results show that the cellulose derivatives interact with mucin. This interaction became stronger if cellulose macromolecules contained positively charged groups and an electrostatic interaction with the negatively charged mucin particles occurred. Under certain conditions flocculation of mucin particles by the cellulose polyelectrolyte was observed.     

A search of an optimal carrier for immobilization of chitin deacetylase

Chitin deacetylase is an enzyme that can play an important role in enzymatic deacetylation of chitosan to obtain polymers with a lower degree of acetylation. As this enzyme has never been immobilized up to now, efforts were directed towards determining both the most suitable carrier and the best method of covalent attachment to the selected carrier. In the preliminary experiments several different carriers were tested that were based on acrylic, silica-gel, agarose, dextran or cellulose materials. The best results were obtained for cellulose-based Granocel matrix. DEAE- and NH₂-Granocel activated with divinyl sulfone or glutaraldehyde were chosen for optimization of the immobilization procedure and the carrier’s superstructure. It was found that covalent binding of chitin deacetylase on DEAE-Granocel-2000 via divinyl sulfone offers preparations with the highest activity and stability. The characteristics of the selected preparation and comparison with the native enzyme show that optimal conditions are close to those for the free enzyme: the optimal pH is 4.0 for both enzymes and the optimal temperatures are 55 °C and 50 °C for native and immobilized forms, respectively. The kinetics of chitosan deacetylation for both enzymes follow the Michaelis–Menten relationship, but significant differences in the values of the equation parameters were observed.     

LC Separation of Fatty Acid Ceramides Using a Two Column System

Type II ceramides were separated according to the length of their fatty acid alkyl chain using two column system. The packing of the first column was non-polar adsorbent prepared by coating of macroporous spherical silica with cationized poly(vinyl alcohol) of low substitution degree. Commercial normal phase column Lichrosorb Si 60 was used as a second column in this system. Elution was performed in an isocratic mode using methanol:chloroform 50:50 (v/v) as an eluent.     

Immobilized Stationary Phases for Hydrophobic Interaction Chromatography of Proteins

Immobilized stationary phases for hydrophobic interaction chromatography (HIC) of proteins are prepared by coating macroporous silica Daisogel of different porosity with hydrophobized cellulose derivatives. The polymer adsorbed on the silica surface afterwards was cross-linked with bifunctional compounds. A uniform polymer nanocoating was indicated using the nitrogen gas adsorption method BET and scanning electron microscopy. The absence of non-specific protein sorption of the synthesized adsorbents shows that the developed polymeric coating isolates silica surface from contact with the sorbate. The retention data of bovine serum albumin (BSA) in the HIC mode on different synthesized adsorbents were evaluated. It was shown that sorption capacity of such phases may vary over a wide range and depends mainly on the substitution degree of the immobilized polymer. The dynamic sorption capacity of BSA was up to 63 mg mL⁻¹. The results proved that the new stationary phases have significant promise for the separation and purification of proteins in the HIC mode.

     

Influence of Surface-Active Compounds on Starch Dispersion in Water. Part I. Starch Pasting

The influence of native lipids and additives of surface-active compounds on starch paste rheology was investigated. The aim of the study was to gain better understanding of mechanisms involved in starch gelatinization and how these structure changes of granules later affect rheological properties of pastes and gels. Starches from three main sources—potato, maize, and wheat—were tested; sodium dodecylsulfate, oleate, and benzalkonium chloride were employed as additives. Starch pasting was examined by a rheometer to get a viscosity profile, also pastes were analyzed by differential scanning calorimetry, for particle size using a light scattering technique. Results revealed that there was a competition between native lipids and added surfactants for amylose complexation. Complexes formed during gelatinization were strongly affecting granule swelling and dissolution of starch polymers, and viscosity of pastes was mainly dependent on the particle size of a disperse phase in the paste. Addition of strong ionic surfactants to cereal starches resulted in smaller granular remnants and, therefore, decreased viscosity, while the weak anionic surfactant promoted an increase in the particle size and paste viscosity for both cereal and tuber starches. The mechanism of the effect of surfactants on the particle size in pastes is discussed.