Improvement of biogas production from vinasse via covalently immobilized methanogens.

Improvement of biogas production was realized by covalent immobilization of a methanogenic consortium onto a granulated polymeric support [poly(acrylonitrile-acrylamide)]. The growth kinetics of the immobilized consortium was investigated during a process of vinasse methanation, and a cell concentration increase from 12.3 mg g(-1) support to 52.1 mg g(-1) support was established. The methane yield reached 0.33 m3 kg(-1) CODr, the maximum yield on chemical oxygen demand (COD) removal being 92%. The inhibitory effect of oxygen was reduced by immobilizing the methanogenic consortium.

Crystallization of type I chloramphenicol acetyltransferase: an approach based on the concept of ionic strength reducers.

Chloramphenicol acetyltransferase (CAT) is responsible for bacterial resistance to chloramphenicol. It catalyzes inactivation of the antibiotic by acetyl-group transfer from acetyl CoA to one or both hydroxyl groups of chloramphenicol. Type I CAT possesses some unique properties which are not observed in other CAT variants. Type I CAT overexpressed in Escherichia coli was purified and crystals with a resolution limit of 2.22 A have been obtained using a novel procedure which is based on the concept of 'ionic strength reducers'. The crystals have the symmetry of space group P1 and unit-cell parameters a = 96.46, b = 113.86, c = 114.21 A, alpha = 119.9, beta = 94.1, gamma = 98.6 degrees. These dimensions are consistent with four to six trimers per unit cell, corresponding to a solvent fraction ranging from 65 to 47%.

Multienzyme membranes for biosensors.

Artificial multienzyme complexes were prepared in which enzymes were covalently bound to polysaccharide structures activated with urea and formaldehyde. Double enzyme complexes of glucose oxidase and catalase, a glucose oxidase and invertase, were prepared by immobilization on to cellulose fabric. Also, catalase was covalently bound to soluble dextran. The resulting multienzyme systems were highly active and stable, making them suitable for use in measuring the concentrations of glucose and saccharose in solutions. The measurements were performed using an amperometric oxygen electrode and multienzyme membranes containing glucose oxidase and catalase for the first substrate, as well as glucose oxidase bound to cheese-cloth and a 'liquid' membrane of dextran-bound catalase. To determine the concentration of saccharose, a multienzyme membrane with bound glucose oxidase and invertase was used in combination with a 'liquid' dextran-catalase. The enzyme electrodes exhibited a measuring range of 0.1-5 mol dm-3 and a response time of 2-3 min. The electrodes may be used for measuring saccharose and glucose concentrations both in fermentation broths and food products.

Covalent binding of enzymes to synthetic membranes containing acrylamide units, using formaldehyde.

Synthetic membranes containing 10% acrylamide units were subjected to activation with formaldehyde at pH 7.5 and 45 degrees C. Trypsin, invertase, and urease were bound to this activated membrane and the kinetic properties of immobilized enzymes were studied. The permeability of the membrane for distilled water manifests certain differences depending on the enzyme bound. The membranes with immobilized enzymes stored at 4 degrees C in a moist state showed no change in their activity for 6 months. The membrane with immobilized invertase has preserved its activity even after 20 operations with 2% sucrose solution at 25 degrees C. The proposed method of binding enzymes to synthetic membranes containing acrylamide groups, through the introduction of N-hydroxymethyl groups, possesses several advantages with respect to the activation of the membrane in a one-step reaction with cheap and accessible reagent, high operative stability of the immobilized enzymes, no danger of bacterial rotting, and long shelf life of the membrane.

Properties of chymotrypsin bound covalently to dextran.

The kinetic properties dextran-chymotrypsin conjugate were studied by means of low molecular weight substrates. It was found that KM, kcat and kcat/KM of dextran chymotrypsin for the hydrolysis of benzoyl-L-tyrosine-ethyl-ester did not differ substantially from those of the free enzyme. However, the data found for kcat of dextran-chymotrypsin and free chymotrypsin assayed for the hydrolysis of three tripeptidyl-p-nitroanilide D-Arg-Val-Trp-pNA, D-Arg-Val-Tyr-pNA, Z-Phe-Pro-Phe-pNA, were definitely different. The inhibition of the modified chymotrypsin with soybean trypsin inhibitor was found to be less pronounced than that with the free enzyme. The effect of potassium and magnesium salts on the inactivation of both enzymes was also studied. The effect of dextran matrix on the catalytic properties and the conformational stability of modified chymotrypsin is discussed.

New method for covalent immobilization of proteins to cellulose and cellulose derivatives.

Cellulose and microcrystalline cellulose are treated consecutively with sodium periodate and urea. The interaction of urea derivatives with formaldehyde results in highly reactive groups, capable of further condensation with the amino acid residues of the proteins. The condensation of chymotrypsin, pepsin, and ovomucoid with such activated matrices has been studied in the pH interval 2 to 10. Differences have been found in the binding properties of basic and acid proteins. Satisfactory values have been obtained concerning the relative enzymatic and inhibitory activity of the immobilized products with respect to high- and low-molecular substrates. Chymotrypsin, immobilized on microcrystalline cellulose matrix, is found to manifest better catalytic properties compared with chymotrypsin immobilized on cellulose matrix. A probable sequence of the stages of chemical activation of the matrices and covalent binding of the proteins to them has been proposed. The main advantages of the proposed method consist of the high reactivity of the binding group in a wide pH range, its suitable length, and its easy synthesis.

Structural studies on periodate-oxidized chicken ovomucoid. Spectral behaviour of the tyrosine residues.

About 50% of the carbohydrate moiety of ovomucoid was destroyed by periodate oxidation. The oxidation was carried out for 6 h or 24 h. The data obtained showed that in the carbohydrate chain 2-5 glucosamines and 1-2 neutral sugar residues were decomposed with the consumption of 16 mol and 29 mol of periodate respectively. Periodic oxidation slightly changed the inhibitory activity of the ovomucoid, but altered its spectral properties. An increase of the absorption maximum at 278 nm was noted, as well as a tendency for normalization of phenolic ionization and an increase of the relative fluorescence. The reactivity of tyrosine residues towards tetranitromethane is also changed. It was suggested that even in native ovomucoid the tyrosines could be regarded as 'dissolved' in the 'carbohydrate solvent'. This contact could be achieved by the hydrogen bonds in the formation of which the NHCOCH3 groups of the glucosamine residues play an essential role. Peroxidate oxidation seems to lead to an alteration of the nature of the 'sugar solvent' and disturbs the conformation of the sugar chain.

Ultraviolet difference spectral studies of human serotransferrin and lactotransferrin.

In order to determine the conformational relationship of iron binding of human serotransferrin and lactotransferrin, ultraviolet difference spectral studies were performed in the presence of guanidine chloride and perturbants as deuterium oxide, ethylene glycol, glycerol and polyethylene glycol. In the presence of guanidine chloride solution the molar absorption differences at 292 nm of iron-saturated forms versus iron-free forms of human serotransferrin and lactotransferrin are respectively -16000 +/- 1000 and -14000 +/- 775. These modifications may be attributed to the involvement of tryptophan residues in the iron-binding sites of the two proteins. However, the results do not demonstrate that these tryptophan residues are bound directly to iron. Difference spectral studies in the presence of perturbants show that the apparent exposed tryptophan and tyrosine residues are higher with shorter range perturbants in iron-free forms of both transferrin molecules. The most important modification of exposed tyrosine residues has been noticed upon removing iron from human lactotransferrin than from serotransferrin.