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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.76, #1)
Modification of β2 I by glutardialdehydeI by glutardialdehyde by Margaret Galazka; Michelle Tang; Vincent A. DeBari; Joseph D. Kohles; Jung K. Lee; Lynn B. Keil; Matthew Petersheim (pp. 1-13).
Autoantibodies from patients with antiphospholipid syndrome (APS) recognize an epitope on β2glycoprotein I (β2GPI) only when native β2GPI is adsorbed on surfaces composed of anionic phospholipids or oxidized polystyrene, β2GPI was modified with the crosslinking agent, glutardialdehyde (GDA), which induced exposure of the anti-β2GPI epitope at GDA: β2GPI mol ratios in the range of 500–2000. A second crosslinking agent, dimethyl-suberimidate (DMS), did not expose the epitope, which may be a consequence of its having less tendency than GDA to form intermolecular links. SDS-PAGE experiments demonstrate that GDA does promote extensive intermolecular crosslinking of β2GPI, and DMS does not. Formaldehyde also reacts with the lysine residues of β2GPI, but does not expose the epitope. The circular dichroism spectra of native and modified β2GPI confirm that GDA induces changes in conformation that are qualitatively different from those caused by formaldehyde. These data provide evidence that binding of lysine residues is not a sufficient condition for exposure of the autoepitope, and also support the likelihood that β2GPI antibodies bind only to aggregates of the protein. Thus, by synthesizing an active holoantigen of β2GPI, conditions were defined that are necessary for binding of human autoantibodies. The authors also suggest that treatment of phospholipid-binding proteins with chemical agents might provide a strategy to modify their structure and permit exposure of epitopes, resulting in synthetic antigens for therapeutic and diagnostic use.
Keywords: Antiphospholipid syndrome; β2glycoprotein I; autoimmune diseases; protein crosslinking; circular dichroism; glutardialdehyde
Anaerobic treatment of low-strength brewery wastewater in expanded granular sludge bed reactor by Mario T. Kato; Salih Rebac; Gatze Lettinga (pp. 15-32).
Anaerobic treatment of low-strength brewery wastewater, with influent total chemical oxygen demand (COD) (CODin) concentrations ranging from 550 to 825 mg/L, was investigated in a pilot-scale 225.5-L expanded granular sludge bed (EGSB) reactor. In an experiment in which the temperature was lowered stepwise from 30 to 12°C, the COD removal efficiency decreased from 73 to 35%, at organic loading rates (OLR) of 11–16.5 g COD/L/d. The applied hydraulic retention time (HRT) and liquid upflow velocity (V up) were 1.2 h and 5.8 m/h, respectively. Under these conditions, the acidified fraction of the CODin varied from 45 to 90%. In addition to the expected drop in reactor performance, problems with sludge retention were also observed. In a subsequent experiment set at 20°C, COD removal efficiecies exceeding 80% were obtained at an OLR up to 12.6 g COD/L/d, with CODin between 630 and 715 mg/L. The values of HRT and V up applied were 2.1–1.2 h, and 4.4–7.2 m/h, respectively. The acidified fraction of the CODin wasabove 90%, but sludge washout was not significant. These results indicate that the EGSB potentials can be further explored for the anaerobic treatment of low-strength brewery wastewater, even at lower temperatures.
Keywords: Anaerobic treatment; expanded granular sludge bed reactor; brewery wastewater; acidified low-strength wastewater; low temperature
Purification and properties of three cellobiases from Aspergillus niger A20 by Mohamed A. Abdel-Naby; Mona Y. Osman; Ahmed F. Abdel-Fattah (pp. 33-44).
Three cellobiases, here called cellobiase A, B, and C, from the culture filtrate of Aspergillus niger A20, were purified by precipitation with ammonium sulphate, gel filtration through Sephadex G-75, and column chromatography of DEAE-cellulose. The purified enzymes were homogeneous on polyacrylamide disk electrophoresis. The mol wt of the purified enzymes were estimated by SDS-gelelectrophoresis to be 88,000, 80,000, and 71,000 for cellobiases A, B, and C, respectively. The enzymes were active at pH 4.5 and 55–60°C. The pattern of their aminoacid compositions showed high contents of aspartic acid, glutamic acid, threonine, serine, and glycine. The apparent Km values for cellobiose were 0.9, 1.63, and 1.0 mM for cellobiases A, B, and C, respectively. Calcium ions stimulated cellobiases B and C, and Co2+ and Mg2+ ions stimulated cell obiase A. The purified enzymes hydrolyzed cellobiose and aryl-β-d-glucosides, but they had no action on sucrose, maltose, and cellulose. The three cellobiases catalyzed transglycosylate reaction, and the major product formed from cellobiose was tetramer of glucose.
Keywords: Aspergillus niger ; cellobiase; purification; properties
Effect of thermosensitive matrix-phase transition on urease-catalyzed urea hydrolysis by Nikolay L. Eremeev; Alexandr V. Kukhtin; Eugenia A. Belyaeva; Novella F. Kazanskaya (pp. 45-55).
Temperature dependencies of kinetic and equilibrium parameters of urea hydrolysis catalyzed by native urease and the urease immobilized in a thermosensitive poly-N-isopropylacrylamide gel have been studied. The swelling ratio of the collapsed urease-containing gel is shown to increase in the presence of urea. Below a lower critical solution temperature (LCST) of the polymer, the immobilized u reaseactually has thesame catalytic properties as the native enzyme. At temperatures above LCST, the observed catalytic activity of the immobilized enzyme depends chiefly not only on the thermoreversible matrix state, but also on gel water content.
Keywords: Enzymekinetics; urease; urea hydrolysis; thermoreversible hydrogels; phase transition; enzyme activity thermoregulation
Studies on alkaline protease produced by Bacillus sp. NG312 by S. Jasvir; Navdeep Gill; Gina Devasahayam; Debendra K. Sahoo (pp. 57-63).
An alkalophilic hyperproducer of alkaline protease, Bacillus sp. NG312, was isolated, and the enzyme showed maximum activity at pH 11.0 and 60°C. The temperature optimum was increased by 10°C in presence of Ca2+. The crudeenzyme was found to have half-life of 11 d at 37°C and maximum stability at pH 9.0–10.0. It also exhibited very good stability in presence of detergent components and some locally available commericial detergent powders.
Keywords: Alkaline protease; Bacillus ; characterization; detergent compatibility