Bactericidal action of egg yolk phosvitin against Escherichia coliunder thermal stress.

Chicken egg yolk phosvitin showed a remarkable antibacterial effect against Escherichia coli under thermal stress at 50 degrees C. E. coli cells (10(6)/mL) completely disappeared in 1 mL of L-broth coexisting with 0.l mg/mL phosvitin when incubated at 50 degrees C for 20 min, whereas a considerable amount of cells (10(5)/mL) survived at the same thermal stress without phosvitin. Blocking of the chelating effect of phosvitin by the addition of Ca(2+) ion displayed a protective effect against the bactericidal activity at 50 degrees C. In addition, the antibacterial activity of phosvitin was dramatically reduced by treatment with alpha-chymotrypsin, although the chelating effect remained. The surface properties, such as interfacial tension and emulsifying properties of phosvitin, which are an index of the affinity with the outer membrane, were greatly reduced by the alpha-chymotrypsin digestion. This indicates that the alpha-chymotrypsin-digested membrane-penetrating hydrophobic domains at the N- and C-terminal regions play an important role in antibacterial activity. These results suggest that a significant part of the bactericidal activity of phosvitin against E. coli resides in the synergistic effect of the high metal-chelating ability and the high surface activity under the influence of thermal stress.

Lysyl-tRNA synthetase of Bacillus stearothermophilus molecular cloning and expression of the gene.

The gene of the lysyl-tRNA synthetase of Bacillus stearothermophilus NCA1503 was cloned and sequenced. The gene consists of 1485 bp nucleotides commencing with an ATG start codon and ending with a TAA stop codon, and encodes a polypeptide of 493 amino acids. The recombinant enzymes were expressed in E. coli using an expression plasmid containing the T7 RNA polymerase/promoter.

Lysyl-tRNA synthetase from Bacillus stearothermophilus. Stopped-flow kinetic analysis of enzyme.lysyladenylate formation.

Amino acid activation reaction of the lysyl-tRNA synthetase [L-lysine:tRNALys ligase (AMP forming); EC 6.1.1.6] from Bacillus stearothermophilus was studied fluorometrically by the stopped-flow method. The addition of L-lysine to the enzyme solution caused quenching of the protein fluorescence and the subsequent addition of ATP restored the quenched fluorescence [Takita et al. (1996) J. Biochem. 119, 680-689; Takita et al. (1997) 121, 244-250]. In the stopped-flow analysis, however, the former fluorescence change (quenching) could not be detected, while the latter change (restoration) was detectable. The L-lysine binding process was suggested to be much faster than the ATP binding process, being completed within the dead-time of the apparatus, ca. 3 ms. The hyperbolic dependence of kapp on the initial ATP concentration suggested that the ATP binding to the enzyme.L-lysine complex followed a two-step mechanism. Two L-lysine analogues that exhibit the qualitatively similar behavior to L-lysine in the fluorometric titration, L-lysine hydroxamate and L-lysine amide, were examined similarly. The two-step process was also suggested for these analogues, and the forward rate constant in the rate-determining step for L-lysine amide (221+/-7 s-1) was significantly larger than those for L-lysine (45.7+/-4.6 s-1) and L-lysine hydroxamate (14. 5+/-1.7 s-1) at pH 8.0, 30 degrees C.

Isolation of bovine immunoglobulin G subclasses from milk, colostrum, and whey using immobilized egg yolk antibodies.

Immunoaffinity columns were made with specific egg yolk immunoglobulin (Ig) Y against bovine IgG1 and IgG2 and were used to isolate pure IgG1 and IgG2 from Cheddar cheese whey or colostrum. About 10% of the IgY was specific for IgG, and 3% of the IgY was subclass-specific after hyperimmunization of laying hens with either IgG1 or IgG2. Up to 38% of the potential binding capacity of IgY was obtained after immobilization by reductive amination. The IgY columns were stable, and one column could be reused for more than 50 times for over a year with minimal loss in binding capacity. Milk that was free of either IgG subclass was successfully produced by the selective removal of IgG1 or IgG2 subclasses. Double-immunodiffusion analysis confirmed the isolation of subclasses from whey and colostrum and also confirmed that their removal from milk was specific.

Production and purification of Fab' fragments from chicken egg yolk immunoglobulin Y (IgY).

Methods were described for the production of Fab and Fab' fragments from chicken egg yolk IgY also referred to as IgG by papain and pepsin digestion respectively. Pepsin digestion was found to be suitable for the large scale preparation and purification of Fab'. Optimum yield of Fab' was obtained after peptic digestion of IgY at pH 4.2 for 9 h at low NaCl concentration. This condition led to the complete digestion of pFc' fragment leaving only the Fab' fragment. By combination of ultrafiltration and anion exchange, and conditions which allowed binding of the small amount of contaminants in the digest to the anion exchange column, pure Fab' fragments were easily obtained in the eluent. The advantage of this approach is that a small column could be used to purify large amount of protein, therefore, improving the efficiency of purification. The Fab and Fab' fragments appeared to be similar on the basis of their molecular weights as determined by SDS-PAGE, reaction of identity in immunodiffusion assay and similar antigen binding activities as shown by ELISA.

Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain.

The importance of eggs as a source of specific antibodies is well recognized. Egg yolk contains 8-20 mg of immunoglobulins (IgY) per ml. However, the major problem in isolation is removal of lipids which are present in high concentrations. A method had been developed by employing water dilution to separate the yolk plasma proteins from the granules and lipids. Further purification of IgY from plasma proteins was achieved by a protocol involving salt precipitation and ultrafiltration. The water dilution method (WD) was compared with three other methods, namely, polyethylene glycol (PEG), dextran sulphate (DS) and xanthan gum (Xan) in terms of yield, purity, ease of use, potential scaling up and immunoactivity of IgY. The WD method gave the highest yield, followed by DS, Xan and PEG methods in that order. 9.8 mg IgY/ml egg yolk was routinely obtained from the WD method compared to 4.9 mg IgY/ml egg yolk with the popular PEG method with purities of 94% and 89% respectively. Purification methods had no adverse effect on the immunoactivities of IgY. WD was also found superior in terms of ease of use and large scale production of IgY. WD method therefore provides a simple, rapid and efficient means of purifying IgY with high activity.