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This paper describes and compares three chromatographic methods for the analysis of egg-white proteins. Gel-permeation chromatography allowed the separation of seven peaks from egg white, with an almost total protein recovery. A clean separation of ovomucin and lysozyme from the bulk of the proteins was obtained with this method. Reversed-phase high-performance liquid chromatography led to the fractionation of at least eight peaks. With this chromatographic method, the recovery was relatively poor. Approximately 30% of the ovalbumin was retained in the column after the elution. Finally, eleven chromatographic peaks were separated from egg white by high-performance liquid chromatography on an anion-exchange column. The recovery of proteins was almost total. The latter method afforded higher resolution.

A single-step anion-exchange chromatographic separation of egg white proteins was carried out using a Q Sepharose Fast Flow column. The separation resulted in the isolation of two lysozyme peaks with purities of ca. 99 and 88%, one peak of ovotransferrin purified to ca. 75% and two ovalbumin peaks with purities of ca. 54 and 98%. Recoveries were estimated to be ca. 60, 100 and 83% for lysozyme, ovotransferrin and ovalbumin, respectively. The amino acid compositions of all collected peaks have also been determined. This confirmed the identity of some of the proteins contained in these peaks.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) is an exopeptidase commonly called PYRase, which hydrolytically removes the pGlu-proteins. pGlu also known as pyrrolidone carboxylic acid may occur naturally by an enzymatic procedure or may occur as an artifact in proteins or peptides. The enzymatic synthesis of pGlu suggests that this residue may have important biological and physiological functions. Several studies are consistent with this supposition. PYRase has been found in a variety of bacteria, and in plant, animal, and human tissues. For over two decades, biochemical and enzymatic properties of PYRase have been investigated. At least two classes of PYRase have been characterized. The first one includes the bacterial and animal type I PYRases and the second one the animal type II and serum PYRases. Enzymes from these two classes present differences in their molecular weight and in their enzymatic properties. Recently, the genes of PYRases from four bacteria have been cloned and characterized, allowing the study of the primary structure of these enzymes, and their over-expression in heterelogous organisms. Comparison of the primary structure of these enzymes revealed striking homologies. Type I PYRases and bacterial PYRases are generally soluble enzymes, whereas type II PYRases are membrane-bound enzymes. PYRase II appears to play as important a physiological role as other neuropeptide degrading enzymes. However, the role of type I and bacterial PYRases remains unclear. The primary application of PYRase has been its utilization for some protein or peptide sequencing. Development of chromogenic substrates for this enzyme has allowed its use in bacterial diagnosis.

An improved procedure is described involving gel permeation and anion-exchange chromatography for the purification of four major hen egg white proteins. The procedure involves a first-step purification of ovomucin and lysozyme by gel permeation on a Superose 6 Prep Grade column. In the second step, anion-exchange chromatography on Q Sepharose Fast Flow led to the isolation of ovotransferrin and ovalbumin from a gel permeation chromatographic peak. The purities were estimated as ca. 80, 100, 80 and 100% for ovomucin, lysozyme, ovotransferrin and ovalbumin, respectively. The purification yield was over 60% for each protein. Further characterization of purified lysozyme revealed that it was fully active and homogeneous in relation to the electrospray ionization mass spectrum. The electrospray ionization mass spectrum showed different ovotransferrin species. The amino acid composition of purified ovomucin was compared to those published previously.

Pectate lyases (PL) initiate soft-rot diseases in plants by cleaving pectin which is the major component of the plant cell wall. The present paper reports the first cloning and characterization of a pectate lyase (pel) gene from the Bacillus genus. This gene was isolated from a Bacillus subtilis genomic library constructed in pUC18 as vector and Escherichia coli as host. By Southern hybridization this gene was shown to be present in a single copy in the B. subtilis genome. The nucleotide sequence of a 1.6 kb-pair HindIII restriction fragment, which confers pectate lyase activity to E. coli, indicated a 1,260 bp open reading frame encoding a 420 amino acid polypeptide which includes a 21 amino acid signal sequence. The 45,605 Da deduced protein displays homologies to PLs from Erwinia chrysanthemi. The B. subtilis PL cloned in E. coli was located in the periplasm. It was purified to homogeneity in a one-step procedure from the E. coli periplasmic fluid after overproduction using the pT7 system. Biochemical properties of the purified enzyme were similar to those found for the PL isolated from B. subtilis extracellular media.