Aspartases: Molecular structure, biochemical function and biotechnological applications

Abstract

The experimental finding that a unique enzyme in bacteria mediates an equilibrium between L-aspartic acid, fumaric acid and ammonia was first reported by Quastel and Woolf (Quastel and Woolf, 1926) using cultures of E. coli shifted to resting phase by the addition of propyl alcohol or toluene. Further characterization determined that this enzyme was capable of catalysing the deamination of L-aspartate, and it was named aspartase in 1929 (Cook and Woolf, 1928). Its isolation was successfully achieved from cell extracts of Pseudomonas fluorescens (Virtanen and Tarnanen, 1932), and was subsequently followed by a series of studies performed by Ellfolk describing the initial characterization of this activity which involved its partial purification (Ellfolk, 1953a) and the determination of its substrate specificity (Ellfolk, 1954) and chemical modification (Ellfolk, 1953b). The purification of aspartase from E. coli cells was first reported by Rudolph and Fromm (Rudolph and Fromm, 1971) and involved multiple purification steps. Subsequent refinements in the protocol gradually succeeded in improving both the yield and purity of the enzyme, and an important addition to the purification protocol, dye-ligand chromatography using Red-A agarose and elution with 1mM L-aspartate (Karsten et al., 1985), allowed researchers to obtain pure aspartase in very high yields. The cloning of the structural gene of this enzyme was successful for a variety of bacterial species including E. coli (Guest et al., 1984) and Pseudomonas, figure presented fluorescens (Takagi et al., 1986), and overproduction of the gene product in suitable hosts followed soon after. At present, researchers are able to obtain highly purified samples of aspartase from a variety of organisms using the combination of overproducing strains and dye-ligand chromatography. A comparatively recent development has been the isolation and characterization of aspartases from a number of extremophiles, for example the moderately thermophilic Bacillus species Bacillus sp. YM55-1 (Kawata et al., 1999) and the psychrophile Cytophaga sp. (Kazuoka et al., 2003). The aspartases isolated from these exotic sources possess numerous unique characteristics compared to the aspartases from mesophilic sources. Details will be given in the following sections. © 2007 Springer.