Biotechnology

β-aspartyl compounds are formed by an amidation reaction between the β-carbonyl group of asparagine and amine. Although β-l-Asp-l-Lys and β-l-Asp-l-Arg have a moisturizing effect on the skin and hair, bioactive β-aspartyl compounds are little known. On the other hand, various kinds of bioactive γ-glutamyl compounds such as theanine and glutathione have been reported. Given that the β-aspartyl group and the γ-glutamyl group have similarities in structure, we propose that novel bioactive β-aspartyl compounds can be existed. In order to explore this possibility, it is necessary to establish a method for producing β-aspartyl compounds. In this study, we focus on asparagine synthetase from Streptococcus thermophilus LMG 18311 (StAS) and examined whether β-aspartyl compounds can be synthesized by amidification of l-aspartic acid using StAS.

The expression plasmid was constructed by inserting StAS gene into pET-28a. Escherichia coli BL21 (DE3) transformed with the plasmid was cultured in LB medium in the presence of isopropyl β-D-thiogalactoside at 37°C. The His-tagged StAS was purified by Ni-affinity and DEAE column chromatography to electrophoretic homogeneity. The reaction products were measured by HPLC for enzyme activity. The optimum pH and reaction temperature were found to be 7.5 and 37ºC, respectively. In addition, over 80% of the activity still remained after 10 min preincubation at 30-40°C or pH 5.5-9.0. StAS showed substrate specificity not only towards ammonia but also towards hydroxylamine and methylamine as amine donors, demonstrating that StAS can be used for the production of various amine-derived β-aspartyl compounds.