Heterologous Expression and Partial Characterization of a New Alanine Dehydrogenase from Amycolatopsis sulphurea

Abstract

A novel alanine dehydrogenase (AlaDH; EC.1.4.1.1) was isolated from Amycolatopsis sulphurea and the AlaDH gene was cloned into a pET28a(+) plasmid and expressed in E. coli BL21 (DE3). The molecular mass of this enzyme was calculated as 41.09 kDa and the amino acid residues of the pure protein indicated the presence of N terminus polyhistidine tags. Its enzyme kinetic values were K_m 2.03 mM, k_cat 13.24 (s^-1), and k_cat/K_m 6.53 (s^-1 mM^-1). AlaDH catalyzes the reversible conversion of L-alanine and pyruvate, which has an important role in the TCA energy cycle. Maximum AlaDH activity occurred at about pH 10.5 and 25 °C for the oxidative deamination of L-alanine. AlaDH retained about 10% of its relative activity at 55 °C and it remained about 90% active at 50 °C. These findings show that the AsAlaDH from A. sulphurea has the ability to produce valuable molecules for various industrial purposes and could represent a new potential biocatalyst for biotechnological applications after further characterization and improvement of its catalytic properties.

Keywords: Alanine dehydrogenase; Amycolatopsis sulphurea; L-alanine; Oxidative deamination.