Over‐production, Purification and Properties of the Uridine‐diphosphate‐N ‐Acetylmuramate: l‐alanine Ligase from Escherichia coli

Abstract

The UDP‐N ‐acetylmuramate:l‐alanine ligase of Escherichia coli was over‐produced in strains harbouring recombinant plasmids bearing the murC gene under the control of the lac or trc promoter. Plasmid pAM1005, in which the promoter and ribosome‐binding site region of murC were removed and in which the gene was directly under the control of promoter trc, led to a 2000‐fold amplification of the l‐alanine‐adding activity after induction by isopropyl‐thio‐β‐D‐galactopyranoside. The murC gene product was visualized as a 50‐kDa protein accounting for approximately 50% of the cell protein. A two‐step purification led to 1 g of a homogeneous protein from an 18–1 culture. The N‐terminal sequence of the purified protein correlated with the nucleotide sequence of the murC gene. The presence of 2‐mercaptoethanol and glycerol was essential for the stability of the enzyme. The Km values for UDP‐TV‐acetylmuramic acid, l‐alanine and ATP/Mg2+ were estimated at 100, 20 and 450 μM, respectively. Under the optimal in vitro conditions a turnover number of 928 min−1 was calculated and a copy number/cell of 600 could be roughly estimated. The specificity of the enzyme for its substrates was investigated with various analogues. The enzyme also catalysed the reverse reaction. Copyright © 1995, Wiley Blackwell. All rights reserved