Over-production, Purification and Properties of the Uridine-diphosphate-N -Acetylmuramate: l-alanine Ligase from Escherichia coli
In: European Journal of Biochemistry, Jg. 230 (1995-05-01), S. 80-87
Online
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Zugriff:
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-beta-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-N-acetylmuramic acid, L-alanine and ATP/Mg2+ were estimated at 100, 20 and 450 microM, 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.
Titel: |
Over-production, Purification and Properties of the Uridine-diphosphate-N -Acetylmuramate: l-alanine Ligase from Escherichia coli
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Autor/in / Beteiligte Person: | Parquet, Claudine ; Blanot, Didier ; Jean van Heijenoort ; Liger, Dominique ; Masson, Anne |
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Zeitschrift: | European Journal of Biochemistry, Jg. 230 (1995-05-01), S. 80-87 |
Veröffentlichung: | Wiley, 1995 |
Medientyp: | unknown |
ISSN: | 1432-1033 (print) ; 0014-2956 (print) |
DOI: | 10.1111/j.1432-1033.1995.0080i.x |
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