N-acetylmuramoyl-l-alanine amidase assay based on specific radioactive labeling of muropeptide l-alanine: Quantitation of the enzyme activity in the autolysin deficient Bacillus subtilis 168, flaD strain
✍ Scribed by Philippe Margot; Claude-Alain H. Roten; Dimitri Karamata
- Publisher
- Elsevier Science
- Year
- 1991
- Tongue
- English
- Weight
- 468 KB
- Volume
- 198
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A sensitive and highly reproducible assay for N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) was devised, based on specific and homogeneous L-[14C]alanine labeling of the substrate, the peptidoglycan. The method involves partial purification of both the enzyme and the substrate and monitoring the muropeptide cleavage by coupling fluorodinitrobenzene to freed L-alanine NH2 groups. After acid hydrolysis of the substrate, the resulting DNP-L-alanine and L-alanine are separated by TLC, and radioactive counts in relevant spots are determined. Application of the method to the autolysin-endowed strain and an autolysis-deficient flaD-bearing mutant has revealed (i) that the N-acetylmuramoyl-L-alanine amidase behaves like an endoenzyme with an apparent Kcat(s-1) of 40, and (ii) that the residual enzyme activity in the flaD bearing strain amounts to 2.5 (+/- 0.1)% of that of the parent strain.