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

Abstract

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 NH 2 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 K cat(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.