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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


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



Analytical Biochemistry 198(1): 15-18



ISSN/ISBN: 0003-2697

PMID: 1686374

DOI: 10.1016/0003-2697(91)90499-j

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 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 that the N-acetylmuramoyl-L-alanine amidase behaves like an endoenzyme with an apparent Kcat(s-1) of 40, and that the residual enzyme activity in the flaD bearing strain amounts to 2.5 (.+-. 0.1)% of that of the parent strain.

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Accession: 007581403

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