Substrate for L-alanine aminopeptidase. L-alanine aminopeptidase is an enzyme localized in the bacterial cell wall which cleaves the amino acid L-alanine from various peptides. Significant activity is found almost only in Gram-negative microorganisms, all Gram-positive or Gram-variable microorganisms so far studied display no or very weak activity. The aminopeptidase test thus provides a reliable method for determining the Gram behaviour of microorganisms. L-alanine-4-nitroanilide is split by L-alanine aminopeptidase into L-alanine and 4-nitroaniline. The latter compound causes the bacterial suspension to turn yellow.