Contribution of endopeptidases to the formation of nonprotein nitrogen during ensiling of alfalfa

The experiment was conducted to investigate endopeptidase classes in alfalfa (Medicago sativa L.) leaves and to determine their contribution to the formation of nonprotein N (NPN) components during ensiling. Four classes of inhibitors (i.e., phenylmethanesulfonyl fluoride (PMSF, serine peptidase inhibitor), 1,10-phenanthroline (metallo peptidase inhibitor), pepstatin A (aspirate peptidase inhibitor) and E-64 (cysteine peptidase inhibitor)) were examined. To determine the contribution of each endopeptidase to the formation of NPN products, green alfalfa extract was fermented to imitate the proteolytic process of ensiled alfalfa and to ensure that each class of endopeptidase inhibitor would have immediate contact with the alfalfa proteases in the alfalfa. Four classes of endopeptidases, namely serine, metallo, aspartic and cysteine peptidase, were shown to be in alfalfa leaves, each playing a different role in alfalfa protein degradation. Metallopeptidase inhibitor effectively inhibited the formation of NH3-N during ensiling of alfalfa. Among the four classes of endopeptidases, the metallopeptidase was the principal peptidase in the formation of free amino acid N (AA-N) while aspartic peptidase made little contribution to AA-N. Serine and metallo peptidase contributed to the degradation of peptides into free AA, and degradation of protein into oligopeptides was mainly due to aspartic and cysteine peptidases. Metallo and cysteine peptidases were the principle peptidases for hydrolyzing forage protein into NPN during ensiling, and treatment with the mixture of four inhibitors reduced the total NPN content in the fermented alfalfa extract to about 44% of that in the control after 14 d of fermentation.