Contribution of exopeptidases to formation of nonprotein nitrogen during ensiling of alfalfa

Abstract

The experiment was conducted to investigate the exopeptidase classes in alfalfa (Medicago sativa L.) leaves, and to determine their contribution to the formation of nonprotein nitrogen (NPN) components during ensiling. Six classes of inhibitors that included bestatin (aminopeptidase inhibitor), potato carboxypeptidase inhibitor (PCI, carboxypeptidase inhibitor), 1,10-phenanthroline (dipeptidase inhibitor), diprotin A (dipeptidyl-peptidase inhibitor), butabindide (tripeptidyl-peptidase inhibitor), and dipeptide PheArg (peptidyl-dipeptidase inhibitor) were used. To determine the contribution of each exopeptidase to the formation of NPN products, aqueous extracts of fresh alfalfa were fermented to imitate the proteolytic process of ensiled alfalfa and to ensure that each class of exopeptidase inhibitor would have immediate contact with the proteases in the alfalfa extract. Five classes of exopeptidases; namely, aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase, were shown to be present in alfalfa leaves, each playing a different role in alfalfa protein degradation. Aminopeptidase, carboxypeptidase, and dipeptidase were the main exopeptidases contributing to the formation of NH 3 -N. Among the 5 exopeptidases, tripeptidyl-peptidase appeared to be the principal exopeptidase in hydrolyzing forage protein into peptides, whereas carboxypeptidase and dipeptidase appeared to be more important in contributing to the formation of amino acid-N. Dipeptidyl-peptidase and tripeptidylpeptidase did not play a role in the formation of NH 3 -N or amino acid-N. Dipeptidase, carboxypeptidase, and tripeptidyl-peptidase were the principal exopeptidases for hydrolyzing forage protein into NPN during ensilage, and treatment with a mixture of the 5 inhibitors reduced the total NPN concentration in the fermented alfalfa extract to about 45% of that in the control after 21 d of fermentation.

Characteristics of proteolytic microorganisms and their effects on proteolysis in total mixed ration silages of soybean curd residue

“…In addition, the initial hydrolysis of protein and the subsequent degradation of peptides into free amino acids might also result from the exopeptidases activities in TMR silages. According to Tao et al [11], the endoand exopeptidases both contribute to the protein hydrolysis and the tripeptidyl-peptidase was a principle exopeptidase for protein degradation in alfalfa silage. So, there is also a necessity for another determination of the microbial exopeptidases in TMR silages in a further study to conclusively link the observed proteolysis with the specific bacteria species which have the capability of producing exopeptidases during the ensiling process.…”

“…So far, the plant proteinases and peptidases have been principally clarified, and their relative contributions to the formation of various NPN compounds have been well demonstrated in silages. According to Guo et al [10] and Tao et al [11], protein hydrolysis mainly resulted from the plant exo-and endopeptidases, and the principle exo-and endopeptidases hydrolyzing the forage protein were di-, tripeptidyl-and carboxypeptidases, and metallo and cysteine peptidases, respectively. McKersie and Buchanan-Smith [12] also demonstrated that carboxypeptidase, aminopeptidase, and acid proteinase were important in the degradative process of protein in ensiled alfalfa.…”

Effects of four short‐chain fatty acids or salts on the dynamics of nitrogen transformations and intrinsic protease activity of alfalfa silage

“…The presence of many different proteases in plant tissues is an advantage to the plant given the range and structural complexity of the proteins to be degraded . Tao et al identified five types of exopeptidases in fresh alfalfa, and they concluded that each of them played a different role in alfalfa protein degradation and formation of different N constituents during fermentation of alfalfa green extract. In the present study, although all of protease activities declined as the ensiling progressed, each of the protease activities declined independently, as observed by McKersie and Buchanan‐Smith .…”

Effects of plant enzyme inactivation or sterilization on lipolysis and proteolysis in alfalfa silage

“…Numerous studies have been conducted to investigate contribution of microbe-originated enzymes and plant enzymes to formation of NPN in silage. The dominant role that plant enzymes play in proteolysis during ensiling has been proven by a classical method of sterilizing forage at ensiling (Heron et al, 1986) or by using specific inhibitors of endo-and exopetidases (Guo et al, 2011;Tao et al, 2011). Plant enzyme inactivation before ensiling further validated the leading role of plant enzymes in proteolysis and could effectively reduce both the extensive proteolysis and lipolysis which occur in ensiled alfalfa.…”