Contribution of endo- and exopeptidases to formation of non-protein N during ensiling of alfalfa Dr. Xusheng Guo,1 W. Cheng,1 L. Tao,2 Yu Zhu,2 F.Y. Yang 2 & H. Zhou2 1.School of Life Science, Lanzhou University 1. International Centre for Tibetan Plateau Ecosystem Management (ICTPEM), Lanzhou University 2. Institute of Grassland Science, China Agricultural University Hämeenlinna, Finland 2-4 July 2012 Introduction Alfalfa (Medicago Sativa L.) is well known for its high nutritive value However, after ensiling: N use efficiency Extensive ppyroteolysis Reduce True protein NPN(Peptide, FAA, NH3-N etc.) Silage DM intake (44-87% of Total N; Muck, 1987) Silage Fermentation Proteolysis in ensiled forage mainly results from plant proteases (Ohshima and McDonald, 1978; McKersie, 1981; Heron et al., 1988). Proteases (peptidases) are divided into 2 classes (NC-IUBMB, 1992): Exopeptidase Endopeptidase Objectives Proteases (peptidases, E.C.3.4） Endopeptidases Exopeptidases ？！ Ser ine pep tidase (E .C .3 .4 .21) Aminopeptidase (EC 3.4.11) Carboxypeptidase (EC 3.4.16) Cysteine peptidase (E.C.3.4.22) Dipeptidase (EC 3.4.13) Aspartic Peptidase (E.C.3.4.23) Dipeptidyl-peptidase (EC 3.4.14) Metallopeptidase (E.C.3.4.24) Tripeptidyl-peptidase (EC 3.4.14) Peptidyl-dipeptidase (EC 3.4.15) Aims of our research were: 1. To clarify the classes of exo- and endopeptidases that are involved in proteolysis within ensiled alfalfa. 2. To determine the contribution of these peptidases to the formation of different NPN compounds (peptide-N, FAA-N, and NH3-N) during ensiling Materials and Methods Trial 1: Determination of Peptidase Classes in Fresh Alfalfa Crude Enzymes Extraction (Feller et al ., 1977 ) Crude enzyme extract 5g ground in Desalted at 4 liquid N Centrifugation Ԩ in Sephadex 4Ԩ, 20, 000 20ml, pH7.5, G-25 columns 25 ml Tris-HCl r/min for 10 min Residual Crude Specific Incubation Enzyme Incubate at Measure ＋ peptidase Fischer enzyme ＋ inhibitors 20 C for 0.5 h substrates 37Ԩ for 1 h etal.,1998 activities For each of the exo- Compared to control (Without and endopeptidase inhibitor) Materials and Methods Table 1. Endopeptidases and specific inhibitors Table 2. Exopeptidases and specific inhibitors Endo- Inhibitors Concentration Exopeptidase Inhibitors Concentration peptidase used used Aminopeptidase Serine PMSF* 5 mM Bestatin 0.05 mM Carboxy-peptidase PCI* 0.825 μM Pepstatin A 1mM1 mM Aspartate 1,10- Dipeptidase 10 mM phenanthroline 1,10- 10 mM Dipeptidyl-peptidase Driprotin A 0.5 mM Metallo phenanthroline TiTripep tidltidyl-peptidase Bu ta bin dide 0. 025 m M dipeptide 0.25 mM Cysteine E-64* 1 mM Peptidyl-dipeptidase Phe-Arg *PMSF, phenylmethanesulfonyl fluoride. *E-64, L-trans-epoxysuccinyl-leucylamide- (4-guanidino)-butane. *PCI, potato carboxypeptidase inhibitor Materials and Methods Trial 2: Determination of concentration of each inhibitor required for different peptidases Table 3. Inhibitor concentrations designed for each of the endo- and exopeptidase inhibitors The experimental method is same as Trial 1 Materials and Methods Trial 3: Determination of contribution of endo- and exopeptidases to formation of NPN during ensiling of alfalfa Aqueous Extracts of Whole-crop Alfalfa were fermented to imitate the fermentation of ensiled alfalfa forage Fermented at 30Ԩ Aqueous ＋ Specific Analysis of Extracts inhibitors NPN and its 1, 2, 3, 5, 7, 14, and 21 d constitutes Endopeptidase Exopeptidase Concentration used Concentration used inhibitor treatments inhibitor treatments Control - Control - PMSF (A) 5 mM Bestatin(A) 0.1 mM Pepstatin A (B) 0.1 mM PCI (B) 0.413 mM 1,10-phenanthroline (C) 5 mM 1,10-phenanthroline (C) 50 mM E-64 (D) 0.1 mM DiDriprot in A (D) 2 mM A+B+C+D - Butabindide (E) 0.5 mM A+B+C+D+E - Results and discussion Guo et al ., Anim Feed Sci Technol .168 (2011) ,42 -50; Tao et al . J Dairy Sci . 94 (2011) ,3928 -3935. 1. Identification on classes of exo- and endopeptidases in alfalfa Table 1. Changes in proteolytic activity of exopeptidases in crude enzyme extract of alfalfa after peptidase inhibition treatments Table 2. Changes in proteolytic activity of endopeptidases in crude enzyme extract of alfalfa after peptidase inhibition treatment 1Proteolytic activity was express as absorbance of the incubation samples at 570nm directly and the proteolytic activity in the fresh alfalfa extract was considered as 100 percent. Results and discussion 2. Contribution of exopeptidases to formation of NPN during fermentation of alfalfa extract B C A Carboxy- and dipeptidases TN TN control gg control Control -N mg/g 3 NH tide-N, mg/g tide-N, mg/g TN mino acid-N, mg/ pp AA Pe Tripeptidyl-peptidase Day of fermentation Day of fermentation Day of fermentation Figure 1. Concentrations of NH3-N (A), AA-N (B), and peptide-N (C) in the fermented alfalfa extract treated with different specific enzyme inhibitors of exopeptidases Table 3. NPN, Peptide-N, AA-N and NH3-N contents (mg/g total N) in fresh and fermented alfalfa extracts with different specific enzyme inhibitors (exopeptidases) for periods of up to 14 days Inhibitor classes Inhibitor NH3-N AA-N Peptide-N NPN Fresh alfalfa extracts <0.001 28.0 209.0 237.0 Fermented extracts Control - 26.0a 321.1c 273.4cd 620.5a Bestatin Aminopeptidase 8.4b 273.7d 317.2b 589.3a PCI* Carboxypeptidase 12.2b 236.3e 291.9bc 540.4b 1,10-phenanthroline Dipeptidase 0.0c 219.8e 258.1d 472.9c DriprotinA Dipeptidyl-peptidase 28.6a 346.9b 211.5e 587.0a Butabindide Tripeptidyl-peptidase 24. 3a 381. 6a 133. 0f 538. 9b Combined inhibitors Five of the peptidases 0.0c 109.8f 350.5a 460.3c SEM 2.40 77.2 37.4 62.4 a-fMeans within a column with different suppperscripts differ ( P < 0.05). * PCI, potato carboxypeptidase inhibitor. SEM, standard error of the mean. Five exopeptidase inhibitors could reduce reduce the NPN content in the fermented alfalfa extract to about 42% of that in the control after 14 d of fermentation 3. Contribution of endopeptidases to formation of NPN duringgg ensiling of alfalfa Control Control Metallo peptidase Control Control Peptide-N Aspartate peptidase Day of fermentation Day of fermentation Figure 1. Concentrations of NH3-N (A), free amino acid-N (B), and peptide-N (C) in the fermented alfalfa extract treated with different specific enzyme inhibitors of endopeptidases (mg/g TN) Table 4. NPN, Peptide-N, AA-N and NH3-N contents (mg/g total N) in fresh and fermented alfalfa extracts with different specific enzyme inhibitors (endopeptidases) for periods of up to 14 days a-f Means within a column with different superscripts differ (P < 0.05). *PMSF, phenylmethanesulfonyl fluoride. SEM, standard error of the mean. Four inhibitors reduced the total NPN content in fermented alfalfa extract to about 44% of that in the control after 14 d of fermentation. Conclusions Five exo- and 4 endopeptidases were presented in alfalfa leaves Dipeptidase, carboxypeptidase and tripeptidyl-peptidase were the principal exopepdidases contributing to the proteolysis in ensiled alfalfa Metallo and cysteine peptidases are thought to be the principle endopppeptidases for p rotein deg radation during ensiling of alfalfa Exo- and endopeptidase were comparable in contributing to formation of NPN during ensiling of alfalfa . Thank you very much for your attention ! .