Contribution of endo- and 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

in ensiled forage mainly results from plant (Ohshima and McDonald, 1978; McKersie, 1981; Heron et al., 1988).  Proteases (peptidases) are divided into 2 classes (NC-IUBMB, 1992):

Exopeptidase Objectives

Proteases (peptidases, E.C.3.4）

Endopeptidases Exopeptidases ？！ Ser ine peptid ase (E .C .3 .4 .21) (EC 3.4.11) (EC 3.4.16) Cysteine peptidase (E.C.3.4.22) (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 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 Extraction (Feller (Feller et al 1977et al., 1977 )

Crude 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 ) Materials and Methods

Table 1. Endopeptidases and specific inhibitors Table 2. Exopeptidases and specific inhibitors Endo- Inhibitors Concentration 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 TiTripeptidyl tidl-peptidase But abi ndid e 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 Alfalf fermentation of ensiled alfalfa forage

Fermented at 30Ԩ Aqueous ＋ Specific Analysis of Extracts inhibitors a were fermented to imitate the 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 DiDriproti n A (D) 2 mM A+B+C+D - Butabindide (E) 0.5 mM A+B+C+D+E - Results and discussion Guoet et al Animal., Anim Feed SciFeed Technol Sci Technol 168 (2011).168 42 (2011),42-50;Tao Tao et et al J Dairyal. J Dairy Sci 94 (2011)Sci. 94 3928 (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 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 !