According to the Nomenclature Committee of the

According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB, 1992), peptidases are divided into two classes, namely exopeptidases and endopeptidases, based on their actions on substrates and their active sites, respectively. Endopeptidases include four main peptidases, namely serine peptidase (E.C.3.4.21), cysteine peptidase (E.C.3.4.22), aspartic peptidase (E.C.3.4.23) and metallopeptidase (E.C.3.4.24). So far, the enzymes involved in proteolysis during alfalfa ensiling have not been systematically and well characterized. There have been few studies which have addressed contributions of different endo and exopeptidases to proteolysis, and to the formation of NPN compounds in ensiled alfalfa.
Materials and methods
Results
Discussion
Conclusions
Acknowledgements Financial supports from the National Natural Science Foundation of China (30800800), the Ph.D. Programs Foundation of Ministry of Education of China (200807301008) and the National Key Technology R&D Program in the 12th Five year Plan of China (2010BAD17B02) are gratefully acknowledged. The authors would like to express their highly appreciation to Mr. Malcolm Gibb (Formerly Environment and Grassland Research Institute, UK) for revision of the manuscript.
Introduction Carica papaya is widely cultivated in tropical and subtropical regions all around the world. Apart from the edible fruits, enzymes stored in its lactiferous norepinephrine bitartrate can be produced and have found several applications (de Oliveira & Vitória, 2011). When these cells rupture, the coagulation of latex occurs. This represents an important defence mechanism of the plant against pathogens and other harmful attacks. In addition, the latex of C. papaya is a rich source of cysteine endopeptidases, including papain, glycyl endopeptidase, chymopapain and caricain, constituting more than 80% of total enzymes (Azarkan, El Moussaoui, Van Wuytswinkel, Dehon, & Looze, 2003). Papaya latex was used for preparing protein hydrolysates with bioactivities (Kittiphattanabawon et al., 2012, Ngo et al., 2011). Due to the abundance of glycine in gelatin molecules, glycyl endopeptidase, a major component which constitutes almost 30% of total protein in the latex of C. papaya, can serve as a potential protease, which preferably cleaves the peptide bonds in gelatin. However, undesirable off-odour of crude papaya latex leads to the offensive odour or flavour in the resulting gelatin hydrolysates, thereby causing consumer rejection. The volatile compounds of various papaya cultivars have been extensively investigated (Pino, Almora, & Marbot, 2003). Twenty-five odorants were considered as odour-active compounds and contribute to the typical papaya aroma. The pungent-sour and green-note odours found in the green fruit are due to benzyl isothiocyanate and some C6 compounds (e.g., 1-hexen-3-one), respectively (Pino, 2014). Ulrich and Wijaya (2010) found that stinky and smokey odours were caused by butanoic acid and benzyl isothiocyanate. Therefore, the crude enzyme obtained from green fruit latex might contain those odorous compounds. When papaya latex proteases were employed, compounds contributing to offensive off-odour were present in the resulting gelatin hydrolysate, thus obstructing the extensive utilisation and consumption of hydrolysate. Several separation techniques have been applied for protein concentration and purification. Membrane-aided filtration and other techniques can be effective in protein separation. However, the adverse effects of operating conditions can be associated with enzyme denaturation (Nakkeeran and Subramanian, 2010, Krstic et al., 2007). Aqueous-two-phase system (ATPS) is a good choice, which offers mild and non-disruptive purification conditions for biomolecules, especially enzymes (Prinz et al., 2012). ATPS has been widely employed as an effective and economical process for the separation, purification and concentration of enzymes (Ketnawa and Rawdkuen, 2011, Rawdkuen et al., 2011, Subathra et al., 2012). ATPS can remove the undesirable compounds present in the system including unidentified polysaccharides, interfering protein and contaminants (Dubey & Jagannadham, 2003). Chaiwut, Kanasawud, and Halling (2007) used ATPS followed by salt precipitation for isolation of glycyl endopeptidase from papaya latex. Therefore, ATPS can be used to fractionate glycyl endopeptidase in papaya latex and remove offensive odorous compounds under appropriate condition. As a consequence, a more active fraction without undesirable odour could be prepared from papaya latex and further used as a processing aid in production of gelatin hydrolysate. The aims of the present study were to fractionate glycyl endopeptidase from papaya latex of Red Lady and Khack Dum cultivars grown in Thailand, using ATPS and ammonium sulphate precipitation, and to determine odorous compounds in the obtained fraction. Additionally, antioxidative activities and odorous compounds of hydrolysate prepared using glycyl-endopeptidase-rich fraction were determined.