| dc.description.abstract | There are 300 million tons of agricultural wastes produced every year in Taiwan. The typical agricultural wastes in Taiwan are rice husk, rice bran, mushroom residua, coconut and bamboo fiber. The temperatures during the composting can reached 60-80°C. The enzymes released by microbes during composting play a key role in the biological and biochemical transformation of the organic matters. These enzymes usually are more stable and with higher activity than normal enzymes at high temperature. Cellulases, for example, secreted by microorganisms during high temperature composting play an important role in the biochemical transformation of cellulose. They are considered to be potential in applications on biofuel production. Although extracellular enzymes are important, these proteins are difficult to be isolated due to they are often associated with humic substances in the environmental samples. The focus of this study is on the methodology in order to improve the efficiency of the extracellular protein extraction from composts. A metaproteomics method was developed to investigate the presence of expressed enzymes at the peak temperature during composting. The mechanical and chemical treatments were applied on two composts which contain different composition of agricultural waste and manure. The extracelluar proteome was collected by centrifugation and filtration to remove the compost debris and microbial cells, respectively. The following steps included the humic substance removal, extracellular protein extraction, purification, and separation (by 1D and 2D-SDS-PAGE). Then, the proteome was digested by trypsin followed by analysis of the peptide sequences via ESI-MS/MS. sixty-three proteins, including thirty-six lignocellulytic enzymes, were identified from this study. The results provided the composition of proteins in the composts when the cellulase activity is at highest stage. Particularly, protein expressed from uncultivated microbes can be investigated. By this approach, two novel lignin-degrading enzymes, laccases, were identified from a two-week compost at 70°C (30% rice husk, 10% rice bran, 40% mushroom waste and 20% coconut fiber). They are highly likely from Spongipellis sp.. Our results represent the first metaproteomic study expressed protein profiles of natural microbial communities in compost environment.
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