| 摘要: | 台灣每年產生300萬噸之農業廢棄物，包括稻穀、米糠、菇類太空包和椰纖等。堆肥過程中微生物將此類大型有機物農業廢棄物降解，而其過程能達60-80℃的高溫。其中能在如此高溫生長之嗜(耐)高溫微生物所分泌之各種酵素，在堆肥過程中扮演關鍵的角色，尤其是木質纖維水解酵素，此類耐高溫酵素，未來具應用於生質酒精生產之潛能。然而環境中之分泌性酵素，會與環境中的有機質如腐植質結合，使其穩定且不易受其他微生物降解。所以本試驗目標為建立堆肥之胞外蛋白質分析方法，即建立一套有效分析堆肥之胞外蛋白體之方法 (宏蛋白體學，metaproteomics)。對二種不同成份組成分之堆肥，進行堆肥胞外蛋白質之萃取，過程中利用不同物理和化學方法，將蛋白質和有機質分離。堆肥樣品首先經過離心及過濾將雜質與細胞去除，接著探討樣品中腐植質之去除效率、堆肥胞外蛋白質最適之緩衝溶液及蛋白質沈澱方法，再以一維和二維電泳將蛋白質體分離，分離後之蛋白質經胰蛋白酶消化後以電噴灑游離/四極柱飛行時間質譜儀（ESI-Q-Tof）鑑定其身分。試驗結果共鑑定到63個蛋白質，包括36個木質纖維水解酵素，其中10來自於實驗室中不可培養之菌種。結果證實以本實驗建立之堆肥胞外蛋白體分析方法，可探討堆肥於其酵素活性高點之胞外蛋白質體，且對於在試驗室中不可培養之菌種，也可以此宏蛋白體學的方法進行探討。其中從一星期之堆肥（30% 稻殼，10% 米糠，40%廢棄太空包和20%椰纖）中，以溶液消化時鑑定二個Laccase與到尚未有相關文獻報導之物種Spongipellis sp. FERM P-1817之laccase非常相近。Laccase可降解木質素，木質素之去除為生產生質酒精前處理之關鍵，有效之前處理可減低生產纖維酒精成本五至十倍，未來值得深入探討。本研究為第一個以宏蛋白體學的方法對堆肥中微生物蛋白質體進行研究，並能成功鑑定到其中之蛋白體及木質纖維水解酵素。 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. |
