Studies of anaerobic fungus Neocallimastix patriciarum J11 cellulosome system
Date Issued
2014
Date
2014
Author(s)
Wang, Hui-Chang
Abstract
In this study, we purified the cellulosome of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The cellulosome with molecular weight more than 669 kDa, and the cellulase and xylanase activities were detected by polyacryamide gel electrophoresis and zymogram. The cellulosome is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight and four of these constituents have demonstrated cellulase and xylanase activites on zymogram analysis, respectively. Western hybridization analysis revealed that the fungal dockerin could bind to the protein with molecular weight of 79 kDa, and did not need calcium ion for mediation. This postulated fungal scaffoldin responded to the gathering of the cellulolytic components. Binding titration revealed that the high conserved Trp30 residue in fungal dockerin could participate in binding with scaffoldin by the hydrophobic interaction. The levels and subunit ratio of the cellulosome from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the cellulosome were consistent. In this study, we identified six components of N. patriciarum J11cellulosome using liquid chromatography/mass spectrometry. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through LC/MS/MS, revealing that at least three types of cellulase, including one endoglucanase and two exogluanases, could be found in the N. patriciarum J11cellulosome. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the cellulosome produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase.
Subjects
厭氣性真菌
纖維素分解酶
纖維素分解酶複合體
錨固蛋白質
生質酒精
Type
thesis
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