The components of xylan hydrolases of Neocallimastix patriciarum W5

• Main Authors: Yun-Yin Lo, 羅云吟
• Other Authors: Yo-Chia Chen
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/26852489545496020196

碩士 === 國立屏東科技大學 === 生物科技系所 === 101 === Xylan constitutes the major component of hemicellulose, the major component of the plant wall; xylan-degrading enzymes play a great role in elucidation the structures of complex xylan. Nowadays the xylanase are mostly glycoside hydrolase (GH) 10 and 11 family; however, xylan hydrolysis system of many microorganisms were widely studied, but few studies have the fungi by the xylan hydrolysis system. Ruminal fungi are absolute anaerobic microorganisms distributed in the gastrointestinal tract of ruminant, it preferentially colonize lignocellulose tissue and produce an array of extracellular enzymes that are capable of digesting the major structural carbohydrates of plant cell well. Nevertheless, the rumen fungi able to degrade xylan, but they was not determined by the mechanism. Now the Academia Sinica has been identified the Neocallimastix patriciarum W5 genomic DNA complete sequence, and its xylanase type in comparison is far less than by the other species, but it has high xylanase activity, if doubts xylanase has gene multiplicity. In this study, to understand the rumen fungi GH 10, 11, and 43 of the xylanase groups of functionality and relevance, using analysis from the transcriptome and proteomics approach. We cultured culture N. patriciarum W5 on different carbon sources medium, like as simple sugars (glucose and xylose), the polysaccharide (xylooligosaccharide and xylan), and composites (rice straw). Designed for GH 10, 11, and 43 families primer from the N. patriciarum W5 its xylanase, but GH 11 and 43 families with two different gene fragments, so the design five primer of named as F10, F11-2, F11-2, F43-1, and F43-2. First of all, used real-time PCR to detect gene transcription in different carbon sources, the results show, the F10, F11-2, and F43-2 have transcribed from the all carbon sources; F43-2 gene had overexpression from the carbon sources for glucose and xylan; F11-1 gene is not transcribed in the glucose; F43-1 had not transcription in all the carbon source. And then used denaturing gradient gel electrophoresis (DGGE) observed xylanase gene group whether to the have gene multiplicity. The results showed N. patriciarum W5 has gene multiplicity from the genomic DNA, and then had different multiplicity in different carbon sources. The N. patriciarum W5 had secreted to the extracellular xylanase, found they had one to several different an active region by the having xylan carbon sources in SDS-PAGE. Identification of the protein identity, determine they had GH 10, 11, and 43 families of proteins, and all carbon sources can translation cellulase from the xylan and rice straw.