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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48766

Title: 產黃纖維單胞桿菌幾丁質酶Cellulomonas flavigena NTOU1 Chi50 定位突變對其生化特性之影響
Effects of Site-Directed Mutagenesis on the Biochemical Properties of C.f NTOU1 Chi50 from Cellulomonas flavigena NTOU1
Authors: Huang, Chen-Che
黃振哲
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 產黃纖維單胞桿菌;幾丁質酶;衍生分子
Cellulomonas flavigena;chitinase;derivative molecules
Date: 2016
Issue Date: 2018-08-22T06:31:07Z
Abstract: 產黃纖維單胞桿菌(Cellulomonas flavigena NTOU1)幾丁質酶 C.f NTOU1 Chi63全長分子其衍生分子為C.f NTOU1 Chi50和 C.f NTOU1 Chi38。為改善降解情況及蛋白質表現量不足的問題,本研究採用定位突變和置換不同表現載體的方式進行。 產黃纖維單胞桿菌的幾丁質酶經前人的研究已取得C.f NTOU1 Chi63的全長分子,並在大腸桿菌的表現系統中表現出具有幾丁質酶活性的重組酵素。C.f NTOU1 Chi63以熱處理或是冷藏處存,均會降解成小分子C.f NTOU1 Chi50及C.f NTOU1 Chi38。C.f NTOU1 Chi50也會降解成C.f NTOU1 Chi38。 目前利用分子量大小及胰蛋白酶trysin的切位預測C.f NTOU1 Chi50在R115胺基酸的位置可能與降解作用有關,因此用Dpn1 site-directed mutagenesis的方式進行定位突變,將R115胺基酸突變為A(alanine)、D(aspartate)、V(valine)、F(phenylalanine)、I(isoleucine)、Y(tyrosine),試圖了解R115X突變對C.f NTOU1 Chi50其蛋白質分子降解之影響。
The full-length chitinase gene, C.f NTOU1 Chi63, has been cloned from Cellulomonas flavigena NTOU1 and has been expressed with its enzymatic activity by the heterologous Escherichia coli expression system. Two different prokaryotic expression systems, pETite® and pET20b(+), both have produced the enzymatically active recombinant chitinase C.f NTOU1 Chi63. Derivative molecules of C.f NTOU1 Chi50 and C.f NTOU1 Chi38 were found under the chitinase activity staining after the SDS-PAGE analysis of the recombinant enzymes. The protein purification of the recombinant C.f NTOU1 Chi63 was hampered by the serious protein degradation by unknown factors. In order to dissolve this unknown protein degradation, both a site-directed mutagenesis and an alternative expression system were approached. The C.f NTOU1 Chi50 R115 was chosen as the target amino acid after SDS-PAGE analysis based on the relative molecular weights of the derivat-ive chitinase molecules. The Dpn1 site-directed mutagenesis of C.f NTOU1 Chi50 R115X was performed and its mutants with A(alanine)、D(aspartate)、F(phenylalanine)、I(isoleucine)、V(valine) and Y(tyrosine) mutations were successfully obtained. Biocharacterization of these chitinase mutants were followed and the possible protein degradation mechanisms maybe understood.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010236004.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48766
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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