Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52450
標題: Overexpression of chitosanase and glucosaminidase genes for the production of glucosamine
Chitosanase及glucosaminidase基因之大量表現俾生產glucosamine
作者: 許文輝
關鍵字: Chitosanase;技術發展;生物技術;Glucosamine;Bioconversion;幾丁聚醣?;葡萄糖胺;生物轉換
摘要: 
Glucosamine被發現具有舒緩關節炎的效果,而chitosanase及glucosaminidase具有水解chitosan成為glucosamine的能力。Bacillus sp. NCHU-5的chitosanase (csn)基因經由基因重組,能夠在Escherichia coli BL21(DE3)及B. subtilis WB800中進行胞內或胞外的大量表現,表現出之chitosanase皆可經由Ni-NTA管柱進行純化,經SDS-PAGE的分析,chitosanase的大小約為30 kDa,純化的chitosanase最適反應溫度為40°C,在pH5.6下具有最高的比活性,且水解產物為chitosan dimer、trimer、tetramer或pentamer。另外由Aspergillus fumigatus BCRC30099絲狀真菌染色體DNA中,增幅出glucosaminidase (csx)基因,將此基因於E. coli BL21(DE3)中以T7啟動子進行表現,可得到大小約97 kDa具有活性glycosyl hydrolase之蛋白。經由chitosanase及glucosaminidase的作用,可使chitosan在40°C、pH5.6的環境下有效率地水解生產glucosamine。本研究將利用基因改造、改變培養基組成及誘導的條件來提升glucosamiinidase的產量,並建立chitosanase及glucosaminidase水解chitosan生產glucosamine之最佳條件,以提供工業界所需。

Glucosamine could be used to relief osteoarthritis. Chitosanase is an enzyme that catalyzes the hydrolysis of chitosan to glucosamine. A chitosanase gene (csn) was cloned from Bacillus subtilis NCHU-05 into expression vectors and expressed respectively in Escherichia coli BL21(DE3) and B. subtilis WB800. Expressed recombinant chitosanases were purified by Ni-NTA column chromatography.The molecular mass of chitosanase was estimated to be 30 kDa by SDS-PAGE analysis. Purified chitosanase exhibited optimal temperature of 40C at pH5.6. Thin layer chromatography and LC/MS/MS analysis revealed that the major oligosaccharides from chitosan hydrolyzed by recombinant chitosanase were dimer, trimer, tetramer and pentamer. A glucosaminidase gene (csx) was amplified from the genomic DNA of Aspergillus fumigatus BCRC30099 and cloned into expression vector under the control of T7 promoter. Csx was estimated to be 97 kDa and showed a glycosyl hydrolase activity. Chitosanase and glucosaminidase could efficiently convert chitosan into glucosamine at 40C and pH5.6. Glucosamine production could used glucosaminidase which increase production level by gene modification, medium composition and induction conditions. The optimal conditions for the conversion of chitosan to glucosamine will be established for the industrial applications.
URI: http://hdl.handle.net/11455/52450
其他識別: 100農科-1.1.3-牧-U1(4)
Appears in Collections:分子生物學研究所

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