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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53685

Title: Clostridium cellulolyticum H10 來源之藍藻蛋白合成酶的純化及利用大腸桿菌生產藍藻蛋白
Purification of Cyanophycin Synthetase from Clostridium cellulolyticum H10 and Production of Cyanophycin in Recombinant Escherichia coli
Authors: Huang, Lin-Yu
黃稜育
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 大腸桿菌;藍藻蛋白;藍藻蛋白合成酶;發酵槽
Clostridium cellulolyticum H10;Escherichia coli;cyanophycin;cyanophycin synthetase;fermenter
Date: 2018
Issue Date: 2020-07-03T08:42:46Z
Abstract: 藍藻蛋白 (Cyanophycin Granule Peptide, CGP) 是存在於藍藻菌中,一種非核醣體合成之胺基酸聚合體,其主要以天門冬胺酸 (L-aspartic acid, Asp) 為骨架,而精胺酸 (L-arginine, Arg) 與離胺酸 (L-lysine, Lys) 為側鏈所組成,其分子量介於 25~100 kDa;藍藻蛋白廣泛使用於工業、生物醫學和藥品製造。目前發現藍藻蛋白合成酶 (cyanophycin synthetase, CphA) 是唯一可以催化合成 CGP 的酵素,文獻指出 CphA 的活性與 ATP、鉀離子、鎂離子或是硫醇化合物 (thiol reagent) 有關。 本實驗室先前將源自於 Synechocystis sp. PCC6803 藍藻蛋白合成酶基因 (cphA),選殖入 pET-21b 並轉形至 Escherichia coli 表現,但發現 CGP 之產量不佳。本實驗利用含有源自 Clostridium cellulolyticum H10 之藍藻蛋白合成酶基因 (ClcphA) 之 pET-15b- ClcphA 質體轉形至 E. coli ClearColi BL21(DE3)、BL21 (DE3) 及 BL21 Star (DE3),進行 CGP 之生產。由結果顯示,利用 E. coli BL21 Star (DE3) 表現 ClcphA,於添加 0.2 mM IPTG 之誘導濃度及 20oC 之誘導溫度下誘導 24 小時可最有效的表現 ClCphA,其分子量約為 100 kDa;而培養基中含有不同之胺基酸濃度或與分子伴護蛋白共表現對酵素表現則沒有顯著之影響。另外,本實驗所使用之載體 pET-15b 於 N 端帶有六個胺基酸組成之 His-tag,所表現之 ClCphA 可利用鎳離子親和性管柱層析進行純化,但目前無法偵測到活性。 接著進行藍藻蛋白之純化,以含有 300 mL 培養基之 2 L 錐形瓶,於最適培養條件下進行培養,可獲得佔菌體濕重 0.24% (w/w)、相當於64 mg/L 培養基之可溶 CGP及 0.05% (w/w)、12 mg/L 之不可溶 CGP;利用 6.6 L 發酵槽 (含有 2 L 培養基) 以轉速 400 rpm、通氣量 1 vvm 及最適誘導條件進行培養,最終可獲得提升之可溶CGP產量,佔菌體濕重 1.21% (w/w)、相當於 84 mg/L 培養基,而不可溶 CGP 產量則大約為 0.06% (w/w)、4.4 mg/L。
Cyanophycin (Cyanophycin Granule Peptide, CGP) is a copolymer of aspartate and arginine that forms storage granules. CGP is of biotechnological interest due to its potentially industrial applications. Our laboratory had previously cloned the gene of cyanophycin synthetase (cphA) from Synechocystis sp. PCC 6803 into pET-21b vector, expressed in E. coli to produce cyanophycin, and found that the production was not ideal. In this study, the vector pET-15b-ClcphA with N-terminal His-tag, carrying cphA (ClcphA) gene from Clostridium cellulolyticum H10 was respectively transferred into E. coli ClearColi BL21(DE3), BL21 (DE3), and BL21 Star (DE3) to produce cyanophycin. The results showed that ClcphA expressed in BL21 Star (DE3) has the higher yield, and found that the optimal induction was carried out with 0.2 mM IPTG for 24 h at 20oC. The molecular mass of ClcphA is about 100 kDa analyzed by SDS-PAGE. Although ClcphA was purified by nickel affinity chromatography, enzyme activity couldn’t be detected. After the purification of CGP, soluble CGP comprised 0.24% (w/w) of cell wet matter, equaled to 64 mg/L of medium volume. While insoluble CGP content 0.05% (w/w), 12 mg/L were obtained. However, soluble CGP content could be further increased when incubated in 6.6 L fermenter (included 2 L medium) with 400 rpm, 1 vvm aeration and optimal induction condition. Soluble CGP comprised 1.21% (w/w) of cell wet matter, which was equal to 84 mg/L of medium volume. For insoluble CGP, 0.06% (w/w) and 4.4 mg/L were obtained.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010532013.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53685
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