Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/50978
標題: Bacillus macerans之環狀糊精葡萄糖�A機基轉移�@基因在枯草桿菌中的 表現
Expression of cyclodextrin glucanotransferase gene of Bacillus macerans in Bacillus subtilis
作者: 林大鈞
Lin, Da-Gin
關鍵字: 環狀糊精葡萄糖基轉移
Bacillus macerans
蛋白質表現
枯草桿菌
Bacillus macerans
cyclodextrin glucanotransferase
protein expression
Bacillus subtilis
出版社: 食品科學系
摘要: 中文摘要本研究係以 B. macerans 之 late log phase 染色體 DNA 為模 板,利用聚合鏈反應(PCR)合成一段長度約為 2.1 kb ,含有 ribosome binding site 、signal sequence 之 CGTase 基因。將此酵素 基因選殖於一雙向載體 -- pHY300PLK,轉形至 E. coli 因而得到重組質 體 -- pHG,再轉形至 B. subtilis。pHY300PLK 及 CGTase 基因片段並 無明顯的 promoter 區域,而重組質體 - pHG 卻能分別在 E. coli 及 B. subtilis 中,表現 CGTase 蛋白質,因此推測 pHY300PLK 上有一段 未知的 promoter 區域。將攜帶 pHG 之 B. subtilis 培養於 2 x SG、 LB 及 2 YT 中,發現其在以 2 YT 培養基培養至 24 小時,菌液會具有 最高的 CGTase 酵素活性表現。將培養液離心所得之粗酵素液以β- CD 親合性管柱、 DEAE Sepharose CL 6B 離子交換管柱層析及 Sephacryl S-200 膠體過濾層析等步驟純化分離 CGTase,將純化之 B. subtilis CGTase 進行 N 端胺基酸序列分析得SPDTSVDNKVNF,與文獻之 B. macerans CGTase N 端胺基酸序列相同,此外由澱粉水解及 α-CD 偶合 反應測試之結果,顯示出 B. subtilis 所生產之 CGTase 與 B. macerans 之 CGTase 具有相似的催化活性。自 pQG - D*、pQG - Y* 載 體上,以剪切、置換的方式,導入 E344D、E344Y 等兩種突變酵素至此表 現系統,生產並純化突變酵素,經評估結果顯示此系統能生產足量的野生 型與突變型 CGTase,並將其分泌至胞外,以利生化特性分析的進行。因 此,本研究之系統可作為將來其他突變型 CGTase 之生產與活性分析之依 據。
AbstractA 2.1 kb DNA fragment which encodes the cgt gene (with ribosome binding site and signal sequence)of Bacillus macerans was synthesized by polymerase chain reactions(PCR)and cloned into a shuttle vector -- pHY300PLK(which is not a expression vector). There was no promoter sequence in the cloned cgt gene and vector, but the recombination plasmid, pHG, could express CGTase in B. subtilis DB430. It is proposed that an unknown promoter sequence was located at the upstream of cgt gene on pHG. Higher amount of CGTase was produced as B. subtilis carrying pHG growing on 2YT medium than on 2xSG or LB. B. subtilis CGTase was purified through β-CD affinity chromatography, DEAE Sepharose CL 6B ion exchange chromatography and Sephacryl S - 200 gel filtration to apparent homogeneity. The N-terminal amino acid sequence of B. subtilis CGTase determined was SPDTSVDNKVNF,which is consistent with that of B. macerans CGTase. The B. subtilis CGTase also has the similar starch digesting and α- CD coupling activities as B. macerans CGTase. The results revealed that B. subtilis harboring pHG produces the authentic CGTase of B. macerans. E344D and E344Y were introduced onto this vector system by replacing the corresponding DNA fragment on pHG with that on pQG - D* and pQG- Y* contained the mutated site, respectively. It was determined that the amount of wild type, E344D and E344Y expressed extracellulary from B. subtilis was 55 mg /L, 25 mg / L and 20 mg / L, respectively. These amounts were fairly enough to be applied in the determination the properties of the two mutant CGTases. From above results, we proposed the system could be employed in the production of the mutant CGTases for studying of their catalytic activities in the future.
URI: http://hdl.handle.net/11455/50978
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