Studies of the Trehalose-Producing Related Enzymes and the Production of Trehalose from Starch (I)

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Project title

Code/計畫編號

NSC91-2313-B019-039

Translated Name/計畫中文名

海藻糖生成相關酵素與海藻糖生產之研究(1/3)

Project Coordinator/計畫主持人

Tsuei-Yun Fang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=757842

Year

2002

Start date/計畫起

01-08-2002

Expected Completion/計畫迄

01-07-2003

Bugetid/研究經費

1052千元

ResearchField/研究領域

食品科技(農)

利用聚合脢鏈反應，配合其他基因重組技術，已將Sulfolobus solfataricus的TreX、TreY及TreZ三種基因成功選殖於具有T5、T7或Tac promoter之表現載體。此三種與海藻糖生成相關之酵素基因在T7promoter之大腸桿菌表現系統下，皆具有較佳之重組目標蛋白質。誘導劑IPTG的添加與否並不影脢肝醣枝切脢(GDE)與海藻糖生成脢(TFE)的表現，然而海藻糖脢糊精生成脢(TDFE)在不添加IPTG時有較佳之表3現量。利用熱處理及膠體過濾層析已獲得部份純化之GDE及TDFE。GDE的酵素最適作用溫度約為75℃，最適作用pH為5;TDFE的酵素最適作用溫度約為65至75℃，最適作用pH為5。利用聚合脢鏈反應，配合其他基因重組技術，已將Sulfolobus solfataricus的TreX、TreY及TreZ三種基因成功選殖於具有T5、T7或Tac promoter之表現載體。此三種與海藻糖生成相關之酵素基因在T7promoter之大腸桿菌表現系統下，皆具有較佳之重組目標蛋白質。誘導劑IPTG的添加與否並不影脢肝醣枝切脢(GDE)與海藻糖生成脢(TFE)的表現，然而海藻糖脢糊精生成脢(TDFE)在不添加IPTG時有較佳之表3現量。利用熱處理及膠體過濾層析已獲得部份純化之GDE及TDFE。GDE的酵素最適作用溫度約為75℃，最適作用pH為5;TDFE的酵素最適作用溫度約為65至75℃，最適作用pH為5。 We have successfully cloned TreX, TreY, and TreZ genes from Sulfolobus solfataricus ATCC35092 to three expression vectors possessing T5, T7, and Tac promoters in the upstream of its multiple cloning sites, respectively. The expressions of the TreX, TreY, and TreZ genes were better under the T7 expression system than under the T5 and Tac expression systems. IPTG induction hardly affected the expressions of recombinant GDE and TFE in E. coli BL21 (DE3)-CodonPlus-RIL (pET-15b-TreX) and E.coli BL21 (DE3)-CodonPlus-RIL (pET-15b-TreZ), respectively. The GDE and TDFE have been partially purified by heat treatment and gel filtration. The partially purified GDE exhibited optimum activity at 75.degree.C and pH 5. The partially purified TDFE exhibited optimum activity at 65 ~ 75.degree.C and pH 5.

Keyword(s)

Trehalose
Glycogen debranching enzyme
Trehalosyl dextrins forming enzyme
Trehalose forming enzyme
Sulfolobus solfataricus

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Studies of the Trehalose-Producing Related Enzymes and the Production of Trehalose from Starch (I)

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