Protein Engineering of D-Psicose 3-Epimerase to Enhance Its Thermostability and the Production of D-Psicose from Fructose by Immobilized Cells

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Project title

Code/計畫編號

MOST105-2320-B019-002

Translated Name/計畫中文名

以蛋白質工程提昇阿洛酮糖表異構酶的熱穩定性與利用固定化菌體由果糖生產阿洛酮糖

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=11897936

Year

2016

Start date/計畫起

01-08-2016

Expected Completion/計畫迄

31-07-2017

Bugetid/研究經費

1100千元

ResearchField/研究領域

食品科技(農)

"D-阿洛酮糖 (D-psicose) 為稀有糖類，具特殊生理活性，可應用於食品與膳食補充劑中取代蔗糖作為甜味劑，能降低餐後血糖上升並減少脂肪生成，美國食品藥物管理局於 2012 年將其認可為一般安全性食品，但由於產量稀少價格昂貴，造成許多研究及應用上的限制。生產此稀有糖類，可以D-阿洛酮糖表異構酶 (DPE)將D-果糖轉換成D-阿洛酮糖。我們將 Agrobacterium sp. ATCC 31750 DPE (AsDPE)基因，選殖入大腸桿菌中表現。然而目前已研究之DPE 都有對溫度敏感的問題，我們在先前國科會計畫已經以蛋白質工程技術於之C 端加入ATS (C’ATS-AsDPE)，提昇了AsDPE 的熱穩定性，並將序列中Ile33 突變成 leucine，Ser213 突變成cysteine，而建構出的LCATS-AsDPE 已改善熱穩定性;然而若要以固定化方式重覆使用酵素以降低成本，則其熱穩定性應進一步提昇。本計畫以 PoPMuSic、 ENCoM 及 DUET server 評估可能的突變點，期望能透過蛋白質工程得到理想重組突變AsDPE (提高熱穩定性但酵素活性未降低之突變)，並利用具有理想重組突變AsDPE 的固定化菌體，於高溫下直接以便宜的D-果糖為原料，轉換成D-阿洛酮糖，並測試固定化菌體的重複使用性。本計畫最終目的是藉由理想重組突變AsDPE 的熱穩定性提昇，而能多次重複使用固定化菌體，在較高溫下由果糖生產D-阿洛酮糖，而降低D-阿洛酮糖的生產成本，進而增加D-阿洛酮糖在各方面的應用。" "D-Psicose is a rare sugar which contains special biological activities and can be used in many foods and dietary supplements to replace sucrose as a sweetener. It shows low glycemic response and produces less body fat accumulation, and FDA has approved it as generally regarded as safe in 2012. However, the scarcity and high cost of D-psicose limit the pursuit of more applications and research. The production of this rare sugar can be achieved by converted from D-fructose by D-psicose 3-epimerase (DPE). We have previously cloned the gene of Agrobacterium sp. ATCC 31750 DPE (AtDPE) and expressed the gene in E. coli. All of the currently studied DPEs are temperature sensitive. In previous proposal we have constructed the C’ATS-AsDPE containing an additional ATS sequence at its C-terminal by protein engineering and increasing its thermostability. We also have constructed LCATS-AsDPE by further mutating Ile33 and Ser213 to leucine and cysteine, respectively, to obtain an improved thermostability. However, considering the reuse of the enzyme by immobilized cells to reduce the production cost, the currently improved thermostability is still not enough. In this proposal we will evaluate the possible single point mutations by PoPMuSic, ENCoM and DUET servers, and apply protein engineering to obtain an ideal recombinant mutant AsDPE which has an enhanced thermostability but no declined activity. The immobilized cells which contain the ideal recombinant mutant AsDPE will be used to converting the inexpensive D-fructose to D-psicose at high temperature, and the reuse of the immobilized cells will also be evaluated. Our goal is to obtain the ideal thermostable recombinant mutant AsDPE and reuse the immobilized cells many times at high temperature to reduce the cost of D-psicose production from D-fructose. The reduction in the production cost will accelerate their applications in many different areas."

Keyword(s)

D-阿洛酮糖表異構酶
熱穩定性
D-阿洛酮糖
蛋白質工程
固定化菌體
D-piscose 3-epimerase
thermostability
D-psicose
protein engineering
immobilized cells

DSpace CRIS

Protein Engineering of D-Psicose 3-Epimerase to Enhance Its Thermostability and the Production of D-Psicose from Fructose by Immobilized Cells

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