The Cloning, Expression, Characterization, and Sugar Production of D-Allose-Producing Related Enzymes (III)

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Project title

Code/計畫編號

NSC99-2313-B019-012-MY3

Translated Name/計畫中文名

D-阿洛糖生產相關酵素之基因選殖、表現、特性及產糖條件探討 (3/3)

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=2396866

Year

2012

Start date/計畫起

01-08-2012

Expected Completion/計畫迄

31-07-2013

Bugetid/研究經費

1285千元

ResearchField/研究領域

食品科技(農)
生物技術(農)

D-阿洛糖 (D-allose) 為 D-葡萄糖的 C3 異構物，屬於稀有糖類，有減少組織損傷、降低節狀核嗜中性白血球產生、提升轉殖器官的存活率等功能，近年來因被發現可以抑制多種癌細胞生長，使 D-阿洛糖受到廣泛的重視及研究，但由於產量稀少價格昂貴，造成許多研究及應用上的限制。以酵素法生產 D-阿洛糖是先以 D-塔格糖表異構酶 (D-tagatose 3-epimerase, D-TE) 將果糖轉換成 D-阿洛酮糖 (D-psicose)，再以 L-鼠李糖異構酶 (L-Rhamnose isomerase, L-RhI) 將 D-阿洛酮糖轉換成 D-阿洛糖。此酵素轉換過程不僅可得到 D-阿洛糖，其中間產物 D-阿洛酮糖也是一種多功能且高價的稀有糖類類。 D-TE 為一種酮糖 C3 異構酶，能催化許多酮糖間的轉換，是生成許多稀有糖類的關鍵酵素。 L-RhI 具有將 L-鼠李糖 (L-Rhamnose) 轉換為 L-鼠李酮糖 (L-Rhamnulose) 的能力，近年來發現 L-RhI 尚可轉換多種基質，可應用於量產許多昂貴的糖類。然而已發現之 D-TE 和 L-RhI ，大部分的熱穩定性均不佳，以至於在應用上受到限制。本計畫將以聚合酶鏈反應，配合其他基因重組技術，由高温菌 Thermotoga maritima DSM 3109 及 Thermoanaerobacterium sp. NTOU2 的基因體中，將 D-te 和 L-rhi 基因分別擴增、選殖至表現載體，再轉形到大腸桿菌中表現，以生產熱穩定之 D-TE 及 L-RhI。我們將探討不同的起動子、不同宿主及培養條件對於重組酵素生產之影響，並利用離子交換層析及膠體過濾層析純化酵素。再以純化後之重組酵素進行特性探討及動力學分析，以暸解兩種酵素之反應特性，尋求適當的產糖條件，因而提高D-阿洛酮糖及 D-阿洛糖的產量，以降低其生產成本，進而增加 D-阿洛酮糖及 D-阿洛糖在各方面的應用。 D-allose, a C3 epimer of D-glucose, has been shown to be able to reduce the injury of ischemia reperfusion, to inhibit the segmented neutrophil production, and to increase the survival rate of allograft. Recently, many studies have been focused on the applications and production of D-allose because D-allose was demonstrated to suppress the growth rate of many different types of carcinoma. However, the scarcity and high cost of D-allose limit the pursuit of more applications and research. The enzymatic method for the production of D-allose is a two-step reaction. D-psicose is first produced from D-fructose by D-tagatose 3-epimerase (D-TE), and then isomerized to D-allose by L-rhamnose isomerase (L-RhI). In this enzymatic method, the intermediate D-psicose is also a valuable rare sugar. D-TE, which also catalyzes the epimerization of various ketoses at the C3-position, is expected to be a key enzyme for the production of other valuable rare sugars. L-rhamnose isomerase, which isomerizes L-rhamnose to L-rhamnulose, has also been shown to be able to catalyze many other substrates. This ability has the potential for the mass production of various rare and commercially expensive sugars. In industrial processes, thermostable enzymes can provide a higher reaction rate and process yield, higher solubilities of substrates and products, and fewer contamination problems. However, most of the D-TE and L-RhI mentioned above are thermolabile, and therefore fall short of the expected applications. In this proposal we will clone the D-te and L-rhi genes from the genomes of thermophile Thermotoga maritima DSM 3109 and Thermoanaerobacterium sp. NTOU2, respectively, and these two genes will be expressed in E. coli, respectively, to obtain recombinant D-TE and L-RhI. We will study different combinations of promoters, hosts, and culture conditions to optimize the expression of the D-te and L-rhi genes. We will purify recombinant D-TE and L-RhI by ion-exchange and gel filtration chromatography. The characteristics and kinetic studies of those two enzymes will be studied. Finally, we expect to optimize the reaction conditions of D-allose and D-psicose productions to increase their yields. The reduction in the production cost will accelerate their applications in many different areas.

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The Cloning, Expression, Characterization, and Sugar Production of D-Allose-Producing Related Enzymes (III)

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