Protein Engineering of L-Ribose Isomerase to Enhance Its Industrial Utilization

View Statistics Email Alert RSS Feed

Information

Details

Project title

Code/計畫編號

MOST104-2313-B019-001

Translated Name/計畫中文名

以蛋白質工程提昇核糖異構酶的工業利用性

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=11475391

Year

2015

Start date/計畫起

01-08-2015

Expected Completion/計畫迄

31-07-2016

Bugetid/研究經費

1070千元

ResearchField/研究領域

生物技術(農)
食品科技(農)

"L-核糖 (L-ribose)屬於稀有糖類，可用於合成具有抗病毒及抗癌的核苷類似物 (nucleoside analogues)，包括 Tyzeka、Valtorcitabine、Clevudine、Elvucitabine、Emtriva 以及 Pentacept，使L-核糖受到廣泛的重視及研究，但由於產量稀少價格昂貴，造成研究及應用上的限制。以酵素法生產L-核糖，先以L-阿拉伯糖異構酶 (L-arabinose isomerase, L-AI)，將較便宜的L-阿拉伯糖 (L-arabinose)轉換成L-核酮糖 (L-ribulose)，接著再以L-核糖異構酶 (L-ribose isomerase, L-RI)，將L-核酮糖轉換成L-核糖。我們已將Thermoanaerobacterium saccharolyticum NTOU1 L-AI and Geodermatophilus obscurus DSM 43160 L-RI 的基因選殖、兩酵素完成特性化探討並應用於由L-阿拉伯糖生產L-核糖。與其他已知特性的L-RI 比較， G. obscurus DSM 43160L-RI 雖有較佳的熱穩定性，但對L-核糖的KM 值較大，造成對L-核糖的催化效率較差，若能降低此KM 值，將可提高其對於L-核酮糖及L-核糖轉換的催化效率。而且G. obscurus DSM 43160L-RI 的熱穩定性，雖較其他來源的酵素佳，卻仍無法在60℃下與T. saccharolyticum NTOU1 L-AI 同時進行催化反應。以熱穩定的酵素生產糖類有許多好處，可提高反應速率及產量，並能提高反應物與產物的溶解度以及降低污染的發生。本計畫將配合電腦模擬與蛋白質工程的方式得到理想重組突變 L-RI，以提昇熱穩定性及其對於L-核酮糖及L-核糖轉換的催化效率。我們將製備固定化菌體，使得在固定化菌體內的酵素，在不需純化分離的情況下，可重複使用，也期望能藉此提高反應溫度。本計畫的目的是提昇L-RI 在較高溫下生產L-核糖的活性，並使用含理想重組突變L-RI 固定化菌體提昇L-RI 的工業利用性，由低價L-阿拉伯糖生產L-核糖，進而降低L-核糖的生產成本，最終能增加L-核糖在各方面的應用。" "L-ribose, a rare sugar, can be used as a precursor for the synthesis of L-nucleoside analogs, which are widely used as antiviral and anticancer drugs, such as Tyzeka,Valtorcitabine, Clevudine, Elvucitabine, Emtriva, and Pentacept. Recently, many studies have been focused on the applications and production of L-ribose. However, the scarcity and high cost of L-ribose limit the pursuit of more applications and research. The enzymatic method for the production of L-ribose is a two-step reaction. L-Ribulose is first produced from L-arabinose by L-arabinose isomerase (L-AI), and then can be isomerized to L-ribose by L-ribose isomerase. We have previously cloned the genes of Thermoanaerobacterium saccharolyticum NTOU1 L-AI and Geodermatophilus obscurus DSM 43160 L-RI, characterized these two enzymes, and applied them to produce L-ribose from L-arabinose. Compare to other characterized L-RIs, G. obscurus DSM 43160 L-RI possesses better thermostability, while its KM value against L-ribose is largest. If the KM value against L-ribose can be reduced through protein engineering, the catalytic efficiency again L-ribose will increase. In addition, the thermostability of G. obscurus DSM 43160 L-RI is still not good enough to work together with T. saccharolyticum NTOU1 L-AI at 60℃. In industrial processes, thermostable enzymes can provide a higher reaction rate and process yield, higher solubilities of substrates and products, and fewer contamination problems. In this proposal we will apply computer simulation and protein engineering to obtain an ideal recombinant mutant L-RI, which possesses better thermostability and better catalytic efficiency again L-ribose and L-ribulose. We will also prepare immobilized cells containing L-RI to obtain the enzyme without purification, to be reused for a long time, and to increase the reaction temperature. We would like to use immobilized cells containing an ideal recombinant mutant L-RI to increase its industrial utility and produce L-ribose from the low priced L-arabinose. The reduction in the production cost will accelerate the application of L-ribose in many different areas."

Keyword(s)

L-阿拉伯糖異構酶
L-核糖異構酶
L-阿拉伯糖
L-核糖
L-核酮糖
L-arabinose isomerase
L-ribose isomerase
L-arabinose
L-ribose
L-ribulose

DSpace CRIS

Protein Engineering of L-Ribose Isomerase to Enhance Its Industrial Utilization

Details

Description