Protein Engineering of Psicose 3-Epimerase and Rhamnose Isomerase to Increase Their Enzymatic Activities for the Production of Psicose and Allose at High Temperature

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Project title

Code/計畫編號

MOST103-2313-B019-004

Translated Name/計畫中文名

以蛋白質工程提昇阿洛酮糖表異構酶與鼠李糖異構酶在高溫下生產阿洛酮糖及阿洛糖之酵素活性

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=8347393

Year

2014

Start date/計畫起

01-08-2014

Expected Completion/計畫迄

01-07-2015

Bugetid/研究經費

1324千元

ResearchField/研究領域

生物技術(農)
食品科技(農)

"D-阿洛糖及 D-阿洛酮糖屬於稀有糖類，可應用於食品工業、製藥業、營養產業等，但由於產量稀少價格昂貴，造成許多研究及應用上的限制。生產此兩種稀有糖類，可以 D-阿洛酮糖表異構酶 (DPE)將 D-果糖轉換成 D-阿洛酮糖，再以 L-鼠李糖異構酶 (L-RhI) 將 D-阿洛酮糖轉換成 D-阿洛糖。我們已分別從 Agrobacterium sp. ATCC 31750 與 Thermoanaerobacterium saccharolyticum NTOU1 基因體，擴增 DPE 及 L-RhI 基因，並選殖入大腸桿菌中表現。純化後 DPE 的比活性為目前 DTE-家族酶中比活性最高，但熱穩定性不佳，且純化過程 DPE 出現沉澱，致使酵素活性產量降低。T. saccharolyticum NTOU1 L-RhI 則有良好熱穩定性，為目前酵素活性產量最高之 L-RhI，但此 L-RhI 對 D-阿洛糖的 KM值過大，造成對 D-阿洛糖的催化效率較差，若能降低此 KM 值，將可提高其對於 D-阿洛酮糖及 D-阿洛糖轉換的催化效率。本計畫期望透過蛋白質工程得到理想重組突變 DPE (提昇酵素活性、熱穩定性及酵素活性產量)與理想突變 L-RhI (對於 D-阿洛酮糖及 D-阿洛糖轉換有較佳催化效率)，使此二酵素於高溫下，能直接以便宜的 D-果糖為原料，轉換成 D-阿洛酮糖及 D-阿洛糖。本計畫最終目的是提昇 D-阿洛糖生產相關酵素在高溫下生產 D-阿洛酮糖及 D-阿洛糖的活性，而降低 D-阿洛酮糖及 D-阿洛糖的生產成本，進而增加 D-阿洛酮糖及 D-阿洛糖在各方面的應用。" "D-Allose and D-psicose, which are rare sugars, are able to be applied in the food, medical and nutritional industries. However, the scarcity and high cost of D-allose and D-psicose limit the pursuit of more applications and research. The production of these two rare sugars can be achieved by a two-step reaction. D-psicose is first produced from D-fructose by D-psicose 3-epimerase (DPE), and then isomerized to D-allose by L-rhamnose isomerase (L-RhI). In previous proposal we have cloned the genes of DPE and L-RhI from the genomes of Agrobacterium sp. ATCC 31750 and Thermoanaerobacterium saccharolyticum NTOU1, respectively, and these two genes have been expressed in E. coli, respectively, to obtain DPE and L-RhI. The purified DPE has the highest specificity among the known DTEase family enzymes, while it has poor thermostability and the precipitation occurred during purification has resulted the decrease in the recovery of enzyme activity. The yield of active T. saccharolyticum NTOU1 L-RhI expressed in E. coli was much higher than that of other known L-RhIs. This L-RhI possesses good thermostability, while the KM value against D-allose is largest among all the characterized L-RhIs. If the KM value against D-allose can be reduced through protein engineering, the catalytic efficiency for the isomerization between D-psicose and D-allose will increase. In this proposal we will try to apply protein engineering to obtain an ideal recombinant mutant DPE and an ideal mutant L-RhI. The ideal recombinant mutant DPE should possess a better enzyme activity, better thermostability, and higher activity recovery, while the ideal mutant L-RhI should has higher catalytic efficiency for the isomerization between D-psicose and D-allose. We would like to use these ideal mutant enzymes to produce D-allose and D-psicose from the low priced 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
L-鼠李糖異構酶
D-阿洛酮糖
D-阿洛糖
L-鼠李糖
蛋白質工程
D-piscose 3-epimerase
L-rhamnose isomerase
D-psicose
D-allose
potein engineering

DSpace CRIS

Protein Engineering of Psicose 3-Epimerase and Rhamnose Isomerase to Increase Their Enzymatic Activities for the Production of Psicose and Allose at High Temperature

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