Characterizing a Recombinant L-Ribose Isomerase from Actinotalea Fermentans and Enhancing Its Thermostability(II)

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Project title

Code/計畫編號

MOST106-2320-B019-003-MY2

Translated Name/計畫中文名

源自Actinotalea fermentans的重組L-核糖異構?之特性探討與熱穩定性提昇(2/2)

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=12378186

Year

2018

Start date/計畫起

01-08-2018

Expected Completion/計畫迄

31-07-2019

Bugetid/研究經費

1500千元

ResearchField/研究領域

食品科技(農)

"L-核糖為稀有糖類，可用於合成抗病毒及抗癌藥物，但由於產量稀少價格昂貴，造成研究及應用上的限制。利用酵素法生產L-核糖，可先以L-阿拉伯糖異構酶(L-AI)，將較便宜的 L-阿拉伯糖轉換成L-核酮糖，接著再以L-核糖異構酶(L-RI)，將L-核酮糖轉換成L-核糖。 Actinotalea fermentans ATCC 43279 含有一假設蛋白質與其他三種L-RI 皆有75%以上相似度，推論可能為L-RI (AfRI)。將此可能L-RI 基因合成，並置於大腸桿菌中表現。初步結果顯示，AfRI 酵素活性表現量是其他來源L-RI 活性表現量的4~8 倍以上;而其熱穩定性則與 Geodermatophilus obscurus DSM 43160 L-RI 類似，於40℃保溫2 小時仍有90 %相對活性。為了使AfRI 能搭配熱穩定之Thermoanaerobacterium saccharolyticum L-AI (TsAI)進行催化反應，由L-阿拉伯糖生產L-核糖，AfRI 之熱穩定性應再提昇。本計畫第一年針對重組AfRI 進行大量表現、純化及特性探討;由電腦模擬(computer simulation)產生AfRI 模擬結構，再以PoPMuSic、 ENCoM、MAESTROweb 及 DUET server 評估可能突變點，進行定位突變以獲得突變AfRI 基因。第二年將突變AfRI 基因表現，以獲得熱穩定性提昇之突變AfRI，再將數個熱穩定性提昇之突變AfRI，藉由多重定位突變法組合，獲得熱穩定性提昇更多之多重突變AfRI，使得可以搭配熱穩定TsAI 在較高溫下進行催化反應，由較便宜的L-阿拉伯糖直接生產L-核糖，降低L-核糖的生產成本，最終增加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. Recently, many studies have 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 (L-RI). Actinotalea fermentans ATCC 43279 have a putative protein which has more than 75% similarity comparing to other three L-RIs. Recently, we overexpressed the putative and chemically synthesized L-RI gene of Actinotalea fermentans ATCC 43279 in Escherichia coli. A high yield of active Actinotalea fermentans ATCC 43279 L-RI (AfRI), 23,800 U/L was obtained, and the yield was four to eight times more than those obtained from other expressed L-RIs. AfRI and Geodermatophilus obscurus DSM 43160 L-RI have similar thermostability, they all maintained 90% activity after incubation at 40℃for 2 h. To combine AfRI and thermostable Thermoanaerobacterium saccharolyticum L-AI (TsAI) in the production of L-ribose from L-arabinose, the thermostability of AfRI should be further improved. In the first year of this proposal, we will overexpress, purify, and characterize the recombinant AfRI; and we will evaluate the possible single point mutations of simulated AfRI structure by PoPMuSic, ENCoM, MAESTROweb and DUET servers, and apply site-directed mutagenesis to obtain mutant AfRI genes. In the second year of this proposal, we will express and obtain thermostability-enhanced mutant AfRIs, then apply multiple site-directed mutagenesis to combine and construct an ideal multiple-mutant AfRI which has a further enhanced thermostability but without declined activity. The production of L-ribose from the inexpensive L-arabinose at higher temperature could be achieved by using the thermostable TsAI and the ideal thermostability-enhanced mutant AfRI. The reduction in the production cost will accelerate L-ribose applications in many different areas."

Keyword(s)

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

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Characterizing a Recombinant L-Ribose Isomerase from Actinotalea Fermentans and Enhancing Its Thermostability(II)

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