Expression, Purification, Characterization, and Directed Evalution of L-Arabinose Isomerase (I)

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Project title

Code/計畫編號

NSC96-2628-B019-002-MY3

Translated Name/計畫中文名

阿拉伯糖異構脢之表現、純化、特性探討及定向演化 (1/3)

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=1451596

Year

2007

Start date/計畫起

01-08-2007

Expected Completion/計畫迄

31-07-2008

Bugetid/研究經費

1265千元

ResearchField/研究領域

食品科技(農)

" 塔格糖具有與蔗糖相似的風味及物理特性，是一種天然且低卡路里的酮己糖，已被美國食品藥物管理局認可為GRAS，可以安全地取代蔗糖。此糖可用於第二型糖尿病患者的治療;也可添加於食品中，以減少肥胖及齵齒。目前，發展較經濟的方法生產塔格糖，逐漸受到多方的注意。阿拉伯糖異構酶（L-arabinose isomerase, AI）除了可催化阿拉伯糖之異構化而產生核酮糖之外；因半乳糖與阿拉伯糖的結構相似，也可藉由催化半乳糖之異構化而產生塔格糖。然而，由半乳糖轉換成塔格糖之轉換速率，目前仍遠低於阿拉伯糖轉換成核酮糖之轉換速率。若能改變AI 的基質特異性，而提高AI 對半乳糖的轉換速率，將可提昇塔格糖之產量。另外，假以高溫菌產生之熱穩定AI 生產塔格糖，在高溫下進行反應，可進一步降低微生物污染。本計畫將以聚合酶鏈反應，配合其他基因重組技術，由高溫菌Geobacillus kaustophilus 及中溫菌Bacillus licheniformis 的基因體中，將araA 的基因分別擴增、選殖至表現載體上，再轉形到大腸桿菌中表現，以生產兩種具有不同熱穩定性的AI。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，以獲得塔格糖生產的最適條件。最後，嘗試將以DNA family shuffling 進行定向演化，以改善酵素的基質特異性，而提高塔格糖的產量。 " " The taste and physical properties of D-tagatose are similar to sucrose. D-tagatose is a low-calorie natural ketohexose, and approved as a 「generally recognized as safe (GRAS)」 material byAmerican Food and DrugAdministration. This sugar can replace the usages of sucrose without any safety concerns, and provide many advantages over sucrose. For example, it can be used in the therapies of type 2 diabetics, and be also added into foods to reduce obesity and dental cavity. Developing inexpensive methods to produce D-tagatose have been received more and more attention. L-Arabinose isomerase (AI) mainly catalyzes the isomerization of L-arabinose into L-ribulose, whereas it also catalyzes the isomerization of D-galactose into D-tagatose due to the similar structures of L-arabinose and D-galactose. However, the conversion rate of D-galactose into D-tagatose is much less than that of L-arabinose into L-ribulose. If the substrate specificity of AI can be altered, the conversion rate of AI might be increased when D-galactose is used as substrate, and finally the yield of D-tagatose might be improved. On the other hand, the reaction can be carried out at high temperature to enhance reaction rate and to decrease the risk of contamination of reaction mixture by microorganisms when thermostable AI is used in the production of D-tagatose. In this proposal we will clone the araA genes from the genomes of thermophile Geobacillus kaustophilus and mesophile Bacillus licheniformis, respectively, and two genes will be expressed in E. coli, respectively, to obtain two recombinant AIs, which have different thermostabilities.We will study different combinations of promoters, hosts, and culture conditions to optimize the expression of the two araA genes.We will purify recombinant AIs by ion-exchange and gel filtration chromatographies. The characteristics and kinetic studies of those two enzymes will be studied to gain a better condition for the production of D-tagatose from D-galactose. Finally, directed evolution by DNA family shuffling will be carried out to improve the substrate specificity of AI in order to enhance the yield of D-tagatose. "

Keyword(s)

阿拉伯糖異構酶
半乳糖
塔格糖
DNA family shuffling
L-arabinose isomerase
D-tagatose
D-galactose
DNA family shuffling

DSpace CRIS

Expression, Purification, Characterization, and Directed Evalution of L-Arabinose Isomerase (I)

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