Molecular Mechanism and Characterization of Glucan Modification by Mungbean Starch Biosynthetic Enzymes

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Project title

Code/計畫編號

NSC101-2313-B019-008

Translated Name/計畫中文名

以綠豆澱粉生合成酵素修飾葡聚糖之分子機轉與特性探討

Project Coordinator/計畫主持人

Yuan-Tih Ko

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=2635726

Year

2012

Start date/計畫起

01-08-2012

Expected Completion/計畫迄

31-07-2013

Bugetid/研究經費

1120千元

ResearchField/研究領域

食品科技(農)

本計畫是延伸先前國科會計畫以綠豆 (Vigna radiata; Vr) 澱粉生合成酵素為主題之連續性探討；在種子發育的澱粉累積期間，發現同時具有合成與磷解雙向功能的澱粉磷解酶 (starch phosphorylase, SP) 含量很多，以及在純化澱粉分支酶 (starch branching enzyme, SBE) 時，SP 會與SBE 和其他蛋白質形成複合物；因此，進一步根據綠豆澱粉生合成路徑可能存在一個Glc-1-P pathway 的推論，SBE 和SP 分子間會交互作用，共同生成支鏈聚葡糖分子。提出三年計畫；第一~二年，將合成修飾型基因，重新建構到適當表現載體與轉形到適當宿主，獲得 VrsbeI-modify 和VrsbeII-modify 的穩定株系進行表現適量化，並對個別修飾型重組酵素蛋白質之生化及催化特性進行基礎研究；藉由rSBEs-modify 與 rSP-modify 酵素之不同分子組合或共同在宿主表現 (coexpression) 形成的蛋白質複合物結合特性分析，探討酵素彼此間共同合作產生澱粉分子的機制與建立產物分析方法；接著，第二~三年，將大量製備rVrSP 和 rVrSBE，利用rVrSP 磷解產出的 Glc-1-P 於in vitro 合成不同分子結構葡聚醣與分析產物，進一步對不同產物進行安全性、升糖指數、消化率以及是否為抗性澱粉之功能性評估，以作為食品工業或生醫產業之利用性依據。本研究是導向型基礎研究，利用重組酵素進行體外合成類直鏈澱粉和類支鏈澱粉分子，以可獲得之葡聚糖與類澱粉分子產物形態，驗證 Glc-1-P pathway 在澱粉生合成路徑的存在，期望能將綠豆 SP 和 SBE 對綠豆澱粉合成的貢獻定位出來；本研究將導向SP/SBE 合成產物之特性與功能性開發，使 rVrSBEI、 rVrSBEII、rVrSP 酵素相關株系發展為生技酵素。This is a three-year project of an extended study from previous NSC projects. Based on recent findings, starch phosphorylase (SP), an elongation and phosphorolysis bidirectional enzyme, was expressed in high quantity during seed filling period, and SP, starch branching enzyme (SBE) and some proteins were found in complex forms. An inferential Glc-1-P pathway in mungbean (Vigna radiata; Vr) starch biosynthesis is thus proposed for the molecular interaction and in vitro glucan product investigation among mungbean SP and SBE isoforms. The first two years will construct modified VrsbeI and VrsbeII clones and to optimize their stable protein expression in appropriate host systems. Biochemical properties and catalytic characteristics will be studied for these recombinant enzymes from the modified clones, namely, VrsbeI-modify and VrsbeII-modify. Different molecular assemblies among modified rSBEI, rSBEII and SP, as well as gene coexpression system will be studied for enzyme interaction and establishing product analysis. The second to the third year will study the components, topology and prepare large amount of the synthesized or modified glucan products from the assembled or coexpressed protein complex, using the phosphorolyzed Glc-1-P by SP as a starting material. The functional potential of these glucan product varieties will be evaluated in the aspect of safety, glycemic index, digestibility and whether of resistant starch. In summary, this study is an application-oriented basic research. The in vitro synthesis of the linear amylose-like or the branched amylopectin-like, or enzymatically-modified glucans (EMG) by the recombinant clones will justify the proposed Glc-1-P pathway, and establish the roles of individual enzymes as well as SP/BE cooperation in the starch biosynthesis. The application of EMG will promote rVrSBE and rVrSP becoming industrial enzymes for the use in the food and biomedical biotechnology.

Keyword(s)

澱粉生合成酵素
澱粉分支酶
澱粉磷解酶
酵素性修飾葡聚醣
starch biosynthetic enzymes
starch branching enzyme
starch phosphorylase
enzymatically modified glucan

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Molecular Mechanism and Characterization of Glucan Modification by Mungbean Starch Biosynthetic Enzymes

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