Molecular Structure and Characterization of Modified Α-Glucan Polymers by Enzymatic System

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簡歷

基本資料

Project title

Code/計畫編號

MOST106-2320-B019-005

Translated Name/計畫中文名

以酵素系統修飾葡萄糖聚合物之分子結構與特性探討

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=12229682

Year

2017

Start date/計畫起

01-08-2017

Expected Completion/計畫迄

31-07-2018

Bugetid/研究經費

1120千元

ResearchField/研究領域

食品科技(農)

本計畫動機於在植物發育期間，可雙向反應的澱粉磷解酶 (starch phosphorylase, SP) 與澱粉分支酶 (starch branching enzyme, SBE)會形成複合物而生合成澱粉分子，進而假設體外表現之SBE 和 SP 酵素分子，若以不同比例與時序利用不同的基質成為SBE/SP 酵素性修飾澱粉系統，將獲得不同分子結構的葡聚糖。SP 與SBE 基因來自於綠豆 (Vigna radiata; Vr), 即VrsbeI (綠豆澱粉分支酶I 型，基因註冊碼GenBank accession no.AY667492), VrsbeII (綠豆澱粉分支酶I，GenBank accession no.AY622199)與綠豆澱粉磷解酶Vrsp (GenBank accession no.GQ465004)。前兩年將 VrSBEI、 II 之 N 端與C 端的胺基酸序列在資料庫比對，設計出最適化鏈轉移長度特性的“衍生型Vrsbe”，以人工合成修飾與取代之核酸序列，構建接入表現載體與轉形到E. coli 宿主，分別獲得穩定的株系進行酵素活性表現適量化，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分析。也將重組活性VrSP 酵素以綠豆澱粉適當分子大小為基質，進行磷解反應適量化，產出Glucose-1-phosphate 單體，作為其合成方向的基質，進行不同長度的直鏈葡聚糖合成，獲得作用動力學數據。第二~三年，將製備rVrSP 和 rVrSBEs，運用原生型與不同衍生型VrSBEs 分別作用於不同基質，包括VrSP 產出之不同鏈長直鏈葡聚糖、分離純化出的澱粉顆粒或作用於糊化澱粉，階段性或連續性產生出不同分子大小、分支度與分支鏈長度不同的葡聚糖，進行動力學探討，建立SBE/SP 酵素系統；收集之多型性類直鏈澱粉聚合物、類支鏈澱粉聚合物，類肝糖葡聚糖分子產物，進行益生素潛力、升糖指數、消化率以及抗性澱粉之功能性探討。總之，本研究是應用型的基礎研究。體外合成葡萄糖聚合物和生物性修飾澱粉將促使個別rVrSBEs 和 rVrSP 以及SP / BE 系統提供α-葡聚醣在食品和生藥中的利用。The proposal was motivated by recent findings that starch phosphorylase (SP), a phosphorolysis and elongation bidirectional enzyme, forms complex with starch branching enzyme (SBE) for starch biosynthesis during plant development. Thus, hypothesizing that with different reaction sequence, in vitro synthesis of linear amylose-like or branched amylopectin-like, glycogen-like or enzymatically-modified glucans (EMG) of diverse molecular structure by a SBEs/SP enzyme system from various substrate types would be feasible. The enzyme genes will be from mungbean (Vigna radiata; Vr), namely, VrsbeI (GenBank accession no.AY667492), VrsbeII (GenBank accession no. AY622199) and Vrsp (GenBank accession no.GQ465004). The first two years will search/compare/design the N- and C-terminal sequence features of SBEs in database for various glucan transferring-length catalytic activities. Artificial synthesized nucleotides will perform N- and C-terminal gene replacement of VrsbeI and II (i.e. Vrsbe-derivatives) by molecular recombination approach, then construct into expression vector and optimize each of their stable and active protein expression from E. coli host. Their biochemical properties, catalytic characteristics and product analysis will be established. rVrSP will phosphorolyze mungbean starch into G-1-P monomers which will be used to prepare oligomeric linear glucans by its reversed elongation activity. The last two years, wild-type and derivative VrSBEI, VrSBEIIs will act on various α-glucan substrates, starch granules, and gelatinized glucans to prepare glucan of various size, degree of branching, degree of polymerization for kinetic characterization. Aspects of glycemic index, prebiotic potential, digestibility and comparability to resistant starch will be evaluated. In summary, this study is an applied basic research. The in vitro synthesis and enzymatically-modified starch will justify the roles of individual rVrSBEs and rVrSP enzymes as well as SP/BE enzyme system forα-glucan polymer use in food and medicine.

Keyword(s)

澱粉分支酶
澱粉磷解酶
酵素性修飾葡聚醣
starch branching enzyme
starch phosphorylase
enzymatically modified glucan

DSpace CRIS

Molecular Structure and Characterization of Modified Α-Glucan Polymers by Enzymatic System

基本資料

Description