Modification of enzyme function by carbohydrate chain addition

通过碳水化合物链添加改变酶的功能

• 批准号: 06650913
• 负责人: TERASHIMA Masaaki
• 金额: $ 1.28万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06650913/
• 关键词: alpha-amylase; reaction kinetics; carbohydrate chain; enzyme; protein engineering; 分泌生産

Two chimeric enzymes were engineered from highly homologous rice alpha-amylase isozymes Amy1A and Amy3D.Although these two isozymes show high homology in amino acid sequences, they showed different reaction properties in soluble starch and oligosaccharide hydrolysis. The reaction properties of chimeric enzymes Amy1A/3D and Amy3D/1A were studied. Since one isozyme Amy1A and one chimeric enzyme Amy3D/1D have N-linked carbohydrate chain, effects of carbohydrate chain were extensively studied.(1) Thermostability of Amy3D/1A,which has the carbohydrate chain, were lower than other enzymes (Amy1A,Amy3D and Amy1A/3D). Although our previous study suggests that the carbohydrate chain significantly affects thermostability, Amy3D/1A did not show high thermostability because of a loose conformational packing.(2) Amy3D/1A showed higher reactivity to soluble starch than Amy3D.This result suggested that the carbohydrate chain enhances the enzyme reaction.(3) Tertiary structure near N-terminal end of the enzyme has significant effect on the "stiffness" of the enzyme structure, and affects the hydrolysis efficiency. The barrel structure, on the other hand, affects the hydrolysis efficiency of oligosaccharides. Since the carbohydrate chain is positioned outer surface of the barrel structure near the active cleft, the carbohydrate chain might interact with long substrate soluble starch.Thus, it was suggested that reaction property of alpha-amylase, especially that to soluble starch, can be improved by creating N-glycosylation site at the outer surface of the barrel structure near the active cleft.

两种嵌合酶是由高度同源的水稻α-淀粉酶同工酶Amy1A和Amy3D改造而来。虽然这两种同工酶在氨基酸序列上表现出高度同源性，但它们在可溶性淀粉和低聚糖水解中表现出不同的反应特性。研究了嵌合酶Amy1A/3D和Amy3D/1A的反应特性。由于一种同工酶Amy1A和一种嵌合酶Amy3D/1D具有N-连接的糖链，糖链的影响被广泛研究。(1)具有糖链的Amy3D/1A的热稳定性低于其他酶(Amy1A、Amy3D)和 Amy1A/3D）。尽管我们之前的研究表明碳水化合物链显着影响热稳定性，但由于松散的构象堆积，Amy3D/1A并未表现出高热稳定性。（2）Amy3D/1A对可溶性淀粉的反应性比Amy3D更高。这一结果表明碳水化合物链(3)酶近N端的三级结构对酶结构的“刚性”影响显着，影响水解效率。另一方面，桶结构影响低聚糖的水解效率。由于糖链位于桶结构的外表面靠近活性裂口的位置，糖链可能与长底物可溶性淀粉相互作用。因此，表明可以改善α-淀粉酶的反应特性，特别是对可溶性淀粉的反应特性通过在活性裂口附近的桶状结构的外表面创建 N-糖基化位点。