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.

从高度同源的米α-淀粉酶同工酶AMY1A和AMY3D进行设计了两种嵌合酶。尽管这两种同工酶在氨基酸序列中表现出很高的同源性，但它们在可溶性淀粉和寡聚糖水水解中显示出不同的反应特性。研究了嵌合酶AMY1A/3D和AMY3D/1A的反应特性。由于一个同工酶AMY1A和一种嵌合酶Amy3d/1d具有N-连接的碳水化合物链，因此对碳水化合物链的效果进行了广泛的研究。（1）AMY3D/1A的热稳定性，其具有碳水化合物链的热稳定性，其具有碳水化合物链，比其他酶（AMY1A，AMY1A，AMY1A，AMY13D和AMYY1A和AMYY1A）低。 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酶结构的“刚度”，并影响水解效率。另一方面，枪管结构会影响寡糖的水解效率。由于碳水化合物链位于桶结构的外表面附近，因此碳水化合物链可能与长的底物可溶性淀粉相互作用。因此，建议通过创建靠近活性lectfft的核心基表面的N-糖基化位点来改善α-淀粉酶的反应特性，尤其是可溶性淀粉的反应性能。