Modification of enzyme function by carbohydrate chain addition

利用同源性较高的水稻α-淀粉酶同工酶Amy 1A和Amy 3D构建了两种嵌合酶，虽然这两种同工酶在氨基酸序列上具有较高的同源性，但在可溶性淀粉和寡糖水解方面表现出不同的反应特性。研究了嵌合酶Amy 1A/3D和Amy 3D/1A的反应特性。由于一种同工酶Amy 1A和一种嵌合酶Amy 3D/1D具有N-连接的糖链，因此糖链的作用被广泛研究。(1)Amy 3D/1A的热稳定性低于其他酶（Amy 1A、Amy 3D和Amy 1A/3D），其中Amy 3D/1A含有糖链。虽然我们以前的研究表明，碳水化合物链显着影响热稳定性，Amy 3D/1A没有显示出高的热稳定性，因为松散的构象包装。(2)Amy 3D/1A对可溶性淀粉的反应活性高于Amy 3D，说明糖链的存在对酶反应有促进作用。(3)酶的N-末端附近的三级结构对酶结构的“刚性”有显著影响，从而影响水解效率。另一方面，桶结构影响寡糖的水解效率。由于糖链位于桶结构外表面靠近活性裂缝的位置，糖链可能与长底物可溶性淀粉发生相互作用，因此，在桶结构外表面靠近活性裂缝的位置产生N-糖基化位点，可以改善α-淀粉酶的反应性能，特别是对可溶性淀粉的反应性能。

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.