Modification of enzyme function by carbohydrate chain addition

由高度同源的水稻α-淀粉酶同工酶Amy1A和Amy3D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很高的同源性，但它们在可溶性淀粉和低聚糖水解方面表现出不同的反应性质。研究了嵌合酶Amy1A/3D和Amy3D/1A的反应性质。由于一种同工酶Amy1A和一种嵌合酶Amy3D/1D含有N-连接的碳水化合物链，因此人们对碳水化合物链的影响进行了广泛的研究：(1)含有碳水化合物链的Amy3D/1A的热稳定性低于其他酶(Amy1A、Amy3D和Amy1A/3D)。虽然我们以前的研究表明碳水化合物链对热稳定性有显著影响，但由于构象堆积松散，Amy3D/1A没有表现出高的热稳定性。(2)Amy3D/1A对可溶性淀粉的反应活性高于Amy3D。这一结果表明碳水化合物链促进了酶的反应。(3)酶N端附近的三级结构对酶结构的刚性有显著影响，并影响酶的水解效率。另一方面，桶的结构影响低聚糖的水解率。由于糖链位于筒状结构外表面靠近活性裂隙的位置，因此糖链可能与长底物可溶性淀粉相互作用，因此，可以通过在活性裂隙附近的桶状结构外表面建立N-糖基化位点来改善α-淀粉酶的反应性质，特别是对可溶性淀粉的反应性质。

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.