Cornett Catherine A G, Fang Tsuei-Yun, Reilly Peter J, Ford Clark
Department of Food Science and Human Nutrition, 2114 Sweeney Hall, Iowa State University, Ames, IA 50011-2230, USA.
Protein Eng. 2003 Jul;16(7):521-9. doi: 10.1093/protein/gzg066.
Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N-terminus, catalytic domain + linker + starch-binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIDeltaL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII approximately GAII approximately GAI > RGAIDeltaL approximately RGAI approximately GAE. Insoluble starch hydrolysis rates were GAI > RGAIDeltaL > RGAI >> GAE approximately RGAII > GAII, while insoluble starch-binding capacities were GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE. These results indicate that: (i) moving the SBD to the N-terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.macerans cyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch.
黑曲霉葡萄糖淀粉酶（GA）主要由两种形式组成，即GAI [从N端起，催化结构域 + 连接区 + 淀粉结合结构域（SBD）] 和GAII（催化结构域 + 连接区）。这些结构域经过改组，形成了RGAI（SBD + 连接区 + 催化结构域）、RGAIDeltaL（SBD + 催化结构域）和RGAII（连接区 + 催化结构域），其结构域由功能而非三级结构定义。此外，浸麻芽孢杆菌环糊精葡聚糖转移酶的SBD取代了与之密切相关的黑曲霉GA的SBD，得到GAE。可溶性淀粉水解速率的降低顺序为：RGAII≈GAII≈GAI > RGAIDeltaL≈RGAI≈GAE。不溶性淀粉水解速率为：GAI > RGAIDeltaL > RGAI >> GAE≈RGAII > GAII，而不溶性淀粉结合能力为：GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE。这些结果表明：（i）将SBD移至N端或替换天然SBD会对可溶性淀粉水解产生一定影响；（ii）SBD的位置显著影响不溶性淀粉的结合和水解；（iii）不溶性淀粉水解与其被SBD的结合不完全相关；（iv）将浸麻芽孢杆菌环糊精葡聚糖转移酶的SBD置于连接区末端，而非与酶的其余部分紧密相连，会严重降低其结合和水解不溶性淀粉的能力。
