Chang H Y, Irwin P M, Nikolov Z L
Department of Food Science and Human Nutrition, Iowa State University, Ames 50011, USA.
J Biotechnol. 1998 Oct 27;65(2-3):191-202. doi: 10.1016/s0168-1656(98)00115-1.
Cyclodextrin glycosyltransferase (CGTase) is an industrially important enzyme that produces cyclodextrins (CD) from starch by intramolecular transglycosylation. CGTase consists of five globular domains labeled A through E. To better understand the role of domain E in CGTase catalysis, we have constructed several mutants of Bacillus macerans CGTase. Removing the entire E domain resulted in an inactive enzyme. Adding six amino acids between domains D and E caused a decrease in activity and thermostability. Replacing domain E with the similar starch-binding domain from Aspergillus awamori glucoamylase I caused a drastic decrease in activity, indicating the necessity of correct alignment of bound substrate. Substituting tyrosine residue 634 (Tyr634) with phenylalanine had very little effect on activity or thermostability. Substituting Tyr634 with glycine resulted in a 25% increase of specific cyclization and starch-hydrolyzing activities compared with that of the wild-type enzyme. The latter mutant was less thermostable. The results of this study indicate that domain E is important for the stability and integrity of B. macerans CGTase.
环糊精糖基转移酶（CGTase）是一种在工业上具有重要意义的酶，它通过分子内转糖基作用从淀粉产生环糊精（CD）。CGTase由五个标记为A至E的球状结构域组成。为了更好地理解结构域E在CGTase催化中的作用，我们构建了几种解淀粉芽孢杆菌CGTase的突变体。去除整个E结构域会导致酶失活。在结构域D和E之间添加六个氨基酸会导致活性和热稳定性降低。用泡盛曲霉葡萄糖淀粉酶I的类似淀粉结合结构域替换结构域E会导致活性急剧下降，这表明结合底物正确排列的必要性。将酪氨酸残基634（Tyr634）替换为苯丙氨酸对活性或热稳定性影响很小。将Tyr634替换为甘氨酸导致与野生型酶相比，特定环化和淀粉水解活性增加25%。后一种突变体的热稳定性较低。本研究结果表明，结构域E对解淀粉芽孢杆菌CGTase的稳定性和完整性很重要。
