Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Mol Biotechnol. 2011 Mar;47(3):234-42. doi: 10.1007/s12033-010-9337-7.
Cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 and its recombinant enzyme exhibit three isoforms (I, II, and III) with the same apparent size but different charge. Here, we demonstrate for the first time that the deamidation of labile Asns causes the change in molecular forms of CGTase. The faster increase in number of isoforms was observed upon incubation in deamidation buffer at the more alkaline pH. The increase in levels of isoform II and III over time correlated with the increase in isoaspartate, a unique deamidation product. The predicted labile Asns were individually mutated to Asp, then the selected mutant and wild type isoforms were tryptic digested and labile Asns were investigated by MALDI-TOF. From the results, Asn427 was the most susceptible residue for deamidation, followed by Asn336, Asn415, and Asn567. In addition, Gln389 might also share a role. The advantage of using appropriate CGTase isoform in cyclodextrin production is reported.
来自短小芽孢杆菌 RB01 的环糊精葡萄糖基转移酶 (CGTase)及其重组酶表现出三种同工酶（I、II 和 III），它们具有相同的表观大小但电荷不同。在这里，我们首次证明不稳定天冬酰胺的脱酰胺作用导致 CGTase 分子形式发生变化。在更碱性的脱酰胺缓冲液中孵育时，同工酶的数量更快增加。同工酶 II 和 III 的水平随时间的增加与异天冬氨酸（一种独特的脱酰胺产物）的增加相关。单独将预测的不稳定天冬酰胺突变为天冬氨酸，然后对选定的突变体和野生型同工酶进行胰蛋白酶消化，并通过 MALDI-TOF 研究不稳定天冬酰胺。结果表明，天冬酰胺 427 是最易脱酰胺的残基，其次是天冬酰胺 336、天冬酰胺 415 和天冬酰胺 567。此外，谷氨酰胺 389 也可能起作用。报道了在环糊精生产中使用适当的 CGTase 同工酶的优势。
