Nilsson Carina, Nilsson Frida, Turner Pernilla, Sixtensson Martin, Nordberg Karlsson Eva, Holst Olle, Cohen Arieh, Gorton Lo
Department of Analytical Chemistry, Lund University, P.O. Box 124, 221 00, Lund, Sweden.
Anal Bioanal Chem. 2006 Aug;385(8):1421-9. doi: 10.1007/s00216-006-0570-7. Epub 2006 Jun 28.
In this work, a real-time sampling/analytical method for on-line measurements of two newly discovered cyclomaltodextrinases (CDases) has been developed and evaluated. This novel methodology not only allows the final products to be investigated, but it also reveals enzyme-specific differences in the degradation pathways during the hydrolysis of different substrates, which is a great advantage in the important tasks of investigating the mechanisms of and classifying new hydrolases, and is an advantage that conventional techniques cannot offer. Two different enzymes, one CDase from Laceyella sacchari (LsCda13) and one from Anoxybacillus flavithermus (AfCda13), were investigated during the hydrolysis of alpha-, beta- and gamma-cyclodextrin, and the hydrolysis products were sampled via a microdialysis probe and injected on-line every 30 min into a high-performance anion exchange chromatography system equipped with a pulsed amperometric detector (HPAEC-PAD), where they were identified. The enzymes yielded the same end-products, maltose and glucose, in an approximate molar ratio of 2:1, but they exhibited distinctly different patterns of intermediate product formation before reaching the end-point. LsCda13 had a more random distribution of the intermediate products, whereas AfCda13 showed the distinct intermediate production of maltotriose, which in some cases accumulated.
在这项工作中，已开发并评估了一种用于在线测量两种新发现的环糊精酶（CDases）的实时采样/分析方法。这种新颖的方法不仅可以研究最终产物，还能揭示不同底物水解过程中降解途径的酶特异性差异，这在研究新水解酶的机制和分类的重要任务中是一个巨大优势，是传统技术所无法提供的优势。在α-、β-和γ-环糊精的水解过程中，研究了两种不同的酶，一种来自嗜糖拉西氏菌的CDase（LsCda13）和一种来自嗜热栖热放线菌的CDase（AfCda13），水解产物通过微透析探针采样，并每隔30分钟在线注入配备脉冲安培检测器（HPAEC-PAD）的高效阴离子交换色谱系统中进行鉴定。这些酶产生相同的终产物，即麦芽糖和葡萄糖，摩尔比约为2:1，但在达到终点之前，它们表现出明显不同的中间产物形成模式。LsCda13的中间产物分布更随机，而AfCda13则显示出麦芽三糖的明显中间产物生成，在某些情况下会积累。
