Characterisation of two novel cyclodextrinases using on-line microdialysis sampling with high-performance anion exchange chromatography.
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.
Nilsson
Carina
C
Department of Analytical Chemistry, Lund University, P.O. Box 124, 221 00, Lund, Sweden. Carina.Nilsson@analykem.lu.se
Nilsson
Frida
F
Turner
Pernilla
P
Sixtensson
Martin
M
Nordberg Karlsson
Eva
E
Holst
Olle
O
Cohen
Arieh
A
Gorton
Lo
L
D000837:Anion Exchange Resins / Q000737:chemistry; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003505:Cyclodextrins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D017551:Microdialysis / Q000379:methods*
D000837:Anion Exchange Resins; D002021:Buffers; D003505:Cyclodextrins; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase