Characterisation of two novel cyclodextrinases using on-line microdialysis sampling with high-performance anion exchange chromatography

Carina Nilsson, Frida Nilsson, Pernilla Turner, Martin Sixtensson, Eva Nordberg Karlsson, Olle Holst, Arieh Cohen, Lo Gorton

Research output: Contribution to journalArticlepeer-review

6 Citations (SciVal)

Abstract

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.
Original languageEnglish
Pages (from-to)1421-1429
JournalAnalytical and Bioanalytical Chemistry
Volume385
Issue number8
DOIs
Publication statusPublished - 2006

Bibliographical note

The information about affiliations in this record was updated in December 2015.
The record was previously connected to the following departments: Analytical Chemistry (S/LTH) (011001004), Biotechnology (LTH) (011001037), Division of Occupational and Environmental Medicine (013078001)

Subject classification (UKÄ)

  • Analytical Chemistry

Keywords

  • Laceyella sacchari (LsCda13)
  • enzyme hydrolysis
  • cyclodextrins (CD)
  • carbohydrate analysis
  • Anoxybacillus flavithermus (AfCda13)

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