FullText URL JCA_1440_145.pdf
Author Harada, Airi| Sasaki, Keiko| Kaneta, Takashi|
Abstract Here we describe the application of an in-capillary enzyme assay using micellar electrokinetic chromatography (MEKC) in the determination of enzyme activity in a crude culture medium containing lignin peroxidase released from Phanerochaete chrysosporium (P. chrysosporium). The method consists of a plug–plug reaction between lignin peroxidase and its substrate, veratryl alcohol, the separation of the product, veratraldehyde, from the other components including the enzyme and the culture medium, and the determination of the enzyme activity from the peak area of veratraldehyde produced by the plug–plug reaction. This method is more sensitive than conventional spectrophotometry since the background originates from the enzyme and the culture medium can be removed via MEKC separation. Veratraldehyde was separated at −10 kV in a background electrolyte containing 50 mM tartrate buffer (pH 2.5) and 50 mM sodium dodecyl sulfate (SDS) after a plug–plug reaction in the capillary for 5 min. The calibration curve of veratraldehyde was linear up to 4 pmol (500 μM) with a limit to quantification of 0.026 pmol (3.2 μM) (SN = 10). The activity of lignin peroxidase was directly measured from the peak area of veratraldehyde. The activity of lignin peroxidase released from P. chrysosporium into the medium for 7 days was successfully determined to be 3.40 UL−1.
Keywords Lignin Peroxidase Phanerochaete chrysosporium Micellar electrokinetic chromatography Capillary electrophoresis Enzyme assay
Note © 2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/
Published Date 2016-04-01
Publication Title Journal of Chromatography A
Volume volume1440
Publisher Elsevier Science B.V.
Start Page 145
End Page 149
ISSN 0021-9673
NCID AA10985833
Content Type Journal Article
language 英語
OAI-PMH Set 岡山大学
Copyright Holders http://creativecommons.org/licenses/by-nc-nd/4.0/
File Version author
DOI 10.1016/j.chroma.2016.02.062
Related Url isVersionOf https://doi.org/10.1016/j.chroma.2016.02.062
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