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ID 54058
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Author
Harada, Airi
Sasaki, Keiko
Kaneta, Takashi ORCID Kakenhi
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/
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isVersionOf https://doi.org/10.1016/j.chroma.2016.02.062