Direct determination of lignin peroxidase released from Phanerochaete chrysosporium by in-capillary enzyme assay using micellar electrokinetic chromatography

作者：Harada Airi; Sasaki Keiko; Kaneta Takashi^*

来源：Journal of Chromatography A, 2016, 1440: 145-149.

DOI：10.1016/j.chroma.2016.02.062

• Lignin Peroxidase
• Phanerochaete chrysosporium
• Micellar electrokinetic chromatography
• Capillary electrophoresis
• Enzyme assay

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 50mM 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 4pmol (500 mu M) with a limit to quantification of 0.026 pmol (3.211 mu M) (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.

• 出版日期2016-4-1