title: Direct determination of lignin peroxidase released from Phanerochaete chrysosporium by in-capillary enzyme assay using micellar electrokinetic chromatography.
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 -10kV in a background electrolyte containing 50mM tartrate buffer (pH 2.5) and 50mM sodium dodecyl sulfate (SDS) after a plug-plug reaction in the capillary for 5min. The calibration curve of veratraldehyde was linear up to 4pmol (500μM) with a limit to quantification of 0.026pmol (3.2μM) (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 3.40UL(-1).
authors.affiliation: Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
authors.forename: Airi
authors.initials: A
authors.lastname: Harada
authors.affiliation: Department of Earth Resources Engineering, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Sasaki
authors.affiliation: Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan. Electronic address: kaneta@okayama-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kaneta
mesh_terms: D001592:Benzyl Alcohols / Q000378:metabolism; D002021:Buffers; D002845:Chromatography / Q000295:instrumentation; D003470:Culture Media / Q000737:chemistry; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D010544:Peroxidases / Q000032:analysis* / Q000378:metabolism; D020075:Phanerochaete / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013053:Spectrophotometry
chemical_list: D001592:Benzyl Alcohols; D002021:Buffers; D003470:Culture Media; D012967:Sodium Dodecyl Sulfate; D010544:Peroxidases; C042858:lignin peroxidase; C042197:veratryl alcohol
keywords: Capillary electrophoresis; Enzyme assay; Lignin Peroxidase; Micellar electrokinetic chromatography; Phanerochaete chrysosporium