Harada Airi, Sasaki Keiko, Kaneta Takashi
Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Department of Earth Resources Engineering, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.
J Chromatogr A. 2016 Apr 1;1440:145-149. doi: 10.1016/j.chroma.2016.02.062. Epub 2016 Feb 26.
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).
在此，我们描述了一种采用胶束电动色谱法（MEKC）的毛细管内酶测定法在测定含有从黄孢原毛平革菌（P. chrysosporium）释放的木质素过氧化物酶的粗培养基中酶活性方面的应用。该方法包括木质素过氧化物酶与其底物藜芦醇之间的塞 - 塞反应、产物藜芦醛与包括酶和培养基在内的其他成分的分离，以及根据塞 - 塞反应产生的藜芦醛的峰面积测定酶活性。由于背景源于酶且可通过MEKC分离去除培养基，所以该方法比传统分光光度法更灵敏。在毛细管中进行5分钟的塞 - 塞反应后，在含有50mM酒石酸盐缓冲液（pH 2.5）和50mM十二烷基硫酸钠（SDS）的背景电解质中，于 - 10kV下分离藜芦醛。藜芦醛的校准曲线在高达4pmol（500μM）时呈线性，定量限为0.026pmol（3.2μM）（SN = 10）。木质素过氧化物酶的活性直接通过藜芦醛的峰面积来测定。成功测定出黄孢原毛平革菌释放到培养基中7天的木质素过氧化物酶活性为3.40U L(-1)。
