Isolation of a novel alkaline-induced laccase from Flammulina velutipes and its application for hair coloring.

Laccase is a member of the multi-copper oxidase family and a promising for hair coloring. In this study, we isolated a novel alkaline-induced laccase from the white-rot fungus Flammulina velutipes and studied the possibility to apply the enzyme for hair coloring. Laccase activity detected in the culture supernatant of F. velutipes was found to significantly increase when exchanging the medium to laccase inducing one whose pH was adjusted to 9.0. Three isozymes were detected by activity staining on non-denaturing SDS-PAGE. The major isozyme, Flac1, was purified from the culture supernatant after being induced at pH 9.0 by ion-exchange column chromatography. The N-terminal peptide sequence of Flac1 was determined, revealing clear homology with laccases from other white-rot fungi. Optimum pH of oxidation was found to be around pH 5.0-6.5 regardless of several different substrates used. Oxidation activities of Flac1 to several hair dye agents as substrate showed the higher activity at pH 6.5 than that at pH 9.0. Oxidation activity was also detected at pH 9.0 which was suitable for hair coloring. When the purified Flac1 was applied for hair coloring system without using hydrogen peroxide, effective coloring was observed at the protein amount of 0.25mg/1g of hair used. These results indicated that this alkaline-induced novel laccase isolated from the culture supernatant of F. velutipes might be a useful enzyme for hair color.

A rapid gel electrophoretic chip for serum cholesterol determination.

We present a rapid gel electrophoretic chip, composed of 2.5% (w/v) acrylamide and 1% (w/v) agarose gel, for serum cholesterol determination using a photo lithography technique. After optimizations, we determined the lipoprotein concentration of standard serum using a conventional enzyme method. The serum was diluted, stained and loaded for 15 min onto the chip. After loading, the intensities of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) bands separated at the chip were estimated using an image analyzer. The intensities of these bands corresponded to concentrations obtained from a standard enzyme-based method. The detected LDL-C and HDL-C concentrations were linear up to 146 mg dL(-1) and 53 mg dL(-1) respectively. Finally, we carried out the cholesterol analysis using real biological samples obtained from nine volunteers using our electrophoretic chip. The LDL-C and HDL-C levels detected using our chip correlated well with the results obtained using the conventional enzyme-based method r(2) = 0.98 and r(2) = 0.86 for LDL-C and HDL-C, respectively. Although our sample size is small and confined only to health volunteers, we have demonstrated that this proof-of-concept gel electrophoretic chip can determine lipoproteins, simultaneously.

Mass spectrometric imaging of ginsenosides localization in Panax ginseng root.

We performed mass spectrometric imaging (MSI) to localize ginsenosides (Rb(1), Rb(2) or Rc, and Rf) in cross-sections of the Panax ginseng root at a resolution of 100 microm using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Tandem mass spectrometry (MS/MS) of alkali metal-adducted ginsenoside ions revealed structural information of the corresponding saccharides and aglycone. MALDI-MSI confirmed that ginsenosides were located more in the cortex and the periderm than that in the medulla of a lateral root. In addition, it revealed that localization of ginsenosides in a root tip (diameter, 2.7 mm) is higher than that in the center of the root (diameter, 7.3 mm). A quantitative difference was detected between localizations of protopanaxadiol-type ginsenoside (Rb(1), Rb(2), or Rc) and protopanaxatriol-type ginsenoside (Rf) in the root. This imaging approach is a promising technique for rapid evaluation and identification of medicinal saponins in plant tissues.