In this study, we performed an analysis using isoelectric focusing to clarify the comprehensive composition of the isozymes of Lac derived from lacquer. Isoelectric focusing performed on crude Lac enzymes that had been extracted from acetone powder showed multiple bands indicating Lac activity detected by GU in the ranges of pI 3.50–5.20 and 7.35–9.30 (Fig. 1). However, laccase activity detected by gallic acid showed remarkable staining within pI 3.50–5.85, whereas staining was very weak within pI 7.35–9.30 (Fig. 2). For elucidating the difference between Lac with pI 3.50–5.85 and 7.35–9.30, crude Lac was fractionated and analyzed. When the crude Lac enzymes were separated into the fractions containing the bands within pI 3.50–5.85 and 7.35–9.30, the latter samples were blue in color and the former were yellow (Fig. 6). The yellow fraction showed Lac activity equal to or higher than the blue fraction (Fig. 7). The yellow fraction is conventionally removed during the purification process of lacquer Lac and has thus seldom been analyzed. This paper is the first to report the importance of the yellow fraction as a component of lacquer Lac.
Lacquer Lac was the first multicopper oxidase found and has long been studied in detail to understand the enzyme properties, including structure and functions [6, 10]. Isoelectric points around pI 8–10 have been reported for purified lacquer Lac, and the enzymes are known to exhibit blue color with a maximum absorbance at around 600 nm in the UV–VIS spectra [13].
The blue fraction analyzed in this study had a maximum absorbance at around 600 nm and contained bands within pI 7.35–9.30. These characteristics were consistent with previously reported characteristics of the blue fraction of lacquer Lac. In contrast, the crude Lac enzymes of the yellow fraction had isoelectric points within pI 3.50–5.85 and showed no distinct maximum absorption at around 600 nm in the visible spectrum—characteristics not consistent with those of conventional blue Lac (Figs. 5, 6).
Lacquer Lac has been previously studied for the purpose of analyzing the blue proteins contained in lacquer. Therefore, the non-blue fractions have been removed during the purification of the blue Lac. In many such studies, a purification process that used an ion-exchange resin was set up, and proteins or other components of pI 6.0 or lower were removed [12, 13]. The Lacs from the bands within pI 3.50–5.85 detected in this study should correspond to the fractions that were previously removed.
There was a large difference in Lac activity for the blue and yellow fractions depending on the type of substrate and pH (Fig. 7). When SA, 2,6-DMP, and GU were used as substrates, the yellow fraction showed higher activity than the blue fraction in the pH range of 5.5–7.5. At pH 6–7, especially, Lac activity was mostly derived from the yellow fraction and not the blue fraction. The SA, 2,6-DMP, and GU have been used as versatile substrates to measure the activity of lacquer Lac. The previously reported Lac activity of lacquer acetone powder may have originated from the yellow rather than the blue fraction.
For 3MC and 4MC, the yellow fraction showed higher activity than the blue fraction at pH 4.5–6.5, and the blue fraction showed higher activity than the yellow at pH 7.0–8.0. Compared with SA, 2,6-DMP, and GU, 3MC has a higher structural similarity to urushiol (Fig. 8). The results suggest that in the polymerization of urushiol, the blue and yellow fractions acted complementarily over a wide pH range.
When ABTS was used as the substrate, significant differences in Lac activity were observed compared to other substrates. The optimum pH of lacquer Lac has been reported to be pH 5.0–8.0 when SA is the substrate [6, 18]. In this study, when SA, 2,6-DMP, 3MC, 4MC, and GU were used as substrates, the optimum pH was 5.0–8.0. However, when ABTS was the substrate, both the blue and yellow fractions had optimum pH of 3.0–4.0.
ABTS is widely used as a substrate to measure activity of Lac and peroxidases. The other substrates used had a skeleton similar to the structure of wood components; however, ABTS had a different chemical structure. The Lac of white-rot fungi is considered to have an acidic optimum pH [6]; however, in recent years, studies on yellow and white Lac derived from white-rot fungi have reported that optimum pH of SA is pH 6–7, whereas optimum pH of ABTS is in the range of pH 3–4 [19, 20].
The color development test following isoelectric focusing that used gallic acid confirmed strong color development in multiple bands within pI 3.5–5.85 of the yellow fraction, whereas color development was weak in the five bands within pI 7.35–9.30 of the blue fraction.
The GU color development test performed on the yellow fraction confirmed Lac activity in multiple bands within pI 4.25–5.85, in addition to three bands within pI 3.50–4.25, by extending the reaction time. However, as the reaction time for the Lac activity measurement was shorter than 5 min, it was likely that the three bands that developed color in this short time contributed to the reaction.
All these results indicated that the yellow fraction was at least as important as the blue fraction as a Lac component of lacquer.
Lac is a multicopper oxidase, and the active center responsible for its catalytic function is composed of four copper atoms, which are classified into types I, II, and III [10]. Among them, it has become clear that type I contributes to development of the blue color. An earlier analysis of lacquer Lac reported that removing copper from Lac resulted in decreased enzyme activity and loss of the blue color [21].
In this study, although the bands within pI 3.5–5.2 contained in the yellow fraction were very low in amount and intensity compared to the bands within pI 7.35–9.30, the bands were very clear and were not smeared as often occurs due to degeneration of the protein (Fig. 1). In addition, as discussed earlier, the yellow fraction showed a Lac activity equal to or greater than the blue fraction, with no results indicating deactivation of the enzyme (Fig. 7). Therefore, it is unlikely that the deterioration or degradation of the blue fraction resulted in the deterioration or loss of type I copper and that the loss of blue color gave rise to the yellow fraction. These results indicated that the yellow fraction represented a completely different enzymatic protein from the blue fraction. In white-rot fungi, multiple isozymes of Lac have been reported, including yellow and white Lac, which do not exhibit blue color [17, 22, 23]. Our results indicated that yellow Lac was also present in lacquer.