Comprehensive analysis of the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum

We performed an analysis using isoelectric focusing to comprehensively clarify the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum. When water extracts of acetone powder obtained from lacquer were subjected to isoelectric focusing, five bands within pI 7.35–9.30 and nine bands within pI 3.50–5.25 were detected using Coomassie staining. Similarly, laccase activity staining using guaiacol showed five bands within pI 7.35–9.30 and three bands within pI 3.50–4.25. However, laccase activity staining using gallic acid showed remarkable staining within pI 3.50–5.85, whereas staining was very weak within pI 7.35–9.30. When the water extracts of acetone powder were fractionated into the fractions containing bands within pI 7.35–9.30 and pI 3.50–5.85 by SP-Sepharose column chromatography, the former had a blue color and the latter a yellow color. The laccase activity was measured for each of the fractions in buffer solution in the pH range of 2.5–8.0. When syringaldazine, guaiacol, and 2,6-dimethoxyphenol were used as substrates, the yellow fraction showed considerably higher activity than the blue fraction for pH 5.5–7.5. When 3-methylcatechol and 4-methylcatechol were used as substrates, the yellow fraction showed higher activity for pH 4.5–6.5, and the blue fraction showed higher activity for pH 7.0–8.0. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as the substrate, both fractions showed maximum activity at optimum pH of 3.0–4.0. Conventionally, in research on blue laccase derived from lacquer, the non-blue fraction corresponding to the yellow fraction lower than pI 6 has been removed during the purification process and thus has not been analyzed. Our results indicated that yellow laccase was present in the non-blue components of lacquer and that it may play a role in urushiol polymerization with previously reported blue laccase.


Introduction
The Japanese lacquer tree (Toxicodendron vernicifluum (Stokes) F. A. Barkl.) is one of the family Anacardiaceae that is widely distributed in East Asia [1]. Lacquer is resin and sap extracted from lacquer trees and has been used since Jomon Period as paint for cultural assets and lacquer wares in Japan, making it a natural material of cultural and social significance [2,3].
The components of lacquer include urushiol, water, plant gum, water-insoluble glycoproteins, and enzymes (laccase, peroxidase, and stellacyanin) [4,5]. Urushiol is a generic term for catechol derivatives with an alkyl sidechain that has 15 or 17 carbon atoms at C-3 of the benzene ring; and more than ten types have been reported with different side-chain structures [4,5]. Laccase (Lac) is a phenoloxidase and is found widely in plants such as lacquer trees, fungi such as white-rot fungi, and bacteria [6]. Lacquer hardens and dries to form a film, as a result of urushiol becoming oxidatively polymerized by Lac [4,7], and functions as a coating material [8]. Because drying and hardening properties are used as indicators of lacquer quality, Lac has a direct effect on quality [9].

Open Access
Journal of Wood Science *Correspondence: marin@ffpri.affrc.go.jp 1 Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan Full list of author information is available at the end of the article Lacquer Lac was the first multicopper oxidase discovered and has been studied in detail in regard to the catalytic mechanisms, functions, properties, and structures of the isolated enzyme [10]. In recent years, detailed analyses of the carbohydrate structure and gene structure of Lacs have been reported [11,12]. These studies have focused on two to three Lacs with pI 8-10 purified using Reinhammer's method [13]. However, comprehensive isozyme composition and diversity of lacquer Lac remain to be clarified.
In contrast, a wide variety of Lacs have been reported in white-rot fungi [6]. In addition, in white-rot fungi, ligninolytic oxidizing enzymes, including Lac, are known to have many isoenzymes with the same molecular weight but different pIs and these have been analyzed using isoelectric focusing and two-dimensional electrophoresis [14,15].
In this study, we performed a comprehensive analysis of Lac isozymes derived from Japanese lacquer tree using isoelectric focusing.

Samples
Four samples were used, consisting of "Hatsu-urushi" lacquer collected between mid-June and mid-July 2018 and "Sakari-urushi" lacquer collected between late July and late August, in Ninohe, Iwate Prefecture and Iida, Nagano Prefecture.

Preparation of crude Lac enzymes
Ten milliliters of each lacquer sample was collected in a 50-mL centrifuge tube, and to this was added 30 mL of cooled acetone and mixed well. After remaining standing for three days at − 20 °C, the samples were then centrifuged in a refrigerated centrifuge (13,000 rpm × 10 min), and the precipitate was washed with acetone until the supernatant became colorless. The precipitates were then collected and dried at 6 °C for 3 days to obtain acetone powder. To this was added 15 mL of distilled water, and extraction was performed in a shaker overnight. After centrifugation to remove insoluble matter, 500 g/L of ammonium sulfate was added to the solution, and the mixture was stirred. The solution was again centrifuged to collect blue supernatant, to which ammonium sulfate was added until saturation reached 100% (767 g/L). The sample was centrifuged, and the supernatant removed to collect blue precipitate. To the blue precipitate, 5 mL of distilled water was added and dissolved, and after being desalted with a PD-10 column, the sample was concentrated using VIVA SPIN.

Fractionation of Lac
SP-Sepharose was packed in a column (1 cm × 10 cm) and equilibrated with phosphate buffer (pH 6.0, 10 mM). After crude Lac enzymes were added to the column, 30 mL of the same phosphate buffer was added, and the eluate was collected (yellow fraction). Then, 20 mL of phosphate buffer (pH 6.0, 300 mM) was added and the eluate was collected (blue fraction).

Staining of the gel
Proteins separated in the gel were stained using Coomassie Blue R 250. Guaiacol (GU) and gallic acid were used for Lac activity staining.

Instruments
Multiphor II (Cytiva) was used for electrophoresis. An UV-VIS recording spectrophotometer UV-2400PC (Shimadzu, Kyoto, Japan) was used to measure the enzymatic activity and UV-VIS spectra of lacquer Lac.

Preparation of crude Lac enzymes
Acetone powder was prepared from each lacquer sample, which was mixed with distilled water to obtain a crude Lac enzyme solution. Table 1 shows the locations where the samples were collected, the timings of sample collection, the amounts of samples used, acetone powder yields, the total Lac activities, and the activities per gram of acetone powder. Acetone powder yield and Lac activity varied among the samples.

Isoelectric focusing
The results of isoelectric focusing on the four crude Lac enzyme samples are shown in Fig. 1. Using Coomassie staining, five bands were detected within pI 7.35and 9.30 and nine bands within pI 3.50-5.20. Similarly, when isoelectric focusing was performed, and Lac activity was determined using GU for 30 min, five bands were detected within pI 7.35-9.30 and three bands within pI 3.50-4.25. All five bands detected within pI 7.35-9.30 were found to be Lac. However, of the nine bands detected within pI 3.50-5.20, Lac activity was only found in three bands within pI 3.50-4.25.
Regarding the results of isoelectric focusing, the color development was weak in all bands of Sample 1, which had a low total Lac activity (Table 1); however, the positions and relative intensities of bands were the same in all four samples. These results suggest that the type and composition of isozymes and their compositional ratios were similar among all four samples.
In addition, Lac activity staining using gallic acid, following isoelectric focusing of crude Lac enzymes, showed remarkable color development in the bands within pI 3.50-5.85, whereas color development was very weak in the five bands within pI 7.35-9.30 (Fig. 2). These results showed that Lac with pI 7.35-9.30 and pI 3.50-5.85 had different character on their enzymatic activity.

Spectral changes of 3MC and 4MC oxidized by Lac
Various aromatic compounds, such as SA, 2,6-DMP, and GU, have been used for measuring Lac activity. In addition to these compounds, two others were used as substrates in this study: 3MC, which has a similar chemical structure to urushiol; and 4MC, which has a similar chemical structure to thitsiol. First, 3MC and 4MC were oxidized with Lac to measure their spectral changes during the color development process. Color development by oxidation of 3MC showed a spectrum with a maximum value at 430 nm, and of 4MC a maximum at 400 nm (Fig. 3). Accordingly, Lac activity was assayed by measuring absorbance at 430 and 400 nm for 3MC and 4MC, respectively.

Activity of crude Lac enzymes
The activity of crude Lac enzymes extracted from acetone powder was measured in McIlvaine buffer of pH 2.5-8.0 [16]. The results for using ABTS, SA, and 2,6-DMP as substrates are shown in Fig. 4a, and for 3MC, 4MC, and  Fig. 4b. The optimum pHs for Lac activity were pH 3.0-4.0 for ABTS, pH 6.0-8.0 for SA, and pH 6.0-7.0 for 2,6-DMP. When 3MC, 4MC, and GU were used as substrates, Lac activity was high at pH 5.0-8.0. ABTS showed high Lac activity in acidic conditions, whereas the other substrates showed high Lac activity in the neutral to weakly alkaline conditions.

Fractionation of crude Lac enzymes
As shown in the results described above (Fig. 2), Lac with pI 7.35-9.30 and pI 3.50-5.85 had different character on their enzymatic activity. For analysis of the difference between these two enzymes, crude Lac was fractionated as follows. The Lac samples were separated by SP-Sepharose column chromatography into two fractions: one containing Lac with pI 7.35-9.30 (exhibiting a blue color) and one with pI 3.50-5.85 (exhibiting a yellow color) (Fig. 5). The blue fraction showed maximum absorption at around 600 nm-a characteristic of blue Lac; whereas no maximum absorption was observed around 600 nm for the yellow fraction (Fig. 6).

Isoelectric focusing of the fractionated samples
The blue and yellow fractions were subjected to isoelectric focusing, and the results of Coomassie staining and Lac activity staining by GU are shown in Fig. 5. Coomassie staining showed that the blue fraction contained bands within pI 7.35-9.30, and the yellow fraction contained bands within pI 3.50-5.85. When the duration of GU staining was extended to over an hour, the Lac activity was confirmed widely, corresponding to multiple bands within pI 4.25-5.85 in addition to three bands near pI 4 (Fig. 1b). These results suggested that characteristics of Lac activity differed between the blue and yellow fractions.

Lac activity of the fractionated samples
To compare characteristics of Lac activity between the blue and yellow fractions, Lac activity was measured for each of the substrates within the range of pH 2.5-8.0 (Fig. 7). For comparison of Lac activity of these 2 fractions working in lacquer, Lac activity contained in the same amount of lacquer was assayed. When 3MC was used as the substrate, the yellow and the blue fractions exhibited the highest Lac activity at pH 6.0 and 8.0, respectively. At pH 4.5-6.0, the yellow fraction showed higher activity than the blue fraction, and the blue fraction showed higher activity than the yellow at pH 7.0-8.0. For SA, the yellow fraction showed maximum Lac activity at pH 6.5 and the blue fraction at pH 8.0. In the range of pH 5.0-7.5, the yellow fraction showed higher activity than the blue fraction. For 2,6-DMP, the yellow fraction showed maximum Lac activity at pH 7.0 and the blue fraction at pH 8.0. The yellow fraction showed higher activity at pH 4.5-7.5 than the blue fraction. For 4MC, the yellow fraction showed maximum Lac activity at pH 5.0-6.0, and the blue fraction at pH 8.0. The yellow fraction had higher activity than the blue fraction at pH 4.5-6.0, and the blue fraction had higher activity than the yellow at pH 7.0-8.0. For ABTS, the yellow fraction showed maximum Lac activity at pH 3.5, and the blue fraction at pH 3.0. At pH 2.5-3.5, the blue fraction had higher activity than the yellow. For GU, the yellow and blue fractions showed maximum Lac activities at pH 7.0 and 8.0, respectively. At pH 5.0-7.5, the yellow fraction showed higher activity than the blue.
Although the results shown were for Sample No. 4, which had the highest total Lac activity among all samples (Table 1), similar results were obtained for the remaining three samples.

Discussion
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 whiterot 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.