Abstract
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.
Highlights
The Japanese lacquer tree (Toxicodendron vernicifluum (Stokes) F
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
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)
Summary
The Japanese lacquer tree (Toxicodendron vernicifluum (Stokes) F. The components of lacquer include urushiol, water, plant gum, water-insoluble glycoproteins, and enzymes (laccase, peroxidase, and stellacyanin) [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]
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