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.

中文翻译：

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综合分析日本漆树Toxicodendron vernicifluum漆酶的同工酶组成

我们使用等电聚焦进行了分析，以全面阐明源自日本漆树Toxicodendron vernicifluum的漆酶的同工酶组成。当对从漆料中提取的丙酮粉末的水提取物进行等电聚焦时，使用考马斯染色检测到pI 7.35–9.30内的五个谱带和pI 3.50–5.25内的九个谱带。同样，使用愈创木酚进行的漆酶活性染色显示，pI 7.35–9.30内有五个谱带，pI 3.50–4.25内有三个谱带。然而，使用没食子酸的漆酶活性染色在pI 3.50–5.85范围内显示出显着的染色，而在pI 7.35–9.30范围内则染色非常弱。用SP-Sepharose柱色谱法将丙酮粉的水提物分级为pI在7.35–9.30和pI在3.50–5.85范围内的谱带时，前者为蓝色，后者为黄色。在2.5-8.0的pH范围内，对缓冲液中每个组分的漆酶活性进行了测量。当使用丁香嗪，愈创木酚和2,6-二甲氧基苯酚作为底物时，在pH 5.5-7.5时，黄色级分的活性明显高于蓝色级分。当使用3-甲基邻苯二酚和4-甲基邻苯二酚作为底物时，黄色部分在pH 4.5-6.5下显示较高的活性，蓝色部分在pH 7.0-8.0下显示较高的活性。当使用2,2'-叠氮基双（3-乙基苯并噻唑啉-6-磺酸）作为底物时，两个部分在最佳pH为3.0-4.0时均显示出最大活性。按照惯例，在研究源自漆的蓝色漆酶时，对应于低于pI 6的黄色级分的非蓝色级分已在纯化过程中除去，因此尚未进行分析。我们的结果表明，黄色漆酶存在于漆的非蓝色成分中，并且它可能与以前报道的蓝色漆酶一起在漆酚聚合中起作用。