Zhang Zhiping, Zhang Yue, Xu Boli, Li Tianxiao, Zhang Rongya, Wei Tao, Wen Wu
School of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou, 450002, Henan Province, People's Republic of China.
Technology Center, China Tobacco Sichuan Industrial Co., Ltd,, Chengdu, 610066, Sichuan Province, People's Republic of China.
Appl Biochem Biotechnol. 2025 May 13. doi: 10.1007/s12010-025-05251-y.
A novel laccase, Lac3, was purified from Ganoderma lucidum fermentation broth by salting out, gel filtration chromatography, and Native-PAGE protein recovery. The molecular mass of Lac3 was 58.4 kDa as estimated by SDS-PAGE and exhibited catalytic properties with 2,2'-Biazobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as substrate. The specific enzyme activity of Lac3 was determined to be 313.69 U/mg. The laccase was stable at temperatures < 65 °C and at pH of 2.5-4.5. The pH, temperature optima, K and V of the enzyme for ABTS oxidation were 3.0, 55 °C, 0.077 mM, and 2.98 mM/min, respectively. The metal ions and anions showed inhibitory effects on Lac3 activity except Cu (1 mM). GC-MS analysis showed that various aroma products were generated by Lac3 treatment of alkaline lignin. The Lac3 and lignin model compounds had negative binding energy and hydrogen bonding. The analysis of docking suggested that Asp207, Asn256, and His459 play a key role in substrate binding and catalysis.
通过盐析、凝胶过滤色谱和非变性聚丙烯酰胺凝胶电泳蛋白质回收从灵芝发酵液中纯化出一种新型漆酶Lac3。通过SDS-PAGE估计，Lac3的分子量为58.4 kDa，并且以2,2'-双氮杂双环-3-乙基苯并噻唑啉-6-磺酸（ABTS）为底物表现出催化特性。Lac3的比酶活性测定为313.69 U/mg。该漆酶在温度低于65°C以及pH值为2.5至4.5时稳定。该酶氧化ABTS的最适pH、温度、米氏常数（K）和最大反应速度（V）分别为3.0、55°C、0.077 mM和2.98 mM/min。除了铜离子（1 mM）外，金属离子和阴离子对Lac3活性均有抑制作用。气相色谱-质谱联用（GC-MS）分析表明，Lac3处理碱性木质素会产生各种芳香产物。Lac3与木质素模型化合物具有负结合能和氢键。对接分析表明，Asp207、Asn256和His459在底物结合和催化中起关键作用。
