Ezike Tobechukwu C, Udeh Jerry O, Joshua Parker E, Ezugwu Arinze L, Isiwu Chukwurobe V, Eze Sabinus O O, Chilaka Ferdinand C
Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria.
Heliyon. 2021 Jan 25;7(1):e06080. doi: 10.1016/j.heliyon.2021.e06080. eCollection 2021 Jan.
Various aromatic compounds that are structurally analogous to lignin were tested as possible/preferred substrates for purified laccase from newly isolated white rote fungi, WRF03. The pH optima were tested using different substrates and kinetic studies were conducted at these pH optima. The pH optima in the presence of ABTS, α-naphthol, o-dianisidine, and catechol were 4.5 but 5.0 and 5.5 in the presence of guaiacol and pyrogallol, respectively. The initial velocities obtained from the kinetic study were analyzed using Graph Pad Prism 7 and Lineweaver-Burk plot to obtain kinetic constants ( and ) which were used to calculate substrate specificity. Amongst all the substrates tested, ABTS had the highest specificity-constant (181.51 Ms), and therefore, the most preferred substrate was followed by α-naphthol, -dianisidine, guaiacol, pyrogallol, and catechol. Resorcinol, orcinol, and veratryl alcohol did not display any considerable chemical shift in the presence of WRF03 laccase. Also, oxidation of phenolic substrates appeared to be dependent on the nature of the substituent groups and their relative position on the aromatic nucleus. Since most of these substrates are structural analogs of lignin and many recalcitrant environmental pollutants, the enzyme may find application in delignification, treatment of wastewater containing dyes, and polycyclic aromatic hydrocarbons (PAHs).
测试了各种结构类似于木质素的芳香族化合物，作为新分离的白腐真菌WRF03纯化漆酶的可能/优选底物。使用不同底物测试了最适pH值，并在这些最适pH值下进行了动力学研究。在ABTS、α-萘酚、邻联茴香胺和儿茶酚存在下的最适pH值为4.5，但在愈创木酚和连苯三酚存在下分别为5.0和5.5。使用Graph Pad Prism 7和Lineweaver-Burk图分析动力学研究获得的初始速度，以获得动力学常数（ 和 ），用于计算底物特异性。在所有测试的底物中，ABTS具有最高的特异性常数（181.51 Ms），因此，最优选的底物依次是α-萘酚､邻联茴香胺､愈创木酚､连苯三酚和儿茶酚。在WRF03漆酶存在下，间苯二酚、苔黑酚和藜芦醇没有显示出任何明显的化学位移｡此外，酚类底物的氧化似乎取决于取代基的性质及其在芳香核上的相对位置｡由于这些底物大多是木质素和许多顽固性环境污染物的结构类似物，该酶可能在脱木质素、处理含染料废水和多环芳烃（PAHs）方面得到应用。
