Zimbardi Ana L R L, Camargo Priscila F, Carli Sibeli, Aquino Neto Sidney, Meleiro Luana P, Rosa Jose C, De Andrade Adalgisa R, Jorge João A, Furriel Rosa P M
Department of Chemistry, Faculty of Philosophy, Sciences and Languages of Ribeirão Preto, University of São Paulo, Bandeirantes Avenue, 3900, Ribeirão Preto, SP 14040-901, Brazil.
Department of Molecular and Cell Biology and Protein Chemistry Center, CTC-Center for Cell Therapy-CEPID-FAPESP-Hemocentro de Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Bandeirantes Avenue, 3900, Ribeirão Preto, SP 14040-901, Brazil.
Int J Mol Sci. 2016 May 5;17(5):672. doi: 10.3390/ijms17050672.
Laccase production by Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions (eight days, 26 °C, 18% (w/w) milled corncob, 0.8% (w/w) NH₄Cl and 50 mmol·L(-1) CuSO₄, initial moisture 4.1 mL·g(-1)), the laccase activity reached 138.6 ± 13.2 U·g(-1). Lacps1 was a monomeric glycoprotein (67 kDa, 24% carbohydrate). Optimum pH and temperature for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0-8.0, and after two hours at 55-60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 μmol·L(-1), maximum velocity of 413.4 ± 21.2 U·mg(-1) and catalytic efficiency of 3140.1 ± 149.6 L·mmol(-1)·s(-1). The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.
采用中心复合旋转设计的响应面法，对血红密孔菌RP15在麸皮和玉米芯固态发酵条件下产漆酶的工艺进行了优化。对一种漆酶（Lacps1）进行了纯化和表征，并评估了纯Lacps1和粗培养提取物对合成染料脱色的潜力。在最佳条件下（8天，26℃，18%（w/w）磨碎玉米芯，0.8%（w/w）NH₄Cl和50 mmol·L⁻¹ CuSO₄，初始水分4.1 mL·g⁻¹），漆酶活性达到138.6±13.2 U·g⁻¹。Lacps1是一种单体糖蛋白（67 kDa，24%碳水化合物）。2,2'-联氮-双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）氧化的最佳pH和温度分别为4.4和74.4℃。Lacps1在pH 3.0 - 8.0时稳定，在55 - 60℃处理两小时后，具有较高的氧化还原电位（相对于标准氢电极，为0.747 V）。ABTS被氧化时的表观亲和常数为147.0±6.4 μmol·L⁻¹，最大反应速度为413.4±21.2 U·mg⁻¹，催化效率为3140.1±149.6 L·mmol⁻¹·s⁻¹。在25或40℃且无氧化还原介质的情况下，使用纯Lacps1或粗提取物时，溴酚蓝（BPB）、活性艳蓝R和活性蓝4（RB4）的最大脱色率分别达到90%、80%和60%。这是首次关于血红密孔菌漆酶对BPB和RB4脱色的研究。数据表明其在处理含工业染料废水方面具有良好潜力。
