Bagewadi Zabin K, Mulla Sikandar I, Ninnekar Harichandra Z
Department of Biochemistry, Karnatak University, Dharwad, Karnataka 580 003, India.
Department of Biotechnology, KLE Technological University Hubballi, Karnataka 580 031, India.
J Genet Eng Biotechnol. 2017 Jun;15(1):139-150. doi: 10.1016/j.jgeb.2017.01.007. Epub 2017 Feb 14.
In this study we report the purification of laccase produced by strain HZN10 (using wheat bran under solid state fermentation) and its application in decolorization of synthetic dyes. Extracellular laccase was purified to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with specific activity of 162.5 U/mg and 25-fold purification. Purified laccase was immobilized in various entrapments like calcium alginate, copper alginate, calcium alginate-chitosan beads and sol-gel matrix. Optimization results revealed that the laccase immobilized in sol-gel was optimally active in wide pH range (4.0-7.0) and thermo-stable (50-70 °C) than free enzyme which was optimum at 50 °C and pH 6.0. Kinetic analysis showed of 0.5 mM and 2.0 mM and of 285 U/mg and 500 U/mg by free laccase and sol-gel immobilized laccase respectively with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS] substrate. Free and immobilized laccase was employed for decolorization of three different synthetic dyes (malachite green, methylene blue and congo red). High performance liquid chromatography (HPLC) analysis results revealed that approximately 100% of malachite green, 90% of methylene blue and 60% of congo red dyes at initial concentration of 200 mg/L were decolorized within 16, 18 and 20 h, respectively by laccase immobilized in sol-gel matrix in the presence of 1-hydroxybenzotriazole (HBT) mediator. During the decolorization all three synthetic dyes showed various peaks on HPLC chromatogram indicating different by-products formation. Finally, phytotoxicity analysis results revealed that the by-products of synthetic dyes (formed during decolorization) showed less toxicity against compared to untreated synthetic dyes.
在本研究中，我们报道了菌株HZN10（在固态发酵条件下使用麦麸）产生的漆酶的纯化及其在合成染料脱色中的应用。通过DEAE-琼脂糖和葡聚糖凝胶G-100色谱法将胞外漆酶纯化至均一，比活性为162.5 U/mg，纯化倍数为25倍。将纯化的漆酶固定在各种包埋物中，如海藻酸钙、海藻酸铜、海藻酸钙-壳聚糖珠和溶胶-凝胶基质。优化结果表明，固定在溶胶-凝胶中的漆酶在较宽的pH范围（4.0-7.0）内具有最佳活性，并且比游离酶更耐热（50-70°C），游离酶在50°C和pH 6.0时具有最佳活性。动力学分析表明，以2,2'-联氮双（3-乙基苯并噻唑啉-6-磺酸）[ABTS]为底物时，游离漆酶和溶胶-凝胶固定化漆酶的Km分别为0.5 mM和2.0 mM，Vmax分别为285 U/mg和500 U/mg。游离和固定化漆酶用于三种不同合成染料（孔雀石绿、亚甲基蓝和刚果红）的脱色。高效液相色谱（HPLC）分析结果表明，在1-羟基苯并三唑（HBT）介质存在下，初始浓度为200 mg/L的孔雀石绿、亚甲基蓝和刚果红染料，分别在16、18和20小时内被溶胶-凝胶基质固定的漆酶脱色约100%、90%和60%。在脱色过程中，所有三种合成染料在HPLC色谱图上均显示出不同的峰，表明形成了不同的副产物。最后，植物毒性分析结果表明，合成染料（脱色过程中形成的）副产物对（受试植物）的毒性低于未处理的合成染料。
