Dai Shuang, Yao Qian, Yu Gen, Liu Shan, Yun Jeonyun, Xiao Xiong, Deng Zujun, Li He
Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, College of Life Science and Biopharmaceuticals, Guangdong Pharmaceutical University, Guangzhou, China.
Guangzhou Base Clean Cosmetics Manufacturer Co., Ltd., Guangzhou, China.
Front Microbiol. 2021 May 12;12:633004. doi: 10.3389/fmicb.2021.633004. eCollection 2021.
Laccase is a copper-containing polyphenol oxidase with a wide range of substrates, possessing a good application prospect in wastewater treatment and dye degradation. The purpose of this research is to study the degradation of various industrial dyes by recombinant laccase rlac1338 and the mutant enzyme lac2-9 with the highest enzyme activity after modification by error-prone PCR. Four enzyme activities improved mutant enzymes were obtained through preliminary screening and rescreening, of which lac2-9 has the highest enzyme activity. There are four mutation sites, including V281A, V281A, P309L, S318G, and D232V. The results showed that the expression of the optimized mutant enzyme also increased by 22 ± 2% compared to the unoptimized enzyme and the optimal reaction temperature of the mutant enzyme lac2-9 was 5°C higher than that of the rlac1338, and the optimal pH increased by 0.5 units. The thermal stability and pH stability of mutant enzyme lac2-9 were also improved. With ABTS as the substrate, the k/K of rlac1338 and mutant strain lac2-9 are the largest than other substrates, 0.1638 and 0.618 sM, respectively, indicating that ABTS is the most suitable substrate for the recombinant enzyme and mutant enzyme. In addition, the K of the mutant strain lac2-9 (76 μM) was significantly lower, but the k/K (0.618 sM) was significantly higher, and the specific enzyme activity (79.8 U/mg) increased by 3.5 times compared with the recombinant laccase (22.8 U/mg). The dye degradation results showed that the use of rlac1338 and lac2-9 alone had no degradation effect on the industrial dyes [indigo, amaranth, bromophenol blue, acid violet 7, Congo red, coomassie brilliant blue (G250)], however, adding small molecular mediators Ca and ABTS at the same time can significantly improve the degradation ability. Compared to the rlac1338, the degradation rates with the simultaneous addition of Ca and ABTS of mutant enzyme lac2-9 for acid violet 7, bromophenol blue and coomassie brilliant blue significantly improved by 8.3; 3.4 and 3.4 times. Therefore, the results indicated that the error-prone PCR was a feasible method to improve the degradation activity of laccase for environmental pollutants, which provided a basis for the application of laccase on dye degradation and other environmental pollutants.
漆酶是一种含铜的多酚氧化酶，底物范围广泛，在废水处理和染料降解方面具有良好的应用前景。本研究的目的是研究重组漆酶rlac1338和易错PCR修饰后酶活性最高的突变酶lac2-9对各种工业染料的降解情况。通过初筛和复筛获得了4种酶活性提高的突变酶，其中lac2-9酶活性最高。有4个突变位点，分别为V281A、V281A、P309L、S318G和D232V。结果表明，优化后的突变酶表达量比未优化的酶也提高了22±2%，突变酶lac2-9的最适反应温度比rlac1338高5℃，最适pH值提高了0.5个单位。突变酶lac2-9的热稳定性和pH稳定性也有所提高。以ABTS为底物时，rlac1338和突变株lac2-9的k/K比其他底物时最大，分别为0.1638和0.618 sM，表明ABTS是重组酶和突变酶最适合的底物。此外，突变株lac2-9的K（76 μM）显著降低，但k/K（0.618 sM）显著升高，比重组漆酶（22.8 U/mg）的比酶活（79.8 U/mg）提高了3.5倍。染料降解结果表明，单独使用rlac1338和lac2-9对工业染料[靛蓝、苋菜红、溴酚蓝、酸性紫7、刚果红、考马斯亮蓝（G250）]没有降解作用，然而，同时添加小分子介质Ca和ABTS可以显著提高降解能力。与rlac1338相比，突变酶lac2-9同时添加Ca和ABTS对酸性紫7、溴酚蓝和考马斯亮蓝的降解率分别显著提高了8.3、3.4和3.4倍。因此，结果表明易错PCR是提高漆酶对环境污染物降解活性的一种可行方法，为漆酶在染料降解及其他环境污染物处理中的应用提供了依据。
