重组短小芽孢杆菌CotA漆酶的酶学性质及发酵优化研究

Fermentation Optimization and Enzymatic Properties of Recombinant CotA Laccase from Bacillus pumilus

摘要:

细菌漆酶具有良好的热稳定性和抗碱性环境能力，在工业废水处理和染料脱色方面具有很大的潜力。通过研究短小芽孢杆菌W3菌株的CotA漆酶基因在大肠杆菌中重组表达的酶学性质，对其发酵培养基进行优化，以提高产量。利用分子手段实现CotA漆酶基因在大肠杆菌中的重组表达，并利用BP神经网络建模和萤火虫算法寻优对CotA漆酶发酵培养基进行优化，最后重组表达的CotA漆酶进行染料脱色研究。结果表明，重组菌株表达的CotA漆酶具有良好的热稳定性和耐碱能力，最适反应pH值为3.5，最适反应温度为80 ℃。重组CotA漆酶对底物ABTS具有良好的亲和能力，Km为0.247 mmol/L，Kcat为41.32 s ^-1。在适宜条件下的重组CotA漆酶的表达量为1 915.3 U/L，使用BP神经网络和萤火虫算法优化培养基获得最佳的发酵培养基配方为：蛋白胨1.16 g/dL、酵母粉0.428 g/dL、NaCl 1.06 g/dL、CuSO ₄ 0.274 mol/L和甘油（体积分数）0.931%。优化过的培养基产CotA漆酶酶活为3 088.68 U/L，相比未优化之前提高了61.26%。重组表达的CotA漆酶对孔雀石绿和酸性蓝129脱色效果明显，达到90%脱色率。短小芽孢杆菌W3来源的CotA漆酶具有良好的酶学性质和工业应用前景。利用BP神经网络偶联萤火虫算法寻优是CotA漆酶发酵培养基优化的有效手段。

Abstract:

The CotA-laccase with good thermal stability and resistance to alkaline environments is known as a green catalyst and widely used in industrial dye degradation and wastewater treatment. The enzymatic properties of CotA-laccase cloned from Bacillus pumilus W3 expressed in E. coli was studied. Its yield was increased through optimizing fermentation medium. The recombinant expression of CotA laccase gene in E. coli was achieved by molecular means. The CotA-laccase fermentation medium was optimized by BP neural network and firefly algorithms, and the dye decolorization of recombined CotA-laccase was investigated. The recombinant CotA-laccase had good thermal stability and alkali tolerance. The optimal condition was pH 3.5 and 80 ℃.Recombinant CotA laccase showed goodaffinity for ABTS with a Km of 0.247 mmol/L and a Kcat of 41.32 s-1. The expression of recombinant CotA laccase was 1915.3 U/L under suitable conditions. The compositions of fermentation medium optimized by BP neural network and firefly algorithms were 1.16% of peptone, 0.428% of yeast powder, 1.06% of NaCl, 0.274 mol/L of CuSO ₄ and 0.931% of glycerol. The CotA-laccase activity of the optimized medium reached 3 088.68 U/L, which was 61.26% higher than that before optimization. The significant decolorization effect of recombinant CotA laccase on malachite green and acid blue 129 was observed, reaching 90%decolorization rate. CotA-laccase cloned from Bacillus pumilus W3 has good enzymatic properties, showing promising industrial application prospects.The BP neural network modeling combined with firefly algorithm is a feasible methodto optimize the E. coli fermentation.