【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Cot A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Cot A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Cot A漆酶发酵培养基进行优化,最后重组表达的Cot A漆酶进行染料脱色研究。结果表明,重组菌株表达的Cot A漆酶具有良好的热稳定性和耐碱能力,最适反应p H值为3.5,最适反应温度为80℃。重组Cot A漆酶对底物ABTS具有良好的亲和能力,K_m为0.247 mmol/L,K_cat为41.32 s^-1。在适宜条件下的重组Cot A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/d L、酵母粉0.428 g/d L、Na Cl 1.06 g/d L、Cu SO₄0.274 mol/L和甘油（体积分数）0.931%。优化过的培养基产Cot A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Cot A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Cot A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Cot A漆酶发酵培养基优化的有效手段。更多还原

【Abstract】 The Cot A-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 Cot A-laccase cloned from Bacillus pumilus W3 expressed in E.coli was studied.Its yield was increased through optimizing fermentation medium.The recombinant expression of Cot A laccase gene in E.coli was achieved by molecular means.The Cot A-laccase fermentation medium was optimized by BP neural network and firefly algorithms,and the dye decolorization of recombined Cot A-laccase was investigated.The recombinant Cot A-laccase had good thermal stability and alkali tolerance.The optimal condition was pH 3.5 and 80℃.Recombinant Cot A laccase showed goodaffinity for ABTS with a K_mof 0.247 mmol/L and a K_catof41.32 s^-1.The expression of recombinant Cot A 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 Na Cl,0.274 mol/L of Cu SO₄and 0.931%of glycerol.The Cot A-laccase activity of the optimized medium reached 3 088.68 U/L,which was61.26%higher than that before optimization.The significant decolorization effect of recombinant Cot A laccase on malachite green and acid blue 129 was observed,reaching 90%decolorization rate.Cot A-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.更多还原