| As a necessary catalyst, laccass has been widely used in food, bio-bleaching, pulping and papermaking industry et al. Laccase lose the activity easily in high temperature and pH environments. This is a key factor which limits their applications. In this paper, laccass gens was screened from CotA genes of the Bacillus subtilis LS03 by means of error-prone PCR method. It obtained the mutant laccase with the higher expression quantity, the better thermostability or the stronger tolerance to alkaline environment by directed evolution. Enzymatic property and ability of dye degradation were analyzed and compared of the mutant laccases.The preliminary screening by 96-well plates and secondary screening in large quantity were carried out, which finally choosed five mutant enzymes with increased enzymatic activity: 019, K14, K9, W3, W8. The 019 mutant laccase increased the enzyme activity by 5.65 times. The three-dimensional structure of mutant laccase obtained by homologous modeling indicated that the corresponding amino acid mutational sites of the five kinds of mutant laccase were most located on the surface of the third domain of Cot A laccase. The 260th acid mutational site of the 019 specific laccase amino was located on the surface of "pocket" structure.The wild-type laccase and the 019 mutant laccase was purified by His Trap HP. The O19 mutant laccases increased the enzyme activity by 1.78 times and it has higher expression quantity than wild-type.019 exhibited the higher catalytic efficiency and the stronger affinity with ABTS and SGZ. O19 exhibited the better stability with pH and obvious promotion function with metal ion. The K+ã€Mn2+ã€Co2+ promotion function improved 20% of O19 mutant laccase activity. Mutant laccase in terms of their stability for part of inhibitor and surfactant was improved compared with that of wild-type laccase. But it thermo stability is not good.In terms of their decoloration capability for the four synthetic dyestuffs, the 019 mutant laccases exhibited the better decoloration capability with Remazol Brilliant Blue R and Gongo red. Several single factor experiments were applied to analyze the effects of dye concentration, enzyme concentration and amboceptor concentration on Indigo decoloration. The orthogonal experiment design method was use to further optimize reaction parameters. Desmone ACE concentration is remarkable factor of degradation. The optimal reaction parameters were 75mg/L Indigo,5U/L enzyme and 5mmol/L ACE. In this study equipment was projected which is used to cycle decoloration. It included the pillars of the immobilized laccase ball and constant flow pump. The equipment decoloration ratio could improve 20% than the disperse immobilized laccase ball. |
