Gene Cloning, Expression And Molecular Directed Evolution Of Laccases From Bacillus Sp.

Laccase is a typical green catalyst which is widely applied. So far, the main source of industrialized laccase is fungus fermentation. However, fungal laccase lose the activity easily in high temperature and pH environments. This is a key factor which limit their applications. In contrast, bacterial laccase has short growth circle and can maintain the catalytic activity of enzyme under extreme environments such as high temperature, high salt concentration and alkaline pH, which is more suitable for industrial to use in large scale in these applications. To get large-yield bacterial laccase in short circle, the bacterial laccase genes were cloned and their sequences were heterologously expressed in host cell in this study. It obtained the mutant strains of bacterial laccase with the higher expression quantity, the better thermostability and the stronger tolerance to alkaline environment by directed evolution. Further more, It had investigated the characteristics and spatial structures of mutational laccase, and carries out applied research on the decoloration of synthetic dyestuff. The main research achievements are as follows:(1) The laccase genes were amplified from B. amyloliquefaciens LS05and B. subtilis LS03. The homology was about74%between the laccase genes. The deduced amino acid sequences of B.amyloliquefaciens LS05were77%identical to B.subtilis LS03. The overall length of gene sequence of B. subtilis LS03cot A was1542bp and encoding514amino acid residues. Its predicted molecular weight was58.5kDa. The laccase gene of B. subtilis LS03cot A and expression vector pETDuet-1were connected to construct recombine expression plasmids which were transferred into expression host cell. The recombinant CotA was incubated at lower temperature and placed it in static condition to keep micro-aerobic environment for, which would show the activity of laccase.The expression product existed in the supernate by sonication.62kDa target protein was obtained through Ni-NTA affinity chromatographic purification. The specific activity was1.44U/mg. The recombinant CotA showed a pH optimum reaction for two tested substrates such as ABTS (pH4.0) and SGZ(pH7.0). With SGZ as a substrate, the optimal temperature of the enzyme was60℃which was stable in neutral and alkaline environment. Low concentration of Mg2+, Cu2+and Na+had obvious promotion function on laccase activity. Over60%of laccase activity was maintained in the presence of10%organic solvent. Conventional inhibitors can inhibite the laccase activity The recombinant CotA had good affinity with Syringaldazine.(2) Random mutational sites were introduced to CotA genes of the two kinds of Bacillus sp.by means of error-prone PCR method, which provided diversified maternal homologous genes for the next round of directed molecular evolution of Family Shuffling. Then, the preliminary screening by96-well plates and secondary screening in large quantity were carried out, which finally choosed three mutant enzymes with increased enzymatic activity and thermostability:Cot-46, CotA-120and CotA-183. The CotA-46specific laccase activity was7.49U/mg which increased the enzyme activity by5times. The optimal reaction temperature for CotA-46was70℃which was higher than that of wild-type laccase to10℃. CotA-46exhibited the better stability, the higher catalytic efficiency and the stronger affinity with Syringaldazine. The three-dimensional structure of mutant laccase obtained by homologous modeling indicated that the corresponding amino acid mutational sites of the three kinds of mutant laccase were all located on the surface of enzyme molecules or Loop area, and most of them were located at the third domain of CotA laccase.(3) This research further carried out directed molecular evolution on the domain of B. subtilis LS03CotA, three libraries for domain mutation were built by error-prone PCR. With the enzymatic activity taking Syringaldazine substrate as the screening indicator, it finally obtained LS03-CotA-50and LS03-CotA-76. Homologous modeling analysis showed that mutated amino acids sites were positioned on the second structural domain of the protein, and the third structural domain of laccase, which were far away from catalytic center. So the enzymatic characteristics of domain mutant laccase did not change too much, and the optimal temperature of LS03-CotA-76increased to70℃, with good thermostability and increased catalytic efficiency in comparison with wild-tpye laccase.(4) Mutant laccase in terms of their decoloration capability for the four synthetic dyestuffs with different chemical structure type was improved compared with that of wild-type laccase. CotA-46mutant laccase had the most significant effects. In the absence of mediators, the decoloration ratio of this mutant laccase for nine kinds of synthetic dyestuffs ranged from34%to89%. With the addition of mediators in the decoloration system, the decoloration ratios for the dyestuffs rapidly increased2-3times. When decoloring anthraquinone dyestuff Alizarin Red, the decoloration ratio of CotA-46mutant laccase without mediator was34.6%, which was the same as that of wild-type laccase with the participation of ACE mediator. The maximum decoloration ratio of the simulated dyestuff wastewater could reach40%by CotA-46mutant laccase in acid condition; the simulated dyestuff wastewater system of laccase-mediator decoloration determined was that under neutral and alkaline environment, the maximum decoloration ratio could reach87%...