Gene Cloning And Expression Of A Manganese Catalase

Catalase can catalyze the decomposition of hydrogen peroxide into water and oxygen. It is widely uesed in food, paper and pharmaceutical industries, particularly in the textile industry, due to it can reduce pollution and improve the printing quality. The manganese catalase(MnCAT) of Thermus thermophilus HB27has an important significance, due to its higher thermal stability and alkali characteristic. The main aspects of this study were as following:(1)The gene encoding manganese catalase of T.thermophilus HB27was screened through bioinformatics analysis from the genome of NCBI. The ancient bacterium T. thermophilus HB27was growed in thermophilic and alkaline environment. According to the codon bias of E.coli, the gene encoding MnCAT of T. thermophilus HB27were optimized. Then the optimized gene of MnCAT was synthesised. And the modified gene was inserted into the expression vector pET28a(+) and transformed into E.coli BL21(DE3). And realized the heterologous expression of MnCAT which is from T. thermophilus HB27in E.coli.(2)Then E.coli expression systerm was optimized, including induction temperature, IPTG induction dose and the concentration of manganese ion, and obtained the higher activity of MnCAT. The activity of catalase was25U/mL in supernatant of cell disruption, and the specific activity was78.9U/mL. The enzyme was purified by Ni affinity chromatography and its activity was112.8U/mL. Then its enzyme characterization was analyzed. The optimal temperature and pH of the catalase was70℃and pH10.0, respectively. When the enzyme was incubated at80℃for2hours, the catalase activity was not lost. After incubated at pH9.0～11.0for2hours, its activity retained90%. The results showed that the MnCAT has a good potential and value for industrial development.(3) Meanwhile, the MnCAT gene was inserted into the extracellular expression vector pET20b and transformed into E. coli BL21(DE3). And realized the heterologous expression of MnCAT.(4)The gene of MnCAT was inserted into the expression vector pMA5and transformed into B.subtilisWB600. And realized the heterologous expression of MnCAT in B.subtilis WB600. The activity was80U/mL. At the same time, the gene of MnCAT was inserted into the expression vector pPIC9K and transformed into P.pastorisKM71. And realized the heterologous expression of MnCAT in P.pastoris KM71. The activity was6000U/mL after the optimization.This paper have realized the expression of MnCAT gene of Thermus thermophilus HB27in E.coli, B.subtilisWB600and P.pastorisKM71. The MnCAT was highly active in alkali-and-thermophilic environment, and has a good potential in industry.