Expression And Optimization Of Manganese Catalase

Catalase(Hydrogen-peroxide oxidoreductase EC 1.11.1.6) can catalyze the decomposition of hydrogen peroxide into water and oxygen. The catalase had the advantages of less pollution, lower cost, and higher printing quality. With the enhancement of environmental meaning, catalase are widely used in terms of food, medicine, textile, paper and sewage treatment. Catalase is widely distributed in nature and found in almost all aerobic microorganisms.At present, the catalase from microbe has received greater attention than other sources of catalase for their good property. But the characterization of catalase from animal liver and most known strains still can not satisfy the demands of textile technology completely(pH >10, T >50 °C) or the yield is lower. Therefore, in the paper, the gene encoding manganese catalase from Thermdus thermophilus HB27 was overexpressed in Pichia pastoris and Mn2+ permease MntH from E. coli were co-expressed in E. coli BL21(DE3), comparing the enzymatic property and the fermentation conditions for E. coli BL21(DE3)/pET28a-MnCAT/pACYC-mntH production of catalase were optimized. The main results were listed below:(1) The gene encoding sequence of MnCAT from the high-temperature- alkali ancient bacterium T. thermophilus HB27 was screened by reviewing the literature and analyzing the genome of NCBI by bioinformatics. The mature gene was redesigned with systematic codon optimization to preferentially match codon frequencies of Pichia pastoris. Then the synthesised optimized-gene of MnCAT was cloned into p PIC3.5K and expressed in P. pastoris strain SMD1163. The recombinants were screened in MD and YPD/G418 plates. Finally MnCAT achieved the heterologous expression in P. pastoris. After four steps tratments with(NH4)2SO4 fractionnal precipitation, salting-out DEAE FF anion-exchange chromatography and Gel filtration chromatography, the recombinant MnCAT was purified. The result was that we got 3.36% of the total activity and the MnCAT specific was 1.18×106 U/mg with 717 purification fold. The molecular weight of recombinant MnCAT was estimated to be about 59 kD by SDS-PAGE. The optimal temperature and pH of the recombinant MnCAT was 55 °C and 8.0, respectively. It was stable below 30 °C. In addition, the recombinant MnCAT had great stability on the range of pH 6.0-9.0. The Km and Vmax values for MnCAT at 37 °C and pH 8.0 were 27 mmol/L and 13.4 mmol/(L·min), respectively. The MnCAT was obviously inhibited by Co2+ and Zn2+.(2) The MntH can improve the absorb speed of Mn2+ to reduce the high concentration need for expressing the MnCAT. Mn-catalase from Thermus thermophilus HB27 and Mn2+ permease MntH from E. coli were co-expressed in E. coli BL21(DE3). After four steps tratments with(NH4)2SO4 fractionnal precipitation, salting-out, DEAE FF anion-exchange chromatography and MonoQ ion-exchange chromatography, the recombinant MnCAT was purified. The resμLt was that we got 27.2% of the total activity and the specific of the MnCAT was 1250.7 U/mg with 6.1 purification fold. The molecular weight of recombinant MnCAT was estimated to be about 36 kD by SDS-PAGE. The optimal temperature and pH of the recombinant MnCAT was 90 °C and 10.6, respectively. It was stable above 90 °C. In addition, the recombinant MnCAT had great stability on the range of pH 8.0-10.6. The Km and Vmax values for MnCAT at 37 °C and pH8.0 were 60 mmol/L and 20 mmol/( L·min), respectively. The MnCAT was obviously inhibited by Co2+.(3) Fermentation optimization for the production of Mn-catalase was carried out at the shake flask level. The suitable carbon source and nitrogen source were 7.0 g/L glycerine, 3.75 g/L yeast extract and 11.25 g/L peptone respectively. The optimum induced concentration of IPTG was 0.05 mmol/L under the addition of 1mmol/L Mn2+ in media. Furthermore, the optimal culture temperature and pH were 37 °C and pH 8.0 respectively. Under the optimum conditions, the maximal catalase activity reached 476 U/ml, which was 3-fold higher than that of the control. Finally, these optimum conditions were also was carried out in a 5 L fermentor and the catalase activity increased to 1094 U/ml.In conclusion, the active expression of MnCAT in Pichia pastoris SMD1163 and co-expression of the Mn CAT and Mn2+ permease MntH in E. coli BL21(DE3) was achieved got. By analyzing and comparing the enzyme properties to choose the recombinant Escherichia coli to optimize the fermentation conditions and getting the higher yield MnCAT.