The Study On Enzyme Characterization Of Catalase From Corynebacterium Glutamicum

Catalase catalyzes the decomposition of H₂O₂ to H₂O and O2.It is a ubiquitous enzyme present in both the eukaryotic and prokaryotic organisms.Catalase is widely used in textile,paper,biomedical and food industries due to its high efficiency.However,most catalases used in the textile and paper industries are often subjected to high-alkaline challenges which makes it necessary to develop new type of excellent alkali-tolerant catalase.In this study,we had determining the physiological characterizations of catalase from Corynebacterium glutamicum.The results showed that the catalase played an important role in the resistance to low-pH stress,suggesting its good physiological function.The grow condition of C.glutamicum is in the pH range of 6.0 to 10.0,indicating that it has the alkaline pH tolerance.Thus,it is possible to obtain catalase with high catalytic efficiency and favorable alkali tolerance.Therefore,a catalase from C.glutamicum was heterologously expressed in Escherichia coli,and the expression conditions were optimized.The recombinant catalase was successfully expressed in E.coli,and its optimal conditions were:an IPTG concentration of 0.2 mmol/L,a culturing temperature of 25℃ and a culturing time of 11 h.The recombinant catalase was purified by Ni-chelating affinity chromatography,and the enzymatic properties of recombinant protein were characterized.The purified catalase had a specific activity of 55266 U/mg,and it had a high activity in the pH range of 4.0 to 11.5,with the highest activity at pH 11.0.When treated in pH 11.0 for 3 h,the enzyme retained 93%of its activity,indicating that the enzyme was qualified with a favorable stability under high-alkaline condition.The recombinant catalase had maximal activity at 30℃,and showed a satisfactory thermal stability at a range of 25℃ to 50℃.The apparent Km and Vmax values of purified catalase were 25.89 mmol/L and 185.18 mmol/（min-mg）,respectively.The enzyme activity was increased 10%by 1 mmol/L K+,the metal ions of Mg2+ and Cu2+had little effect in the enzyme activity.Besides,different inhibitors,such as sodium dodecyl sulfate（SDS）,urea,NaN3,β-mercaptoethanol,and EDTA had different degrees of inhibition on enzyme activity.To improve the catalytic efficiency and stability of the catalase,the POPMuSiC software was used to predict the mutant site based on the folding free energy,and combined with the results of amino acid sequences alignment of catalases.A total of 12 mutant strains were constructed by site-directed mutagenesis.Compared to the wild-type enzyme,the crude enzyme activities of P490Q,E80L and D268C mutants were increased by 28.13%,22.72%and 11.59%.The catalase from C.glutamicum shows high catalytic efficiency and high alkaline stability,suggesting its potential utilization in industrial production.And the research on its molecular modification has also laid a foundation for further improving the application performance of the enzyme.