Cloning,Expression And Characterization Of The Catalase Gene From Pseudomonas Stutzeri S12

Catalase can catalyze the decomposition of hydrogen peroxide to produce water and oxygen.Catalase has been widely used in the food industry,such as food preservation,food disinfection,aseptic packaging,and bleaching and decolorizing animal food.However,the source of commercial food catalase is relatively limited,mainly obtained from animal liver separation and purification,aspergillus niger culture under conditions such as ventilation and stirring.Moreover,the enzymatic activity of food-grade catalase is low,and the thermal stability is poor,which cannot meet the needs of some industrial production.Therefore,this study focused on screening the strain with high catalase activity,and expression of its catalase gene in vitro.The method of rational design and orthogonal design Catalase was used to design the mutant with multiple mutations,which successfully improved the specific activity and thermal stability of the enzyme.The main results are as follows:1.Three strains with high catalase activity were screened from the landfill leachate,and the genome of a strain with higher enzyme activity was sequenced.The sequence of 16s rDNA identified the strain as Pseudomonas stutzeri,and the strain was named Pseudomonas stutzeri S12.2.This study is the first in vitro study of the catalase katE gene from Pseudomonas stutzeri,and the optimum temperature of the catalase is 35?,and the specific activity of the enzyme with relatively high ranged from 10 to 40??When the temperature exceeds 40?,the enzyme activity drops rapidly.Therefore,the enzyme is a meso-temperature enzyme that can adapt to low-temperature environments,which provides research ideas for future applications in food preservation such as refrigerated seafood.3.The optimum pH of the catalase KatE in this study is 8.0,and it is relatively stable under alkaline conditions.The 1 mmol/L Na+and Mg2+to the reaction system can promote the activity of the enzyme to a certain extent,which activity of the enzyme increased by 2%and 6%,respectively.The ions Li+,K+,Ba2+ can increase the enzyme activity.The inhibitory effect of Zn2+,Mn2+,Ca2+and Al3+ is not obvious.The ions Ni2+,Cu2+,Co2+have the strongest inhibitory effect on the enzyme activity,and the enzyme activity has all decreased by 99%.Therefore,an appropriate amount of Na+?Mg2+ to the enzyme activity reaction system in actual production applications can help increase the specific enzyme activity of catalase.4.In this study,consensus protein design was used,and 5274 sequences similar to KatE were selected to rationally design the mutations of KatE.The nine mutation sites were selected out and assembled with orthogonal design.These 11 multiple-site mutants were constructed based on the nine mutations.Combined with the orthogonal design,the results of the effects of these 9 mutation sites and their interaction on catalase activity and thermal stability were obtained by only 11 multi-site mutants.As a result,four mutations that could improve the specific activity and thermostability of catalase KatE were screened out.After analyzing these four sites,it is found that the enzyme specific activity of single point mutation Y112F has positive effect on the thermal stability.Additionally,the double point mutations(Y112F and S204N,F135I and T387S)could improve its specific activity and thermal stability,respectively.And,the Tm values of the two double point mutants are(45.46±0.06)? and(45.70±0.05)?,respectively.Therefore,the method combined of the orthogonal experiment and rational protein design is a useful method to improve the activity and thermal stability of proteins.This study screened out a strain Pseudomonas stutzeri with high catalase activity from the landfill leachate,and studied the enzymatic properties of its catalase,and improved its stability and catalyzing efficiency through the orthogonal experiments and rational design methods.The results enriched the genetic research on Pseudomonas stutzeri,laid a foundation for the research of low-temperature catalase,and provided a new method for the production and application of catalase in the food science.