Effect of fusing a self-assembling amphipathic peptide on the enzymatic properties of recombinant catalase

Abstract:

[Objective] In this study,we fused a catalase KatA from Bacillus subtilis with a self-assembling amphipathic peptide to improve its adaptability in industrial production.[Methods]S1vw-PT-katA and katA were cloned into pHT254 to yield the constructs pHT254-S1vw-PT-katA and pHT254-katA,respectively,which were separately introduced into B.subtilis WB800N for expression.The recombinant enzymes were then purified and characterized.[Results] The purified enzymes were acquired from the extracellular crude extract of the engineering bacteria through a four-step procedure consisting of ethanol precipitation,DEAE anion exchange chromatography,hydrophobic chromatography,and gel filtration chromatography.The fused enzyme S1vw-PT-KatA and natural enzyme KatA exhibited maximum activity at 30℃ and pH 11.0.However,the relative activity of the S1vw-PT-KatA incubated at pH 12.0 for 30 min was 77.3%,which was 14.9 times that of KatA under the same conditions.The relative activities of S1vw-PT-KatA incubated at 65℃ and 70℃ for 30 min were 19.8% and 17.5%,respectively,which were 1.8 and 1.7 times that of KatA.The relative activity of S1vw-PT-KatA stored at 4℃ for 14 days was 88.6%,while that was only 44.3% for KatA.Meanwhile,the k_cat/K_m value of S1vw-PT-KatA was 2.3 times that of KatA.[Conclusion]Fusing with a self-assembling amphipathic peptide S1vw can improve the pH stability,thermostability,storage stability,and catalytic efficiency of recombinant KatA.This finding provides a potential strategy for the modification,large-scale production,and application of catalase.