Improving the catalytic activity of difructose anhydride III hydrolase from Arthrobacter chlorophenolicus A6 by modification of two residues

Inulin is an important fructan in nature, which provides energy to organisms by different metabolic pathways. In one of the metabolic pathways, inulin can be converted to difructose anhydride III (DFA-III) by DFA-III-forming inulin fructotransferase (IFTase-III) and DFA-III can be further hydrolyzed to inulobiose by difructose anhydride III hydrolase (DFA-IIIase). Inulobiose is a kind of fructooligosaccharide that can play a role of prebiotics. However, the low activity of DFA-IIIase is the key factor restricting the production and investigation of inulobiose. Therefore, in this work, the enzymatic activity was attempted to be improved by protein engineering. According to the sequence analysis and homologous modeling, several amino acids involved in the active site and the loop region above the active pocket were modified and eight mutants were constructed. The mutants E389A and E91K obtained significantly increased activities, which were 3.63 and 2.57 times that of the wild-type AcDFA-IIIase, respectively. The higher activity of E389A and E91K was possibly ascribed to the higher hydro-phobicity of Ala(389) and tighter catalytic pocket area formed between Lys(91) and His(158), respectively. The work explored how to enhance the activity of DFA-IIIase, laying a foundation for mass production, further investigation, and application of inulobiose.

Automated summary

In this study, protein engineering was used to improve the activity of DFA-IIIase by modifying amino acids involved in the active site and loop region. Two mutants, E389A and E91K, showed significantly increased activity, 3.63 times and 2.57 times higher than the wild-type enzyme, respectively. This work lays a foundation for the production, further investigation, and application of inulobiose.