Coupling a recombinant oxidase to catalase through specific noncovalent interaction to improve the oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid.

ABSTRACT

5-Hydroxymethylfurfural oxidase (HMFO) can catalyze both hydroxyl and aldehyde oxidations. It catalyzes 5-hydroxymethylfurfural into 2,5-furandicarboxylic acid. However, the application of HMFO encountered two problems the expressed HMFO in Escherichia coli. is largely in the form of inclusion bodies, and the by-product of H2O2 has a negative effect on HMFO stability. To solve these problems, recombinant HMFO was generated by fusing the C-terminus to an elastin-like polypeptide (ELP). ELP-HMFO can be expressed with significantly reduced inclusion bodies. ELP-HMFO exhibited improved stability and tolerance toward H2O2. Further recombination is carried out by fusing the N-terminus of HMFO to a glutamic acid-rich leucine zipper motif (ZE). Similarly, recombinant catalase (CAT) is generated by fusing the N-terminus to ELP and fusing the C-terminus to an arginine-rich leucine zipper motif (ZR). ELP-HMFO-ZE can interact specifically with ZR-CAT-ELP, ascribing to the coiled-coil association of ZE and ZR. ELP-HMFO-ZE#ZR-CAT-ELP coordinates the respective catalytic activities of the two enzymes. ELP-HMFO-ZE catalyzes the oxidation of HMF, and the generated hydrogen peroxide is decomposed by ZR-CAT-ELP into H2O and oxygen. During the oxidation of HMF, the cofactor FAD of HMFO is reduced, and molecular oxygen is needed to reoxidize the reduced FAD. The evolved oxygen from the decomposing of H2O2 can just meet the requirement, which can be diffused efficiently from ZR-CAT-ELP to ELP-HMFO-ZE due to the short distance between the two enzymes.