Process optimization and hybrid nanoflower synthesis of 5- hydroxymethylfurfural oxidase for efficient oxidation of HMF and its derivatives to 2,5- furandicarboxylic acid.

2,5-furandicarboxylic acid (FDCA) is a precursor of the biopolymer Polyethylene furandicarboxylate (PEF). 5-hydroxymethylfurfural oxidase (5-HMFO) oxidizes 5- hydroxymethylfurfural (HMF) to FDCA. One such promising biocatalyst is 5-HMFO from Pseudomonas poae (PSPO). The major limitations of free enzymes include high cost, low thermal stability, limited storage stability and lack of reusability. To mitigate these limitations, the protein-inorganic hybrid nanoflowers of PSPO and Mn2+ (PSPO@MnNF) was developed. PSPO@MnNF exhibited a flower-like morphology, as observed under scanning electron microscopy. Molecular dynamics simulations were conducted to elucidate the coordination patterns and binding interactions between PSPO and the Mn2+ and Co2+ ions. PSPO was upscaled in a 5 L fermenter yielding 1.004 ± 0.2 g/L of recombinant protein. Analytical and biochemical characterization of hybrid nanoflowers showed enhanced storage stability, improved thermostability, higher catalytic efficiency, retaining >95% activity after the fifth reaction cycles for the substrate vanillyl alcohol and >80% activity after two cycles for the substrate HMF. At 4 °C, PSPO@MnNF maintained 95% of its residual activity during 30 days of storage. After immobilization, the kinetic parameters of PSPO@MnNF displayed lower Km for the substrates HMF, FFCA, DFF and vanillyl alcohol (1.620 mM, 2.309 mM, 0.675 mM and 0.792 mM, respectively) compared with free PSPO (2.252 mM, 5.551 mM, 2.503 mM and 1.589 mM, respectively). Hence, the developed PSPO@MnNF could be an efficient biocatalyst for the sustainable oxidation of HMF, FFCA, DFF and vanillyl alcohol.