Engineered Stable 5-Hydroxymethylfurfural Oxidase (HMFO) from 8BxHMFO Variant of Methylovorus sp. MP688 through B-Factor Analysis

Abstract

What is known as Furan-2,5-dicarboxylic acid (FDCA) is an attractive compound since it has similar properties to terephthalic acid. Further, 5-hydroxymethylfurfural oxidase (HMFO) is an enzyme, which could convert HMF to FDCA directly. Most wild types of HMFO have low activity on the oxidation of HMF to FDCA. The variant of 8BxHFMO from Methylovorus sp. MP688 was the only reported enzyme that was able to perform FDCA production. However, the stabilization of 8BxHMFO is still not that satisfactory, and further improvement is necessary for the industrial application of the enzyme. In this work, stability-enhanced HMFO from 8BxHFMO was engineered through employing B-factor analysis. The mutation libraries were created based on the NNK degeneracy of residues with the top ten highest B-factor value, and two of the effective mutants were screened out through the high throughput selection with the horseradish peroxidase (HRP)-Tyr assay. The mutants Q319K and N44G show a significantly increased yield of FDCA in the reaction temperature range of 30 to 40 °C. The mutant Q319K shows the best performance at 35 °C with a FDCA yield of 98% (the original 8BxHMFO was only 85%), and a half-life exceeding 72 h. Moreover, molecular dynamic simulation indicates that more hydrogen bonds are formed in the mutants, which improves the stability of the protein structure. The method could enhance the design of more stable biocatalysts; and provides potential for the further optimization and utilization of HMFO in biotechnological processes.

Current Advances in the Sustainable Conversion of 5‐Hydroxymethylfurfural into 2,5‐Furandicarboxylic Acid

“…The variant of 8BxHFMO from Methylovorus sp . MP688 was engineered to improve its stabilization since it is one of the few reported enzymes able to catalyze the complete oxidation of FDCA from HMF [140] …”

“…MP688 was engineered to improve its stabilization since it is one of the few reported enzymes able to catalyze the complete oxidation of FDCA from HMF. [140] Scheme 4. Multi-enzyme cascade pathway for FDCA synthesis.…”

(Chemo)biocatalytic Upgrading of Biobased Furanic Platforms to Chemicals, Fuels, and Materials: A Comprehensive Review

“…To enhance the thermostability of 8BxHMFO, Nie and co-workers further engineered this variant based on Bfactor analysis. 140 At 35 °C, the half-life of the mutant Q319 K exceeded 72 h, while that of the parent enzyme 8BxHMFO was around 40 h. Molecular dynamic simulation indicated that more hydrogen bonds were formed to stabilize the enzyme upon mutation.…”

“…This variant was able to quantitatively convert 5 mM HMF to FDCA at 25 °C with 2 μM of enzyme within 24 h, which outperformed both the WT enzyme and the variant V367R/W466F. To enhance the thermostability of 8BxHMFO, Nie and co-workers further engineered this variant based on B-factor analysis . At 35 °C, the half-life of the mutant Q319 K exceeded 72 h, while that of the parent enzyme 8BxHMFO was around 40 h. Molecular dynamic simulation indicated that more hydrogen bonds were formed to stabilize the enzyme upon mutation.…”

Progress in the biosynthesis of bio-based PET and PEF polyester monomers

“…Furthermore, molecular dynamics simulations show that the mutant protein structure is more stable due to the formation of more hydrogen bonds. 104 The above approach can be used to design stability-enhancing biocatalysts and will provide new insights into the design of HMFO variants that exhibit superior performance in the oxidation of HMF to FDCA.…”