Catalytic Oxidation Of Furfural And Its Derivatives Into Furan-based Carboxylic Acids By Whole-cells Of Recombinant FAD-containing Oxidase

Furfural(FAL)is a representative biobased platform molecule that can be produced in large quantities by chemical and heat treatment of hemicellulose.It is an excellent organic solvent used in petroleum extraction units and petrochemical refineries to extract diene from other hydrocarbons.Furfural is in an intermediate oxidation state between the corresponding alcohol and the acid.Therefore,it can be reduced to furfuryl alcohol(FOL)or oxidized to furoic acid(FA).Furoic acid is a common raw material that is widely used today in the production of many pharmaceutical,agricultural and industrial chemicals.5-Hydroxymethyl furfural(HMF)is a derivative of furfural.The hydroxyl group is oxidized to obtain5-hydroxymethyl-2-furancarboxylic acid(HMFCA),which can be used as a precursor of various polyester materials to produce fibers and plastics.In addition,HMFCA also has important application value in the medical field,and can be used as a useful intermediate for interleukin inhibitors and drugs.In this study,one selective oxidation method was developed to bioconvert furfural and HMF into the corresponding furan-based carboxylic acids using recombinant E.coli HMFOMUT harboring FAD-containing oxidase.The results were given as follows.Firstly,FAD-containing oxidase was codon-optimized and expressed in E.coli to obtain whole-cell catalyst E.coli HMFO,and then mutated it(V367R-W466F)to improve its catalytic activity.The mutant E.coli HMFOMUT whole-cell could catalyze the biological conversion of furfural and HMF to the corresponding furan-based carboxylic acids.At 30°C and p H 7.0,E.coli HMFO whole cells could convert HMF to HMFCA at a low yield(1.0%),and 2,5-furandicarboxylic acid(FDCA)was obtained in the yield of 81.0%and the selectivity of 84.2%.Using E.coli HMFOMUT whole cells as biocatalysts,HMFCA was achieved in the yield of 84.8%and the selectivity of 84.8%,while 2,5-furandicarboxylic acid was obtained at the yield of 15.2%and the selectivity of 15.2%.It could be found that E.coli HMFOMUT was subjective to produce HMFCA.Secondly,different reaction p H,reaction temperature and metal ions were investigated on the biocatalytic activity of E.coli HMFOMUT.Whole-cells of this recombinant strain harbored good biocatalytic activity in a narrow p H range(p H 6.5-7.0).The optimum reaction temperature and p H were 30?and p H 7.0,respectively.Fe³⁺could promote catalytic activity.Under the optimum reaction conditions.Furan-based carboxylic acids were obtained from HMF(150 m M)and furfural(50 m M)with yields of 96.9%and 100%,respectively.Finally,the kinetic constants of the reaction were measured.Compared with FAL(Km=4.91 m M)and FOL(Km=7.36 m M),the Km value of HMF(Km=4.49 m M)was the smallest,indicating that the enzyme had the greatest affinity with HMF,and that HMF was a more suitable substrate.The results showed that E.coli HMFOMUT had high tolerance toward HMF(150 m M),FAL(50 m M)and FOL(50 m M),which was consistent with the analysis results.In summary,recombinant FAD-containing oxidase mutants capable of transforming furfural and its derivatives into corresponding furan-based carboxylic acids were constructed,and selectively catalytic oxidation of furfural and its derivatives was established,which provided a strategy for industrial production of furan-based carboxylic acids.