Synthesis Of 2,5-furan Dicarboxylic Acid By Whole Cell Catalysis

With the European Union’s"plastic limit"and our"carbon peak,carbon neutral"put forward,bio-based products to replace petroleum products has become a hot topic in the field of research.A variety of biobased platform compounds can be synthesized from biomass resources,among which 2,5-furanodiformic acid（FDCA）can substitute for petroleum-based terylene acid to synthesize polyamides,polyester,polyurethane and other polymer materials,which has great application potential.At present,FDCA is mainly synthesized from 5-hydroxymethylfurfural（HMF）from biomass,and the main synthesis methods include chemical catalysis,enzyme catalysis and whole cell catalysis.Whole-cell catalytic reaction is mild,does not require additional cofactors,and can integrate multi-step catalytic reactions in the same cell,so it has attracted more and more researchers’attention.However,at present,few strains can catalyze whole-cell synthesis of FDCA from HMF,and the tolerance to toxic HMF substrates is generally poor,and most strains are conditioned pathogens,thus limiting the whole-cell catalytic synthesis of FDCA.Therefore,screening of safe strains with high tolerance to HMF is of great significance for the efficient preparation of FDCA.In this study,a strain with high tolerance to HMF was first obtained by screening,then the FDCA synthesis conditions were studied,and finally the synthesis efficiency of FDCA was significantly improved by strengthening the FDCA synthesis pathway.The main research results of this thesis are as follows:（1）Screening and catalytic conditions of FDCA production strain.A strain with catalytic HMF synthesis of FDCA was obtained from paper wastewater by screening,and it was named Bacillus amyloliquefaciens WTF1 by molecular biology identification.Subsequently,the HMF tolerance and FDCA production capacity of B.amyloliquefaciens WTF1 were investigated.The results showed that cell growth was severely inhibited when the HMF concentration was higher than 40 m M,and the FDCA yield was only 0.78m M.In order to improve the FDCA yield,whole-cell catalysis was used for FDCA synthesis.By optimizing the whole-cell catalytic conditions,the optimal reaction conditions were cell concentration OD₆₀₀=100,HMF concentration 75 m M,temperature30°C and p H 7.0.Under this optimal catalytic condition,the yield of FDCA was 32.5 m M and the conversion rate was 43%.The lower conversion rate was mainly due to the large accumulation of the intermediate product 5-hydroxymethyl-2-furancarboxylic acid（HMFCA）.（2）Study of catalytic properties of HMFCA oxidase.In order to promote further conversion of the intermediate product HMFCA to FDCA and improve the efficiency of FDCA synthesis,three possible enzymes catalyzing HMFCA（Hmf H,ADH,and Ma AAO）were selected for study.The heterologous expression of Hmf H,ADH and Ma AAO was purified by vector p ET28a in E.coli BL21（DE3）and their catalytic conditions were investigated.The results showed that the optimum temperature of Hmf H was 30°C and the optimum p H was 7.0;the optimum temperature of ADH was 30°C and the optimum p H was 8.0;the optimum temperature of Ma AAO was 25°C and the optimum p H was6.0.Subsequently,the catalytic performance of the enzymes was investigated,and all three enzymes could catalyze HMFCA to produce 5-formyl-2-furancarboxylic acid（FFCA）,which was then further oxidized to produce FDCA,but the catalytic ability of Hmf H was superior to that of ADH and Ma AAO.（3）To further improve the FDCA production capacity of B.amyloliquefaciens WTF1strain,we firstly tried to express Hmf H,ADH,and Ma AAO separately in B.amyloliquefaciens WTF1 strain using plasmids p STOP,respectively,using the enzymes Hmf H,ADH,and Ma AAO separately expressed in recombinant B.amyloliquefaciens WTF1 strain expressing Hmf H,ADH,and Ma AAO enzymes alone and subjected to whole-cell catalysis,the FDCA yields were 48.2 m M,37.1 m M,and 37.8 m M at a cell concentration of OD₆₀₀ of 100,HMF concentration of 75 m M,temperature of 30°C,and p H of 7.0,respectively;to further improve the FDCA yields,the FDCA yields were increased in B.amyloliquefaciens WTF1 strain,respectively.Subsequently,Hmf H,ADH and Ma AAO were co-expressed in B.amyloliquefaciens WTF1 strain,and FDCA yield reached 54.9 m M and 52.6 m M,respectively.was further increased to 68.1 m M with a transformation rate of 90.8%.