Identification, Properties And Application Of Aldehyde Dehydrogenases From Comamonas Testosteroni In Selective Oxidation Of Furan Aldehydes

5-Hydroxymethylfurfural(HMF)and furfural are important biobased chemicals,which can be synthesized via carbohydrate dehydration.5-Hydroxymethyl-2-furancarboxylic acid(HMFCA)and 2-furoic acid(FCA)are the oxidation products of furan aldehydes above mentioned and have promising applications in polymer,pharmaceutical and food industries.Although significant advances in chemically catalytic oxidation of furan aldehydes into furan carboxylic acids have been achieved,these methods suffer from some problems,such as harsh reaction conditions,environmental infriendliness and unsatisfactory selectivities.Biocatalysis can overcome such drawbacks,which has been a good option to supplement and even replace chemical methods in synthetic chemistry.Especially for upgrading inherently unstable bio-based furans such as HMF and furfural,biocatalysis appears to be a preferred tool compared to chemical catalysis.Using of whole cells as biocatalysts in the redox reactions has attracted more interest than isolated enzymes,because they are more readily prepared,and the enzymes inside the cells are more stable under the protection of cell membranes.In addition,the regeneration of cofactors is easy in vivo.However,the substrates HMF and furfural are well-known potent inhibitors towards microorganisms and enzymes;thus,only limited biocatalysts were reported to show good substrate tolerance,and to enable efficient and selective oxidation of furan aldehydes.In this dissertation,a microbial strain Comamonas testosteroni SC1588 that is highly tolerant to HMF and is capable of selectively oxidizing HMF into HMFCA was isolated from soil samples.The genes of aldehyde dehydrogenases(ALDHs)from C.testosteroni SC1588 that are responsible for HMF oxidation were identified,heterologously expressed,purified and biochemically characterized.Subsequently,whole cells of recombinant E.coli harboring these ALDHs were used for catalytic oxidation of HMF.Moreover,NADH oxidase(NOX)and ALDHs were co-expressed in E.coli to promote the NAD~+regeneration,thus improving the selectivity of target products.An efficient and selective biocatalytic approach for the synthesis of furan carboxylic acids was developed.Based on the study on the substrate inhibition and toxicity against whole cells,it was shown that the HMF-tolerance level of C.testosteroni SC1588 was at least 100 mmol/L.The product HMFCA exerted significant inhibitory and toxic effects on this strain,which might be mainly attributed to the acidity of this compound,because its catalytic performances were highly dependent on pH.The catalytic performances of the strain were improved significantly by both adding histidine and tuning pH.Under the optimal reaction conditions,HMFCA was afforded in a yield of approximately 98%after 36 h at a substrate concentration of 160mmol/L.This strain was capable of selectively transforming furfural,5-methylfurfural and5-formyl-2-furancarboxylic acid(FFCA)to the target carboxylic acids with yields of 90-93%.The genes of aldehyde dehydrogenases including two vanillin dehydrogenases(CtVDH1and CtVDH2),two coniferyl aldehyde dehydrogenases(CtCALDH1 and CtCALDH2)and a3-succinoylsemialdehyde-pyridine dehydrogenase(CtSAPDH)that can catalyze oxidation of HMF were mined from the genome of C.testosteroni SC1588,cloned and heterologously expressed in E.coli.The subunit molecular masses of the five ALDHs were approximately 50kDa.CtCALDH1 was strictly dependent on NAD~+,while other four ALDHs were both NAD~+-and NADP~+-dependent.Optical pH and temperature of these ALDHs were 7.0 and25-35oC,respectively.Nonetheless,all the ALDHs exhibited unsatisfactory stability in vitro.The recombination ALDHs had different substrate specificity.CtCALDH1 and CtCALDH2exhibited low activities towards most aromatic aldehydes,and CtCALDH1 could not accept FFCA,5-methylfurfural and 4-formylbenzoic acid as substrates.CtVDH1 and CtVDH2exhibited satisfactory activities towards most substrates,while CtSAPDH displayed moderate activities.In general,benzylaldehydes appeared to be preferable substrates of these ALDHs compared to their structural analogs(furan aldehydes).Of recombinant E.coli strains expressing ALDHs tested,E.coli/pET-CtVDH1 displayed the highest catalytic efficiency in biocatalytic oxidation of HMF.Upon the reaction of 12 h,HMF of 200 mmol/L was completely converted,and HMFCA was affored in the yield and selectivity of 92%and 89%,respectively.HMFCA of 448 mmol/L was produced in 41.5 h by a fed-batch strategy.E.coli/pET-CtVDH1 could also biotransform crude HMF(70%of the purity)into HMFCA.In addition,various aldehydes such as furfural,FFCA,5-methoxymethylfurfural(MMF),5-methylfurfural and benzaldehyde were selectively transformed into the corresponding acids with yields of 83-95%by this biocatalyst.NOX was co-expressed with CtVDH1,CtVDH2,CtCALDH2 and CtSAPDH in E.coli,respectively.The catalytic performances of these recombinant cells in the oxidation of HMF and its structural analogs(aromatic aldehydes)were investigated.It was found that E.coli-CtVDH1-NOX showed the best catalytic performance in the oxidation of HMF,among these biocatalysts examined.HMF was converted into HMFCA with the yield and selectivity of both 95%within 9 h when the substrate concentration was 250 mmol/L.Of whole-cell biocatalysts tested,E.coli-CtVDH2-NOX gave the highest catalytic efficiency toward furfural and MMF.FCA was obtained in a yield of 90%within 24 h when the furfural concentration was 240 mmol/L.FCA of 177 mmol/L was produced in 6 h by a fed-batch strategy,and its productivity was 3.3 g/L h.Moreover,MMF was converted into5-methoxymethyl-2-furancarboxylic acid(MMFCA)in a yield of 99%within 9 h when its concentration was 240 mmol/L.MMFCA of 287 mmol/L was produced in 8 h by a fed-batch strategy,and its productivity was 5.6 g/L h.In this study,the catalytic performances of whole cells in selective oxidation of furan aldehydes as well as the enzyme properties of ALDHs were uncovered,which enriches the theoretical knowledge for biocatalytic valorization of biobased furan aldehyde.In addition,a green,efficient and highly selective biocatalytic approach for the synthesis of furancarboxylic acids was developed.Biocatalysis will creat new opportunities for the development of clean"biorefinery".