Valorization Of Biobased Furans By Reductive Aminase AspRedAm

5-Hydroxymethylfurfural（HMF）and furfural are important bio-based platform compounds.N-Alkylfurfurylamine is a structural unit of many biologically active compounds and can be obtained via reductive amination of bio-based furan.Currently,N-Alkyl furfuramines are mainly prepared by chemical catalysis.Although great advances in chemical methods have been achieved,they are suffering from the problems such as self-reduction of carbonyl groups,poor stability of imine or enamine intermediates,and over-alkylation of the desired amines.The above-mentioned issues could be addressed by biocatalysis.Among many biocatalysts that enable the formation of C-N bonds,reductive aminases are preferred.Compared to transaminases and amine dehydrogenases that can only catalyze the synthesis of primary amines,reductive aminases are capable of synthesizing secondary amines.To date,biocatalytic reductive amination of bio-based furans to N-Alkylfurfurylamines has not been reported.The reductive aminase from Aspergillus aryzae（Asp Red Am）has a broad substrate spectrum.In this thesis,therefore,the feasibility of the reductive amination of bio-based furans by Asp Red Am was explored;based on molecular docking analysis,semi-rational molecular modification of Asp Red Am was conducted to improve its catalytic efficiency in the synthesis of bio-based N-Alkylfurfurylamines;the catalytic promiscuity of Asp Red Am capable of reducing carbonyl groups was studied.The main results obtained in this study are summaried as follows:1.Reductive amination of biobased furans by Asp Red Am and its modification.Asp Red Am not only catalyzes the reductive amination of HMF with n-propylamine,but also exhibits the catalytic promiscuity of reducing HMF.The optimal reaction temperature,n-propylamine/HMF molar ratio and enzyme concentration in Asp Red Am-catalyzed reductive amination of HMF with n-propylamine were 25℃,10,and 0.8 mg/m L,respectively.Under the optimal reaction conditions,the biocatalytic synthesis of 5-[（Propylamino）methyl]-2-furanmethanol was successfully scaled up.After 12 hours,the substrate was completely converted,and the selectivity toward the desired product was 99%;the target product was obtained with an isolated yield of 92%and a purity of 90%.Based on molecular docking analysis,the sites N93,I118,D169,L173,M119,W210 and V121 were identified for mutation.When Asn93 was mutated to Ala or Ser,the mutants completely lost reductive amination activity,but its promiscuous reduction activities were significantly improved.After Trp210 was mutated to Phe,the enzyme activity of the variant W210F was 2.1-4.7 times higher in the reductive amination of furfural than that of wild type.Molecular docking analysis shows that the smaller side chain of Phe210can provide more space for substrate binding,thus allowing the substrate binding in a proper manner.The synthesis of N-Propyl-2-furfurylamine by the variant W210F was scaled up;after12 h,the substrate furfural was completely converted;N-Propyl-2-furfurylamine was isolated with a yield of 97%and a purity of 93%.2.Catalytic promiscuity of Asp Red Am.Asp Red Am shows the NADPH preference in the reductive amination reactions,whereas NADH is preferred in the reduction reactions.Also,the variant N93A showed similar cofactor preference.The optimal p H values of Asp Red Am and the variant N93A were 9.0 and 8.0,respectively.The mutant N93A has high catalytic activity in the p H range of 7-9,with the relative activities of>76%.The optimal temperatures of both Asp Red Am and the variant N93A are 30℃.Upon N93A mutation,the thermal stability of the enzyme has been significantly improved.Asp Red Am is a non-metallic enzyme;except of Al³⁺,other metal ions tested have inhibitory effects on the activities of Asp Red Am and the mutant N93A to some extent.Asp Red Am and the mutant N93A exhibited relatively broad substrate spectra and showed catalytic activities toward most furan aldehydes,aromatic aldehydes and aromatic ketones.The activity of mutant N93A was 3-5 times that of Asp Red Am.Due to the smaller side chain of Ala93,the substrate can bind better in the binding pocket,thereby enhancing the affinity between the mutant N93A and the substrate and resulting in the improved catalytic activity of the mutant N93A.The whole cells harboring Asp Red Am and mutant N93A were used in the reduction of HMF and furfural.The yields of 2,5-Bis（hydroxymethyl）furan（BHMF）and furfuryl alcohol were 45-49%and 85-90%,respectively,after 6 h.In this study,the catalytic promiscuity of the reductive aminase Asp Red Am capable of reducing carbonyl groups was unveiled,and understanding of the catalytic mechanism of reductive aminase was improved.Also,a new biocatalytic pathway for the synthesis of biobased N-Alkylfurfurylamine was constructed.