Selective Oxidation Of Bio-based Furan Aldehydes By Recombinant E.coli_CtSAPDH Cells

Furfural and 5-methoxymethylfurfural?MMF?are important bio-based platform chemicals,which can be synthesized via carbohydrate dehydration.MMF and furfural can be selectively oxidized into 5-methoxymethyl-2-furancarboxylic acid?MMFA?and 2-furoic acid,respectively,due to the presence of the formyl group.These furan carboxylic acids are important intermediates in the synthesis of fragrances,drugs,agricultural chemicals,photoelectric materials and synthetic fibers.Currently,chemical methods remain predominant in the synthesis of MMFA and 2-furoic acid;nonetheless,they suffer from some drawbacks such as environmental unfriendliness,unsatisfactory selectivity,high energy consumption and harsh reaction conditions.These problems may be well overcome in biocatalytic upgrading of bio-based furans;thus,biocatalysis with the nature of greenness and sustainability,will replace chemical methods for green and clean synthesis of 2-furoic acid and its analogs.Recently,we isolated Comamonas testosteroni SC1588 capable of efficiently oxidizing 5-hydroxymethyl-furfural?HMF?.A 3-succinoylsemialdehyde-pyridine dehydrogenase identified from this strain was introduced into Escherichia coli?E.coli_CtSAPDH?.There are few studies on SAPDH presently,which was only found to enable the degradation of nicotine and production of 3-succinyl-pyridine.In this thesis,therefore,the recombinant E.coli_CtSAPDH cells was used for the oxidation of bio-based furan aldehydes.The effects of induction and reaction conditions on E.coli_CtSAPDH-catalyzed oxidation of furfural and MMF were studied.An efficient and selective biocatalytic approach to synthesis 2-furoic acid and MMFA was established.The toxicity of furfural,HMF and MMF and their carboxylic acid derivatives toward the cells as well as their inhibitory effects on the cells was studied.It was found that,of the induction conditions tested,the induction temperature had the greatest influence on the catalytic performances of recombinant cells in the oxidation of furfural;the optimum induction temperature was 30?.The effects of IPTG concentrations and induction periods on the catalytic activities of this recombinant strain were slight.The optimum reaction temperature and pH range were 30?and pH 7.0-8.0,respectively,in E.coli_CtSAPDH cell-catalyzed furfural oxidation.Under optimal conditions,the recombinant cells could tolerate 100mM,as well as 100 mM of HMF.2-Furoic acid and 5-hydroxymethyl-2-furancarboxylic acid?HMFCA?were obtained with 95-98%yields when the substrate concentrations were less than100 mM.This recombinant strain was capable of the production of 147 mM 2-furoic acid within96 h using a fed-batch strategy,providing a space-time yield of 4.2 g·L^-1·d^-1;besides,242 mM of MMFA was produced in 20.5 h,and its space-time yield was approximately 44.2 g·L^-1·d^-1.In addition,the recombinant strain enabled other furan aldehydes and benzaldehydes to be oxidized into the target carboxylic acids,with the yields up to 100%.The whole-cell catalytic synthesis of MMFA was scaled up to 50 mL.It was observed that 200 mM of MMF was completed converted in 6 h,resulting in the production of 174 mM MMFA.The space-time yield of MMFA was up to 108.5 g·L^-1·d^-1.Upon organic solvent extraction,MMFA was obtained with the recovery of 97%and the purity of 85%.In addition,the toxicity of HMF,MMF and furfural toward recombinant cells as well as their inhibitory effects on the cells was compared.The order of the toxicity of these aldehydes as well as their inhibition effects was listed as follows:furfural>MMF>HMF.The toxicity of HMFCA,MMFA and 2-furoic acid toward the cells mainly derived from their acidity.The order of toxicity of these carboxylic acids as well as their inhibition effects was listed as follows:2-furoic acid>MMFA>HMFCA.In this thesis,the application of SAPDH in synthetic chemistry was extended,and the catalytic performances of the recombinant strain were uncovered in the oxidation of furan aldehydes.The toxicity of furan aldehydes toward recombinant cells as well as their inhibitory effects on the cells was unveiled.The present study may not only enrich the relative theoretical knowledge in biocatalytic valorization of bio-based platform chemicals,but also lay the technical foundation for the large-scale synthesis of value-added furan derivatives.