Research And Application Of Pseudomonas Putida In Oxidation Of Biomass-derived Furan Aldehydes

With the rapid development of modern industry,fossil fuel reserves are gradually depleted,global warming and environmental pollution are becoming more and more obvious,and the development and utilization of clean and renewable new energy and alternative energy sources have become the current research hotspot.Lignocellulose is the most abundant biomass resource on earth and the only source of organic carbon.Cellulose and hemicellulose are two major components of lignocellulosic raw material biomass,the hexose and pentose produced by their hydrolysis are further dehydrated to produce 5-hydroxymethylfurfural(HMF)and furfural,respectively.Since HMF and furfural contain functional groups such as aldehyde groups,methylol groups,and unsaturated double bonds,they are very lively in nature and can produce a large number of downstream derivatives,and are considered to be an important bridge connecting biomass and bio-based chemicals.The selective oxidation of HMF and furfural can yield a series of high value-added furan carboxylic acids.For example,the selective oxidation product of aldehyde group of HMF is5-hydroxymethylfurfuric acid(HMFCA),and the oxidation product of furfural aldehyde group is furoic acid.HMFCA and furoic acid are widely used in materials,medicine,food and other industries.More progress has been made in the preparation of various furan carboxylic acids by chemical oxidation,but there are often disadvantages with harsh reaction conditions,unfriendly environment,and products that are prone to excessive oxidation.Compared with chemical oxidation,biological oxidation have advantages with mild reaction conditions,high selectivity,no toxic solvents,and environmentally friendly.However,the research on the selective oxidation of furanaldehydes to various furancarboxylic acids by biocatalysis is still in its infancy.Based on these,in this paper,a Pseudomonas putida KT2440 with various metabolic functions is used as a biocatalyst,and its application potential in bio-based HMF and furfural oxidation is focused on.Firstly,P.putida KT2440 was used as a catalyst to explore its ability to directional oxidation of HMF to form HMFCA,and the biocatalytic system was constructed and optimized to greatly increase the output of HMFCA;secondly,the oxidation ability of P.putida KT2440 in furfural was investigated,based on the induction effect of different inducing agents on the related furanaldehydes oxidase system,the cell’s ability to oxidize furfural was further improved,and a high yield of furoic acid was obtained in a batch feeding mode;finally,in order to mine enzyme proteins related to furanaldehydes oxidation in P.putida KT2440,a series of possible related functional proteins were speculated through biological information analysis and genome speculation,including PP3828,PP3310,PP4234,PP4230 and PP4231,proving the molybdate-dependent oxidase / molybdenum enzymes play a key role in the oxidation of furanaldehydes by P.putida KT2440.The research results are as follows:(1)P.putida KT2440 not only has high substrate tolerance to HMF,but also selectively oxidizes HMF to HMFCA.Based on the optimization of the catalytic system,within a substrate concentration of 150 m M,the conversion of HMF and the yield of HMFCA are close to 100%.When the HMF concentration is 160 m M,the HMFCA yield is 96.9%;when the HMF concentration is 175 m M,the HMFCA yield is 81.5%.In addition to HMF,P.putida KT2440 can also selectively oxidize a series of aldehyde groups of furanaldehydes.(2)P.putida KT2440 not only has high substrate tolerance to furfural,but also oxidizes furfural to furoic acid.Adding 4.5 m M HMF in the mid-log phase of cell proliferation can significantly induce the catalytic ability of intracellular furanaldehydes oxidation-related enzymes in P.putida KT2440;on the basis of the optimized catalytic system,204 m M furfuric acid can be produced by batch feeding,the yield is 88.7%.(3)Based on the analysis of biological information and genomic data,the enzyme proteins associated with furanaldehyde oxidation in P.putida KT2440 were speculated.The gene knockout technique proved that the molybdate transporter is essential for the oxidation of furanaldehydes by P.putida KT2440,thus speculating that the molybdate-dependent aldehyde oxidase is a key enzyme for furanaldehydes oxidation.On these basis,two different aldehyde oxidase gene clusters were speculated and their relationship with P.putida KT2440 furanaldehydes oxidation activity was verified.