A Study On Biocatalytic Baeyer-Villiger Monooxygenation Of Aliphatic Keto Acids

α,ω-Dicarboxylic acids and ω-hydroxycarboxylic acids are important synthetic intermediates that can be used for the synthesis of plastics such as polyester,polyamide,polyurethane and so on.These compounds can be synthesized by biotransformation of variable and renewable vegetable oils in a mild and environmently friendly way instead of the traditional non-renewable chemical synthesis from petroleum.At present,one efficient way to produce α,ω-dicarboxylic acids and co-hydroxycarboxylic acids is to use Baeyer-Villiger monooxygenase(BVMO)to catalyze the oxidation of fatty keto acids from oils and fats into esters.Then the esters can be hydrolyzed into these valuable compounds.The regioselectivity of BVMO directly influences the varieties and proportions of hydrolysates.Due to the low regioselectivity of a reported BVMO from Pseudomonas fluorescens(PfBVMO),the products are variable,which increases the difficulty of the downstream separation and product purification.To improve the utilization of long-chain fatty acids for well-controlled synthesis of α,ω-dicarboxylic acids,a highly regioselective BVMO is needed.Furthermore,it will expand the tool box of BVMOs,helping to clarify the catalytic mechanisms of regioselectivity and laying the foundation for industrial applications.With PfBVMO as the probe and 10-ketostearic acid as the model substrate,PaBVMO with high regioselectivity for long-chain keto acids was identified by gene mining.Then the enzymatic properties were studied.It showed that the optimal pH in Gly-NaOH buffer(100 mM)was 9.0,and the optimal temperature was 25℃.However,the thermal stability of the enzyme was poor as its half-life was 80.6 min at 20℃,56.3 min at 25℃,and 25.5 min at 30℃,respectively.PaBVMO showed a high abnormal regioselectivity(greater than 90/10 for abnormal ester/normal ester)toward long-chain keto acids with the number of carbon atoms in the range of 17-20(10-ketoheptadecanoic acid,10-ketostearic acid,9-ketostearic acid,10-oxononadecanoic acid and 10-oxo-eicosanoic acid).The regioselectivity was significantly higher than that of PfBVMO.These long-chain keto acids are the main components of some vegetable oils,such as palm oil,olive oil and sunflower oil.Therefore,it can be concluded that PaBVMO was very suitable for the biotransformation of vegetable oils,which can significantly reduce by-products and improve the utilization of substrates.After the investigation of substrate scope,it was found that PaBVMO preferred long-chain substrates.Besides,the specific activity of PaBVMO toward keto esters was significantly higher than that toward the corresponding keto acids.The kinetic parameters of PaBVMO for long chain keto acids were determined.The highest catalytic efficiency(kcat/Km)was 7.30 s-1 mM-1 toward 9-ketostearic acid.The lowest one was only 0.22 s-1 mM-1 toward 10-oxo-eicosanoic acid.It was possibly caused by the huge difference in Km,in other words,the difference of affinities of PaBVMO toward substrates.After characterization,we optimized the reaction conditions such as temperature,pH,FAD,cosolvent and Tween 80 during the biotransformation of 10-ketostearic acid.The complete conversion of the substrate(1.0 mM)in 100 mL reaction was achieved,and the molar ratio of the two products was 94/6(abnormal ester/normal ester).After hydrolysis,the sebacic acid was obtained with a yield of 56.8%.This high regioselectivity facilitates separation and purification of products,which highlights the potential of PaBVMO in the production of α,ω-dicarboxylic acids.