Genome Mining,Evolution And Catalytic Performance Of Baeyer-Villiger Monooxygenases In Ketoacid Oxygenation

ω-Hydroxy fatty acid is a kind of important functional chemicals.The presence of the active hydroxyl group in the molecule makes it more reactive.It can be used not only as starting material for surfactants and cosmetic intermediates,but also for synthetic lubrication in the machinery industry.ω-Hydroxy fatty acids can provide the longest monomer carbon-chain for the synthesis of polymeric materials with superior properties;in addition,ω-hydroxy fatty acids can be further converted into other functional chemicals such as α,ω-dicarboxylic acids or ωamino fatty acid.Starting from plant oils and fats,the use of Baeyer-Villiger monooxygenase in the unsaturated fatty acid biotransformation pathway can prepare such high-value added functional chemicals.The advantages of mild conditions,wide source of raw materials and renewable resources are one of the important directions of sustainable development of green chemistry.Baeyer-Villiger Monooxygenase(BVMO)can catalyze the conversion of long-chain aliphatic keto acids derived from plant oils to ester acids,and undergo hydrolysis to produce ωhydroxy fatty acids of significant value.Among them,the oxidation of keto fatty acids into esters by BVMO is a key link in the overall biotransformation path.The regioselectivity of BVMO determines the ratio of two products produced by a single oxygenation reaction on both sides of the carbonyl.The regioselectivity is not ideal,only 74/26(normal ester/abnormal ester).In order to improve the utilization efficiency of ketoacid substrates and simplify the downstream separation and extraction operations,it is urgent to find a BVMO that can catalyze long-chain aliphatic ketoacids with high regioselectivity,and realize the structural controllability of functional chemicals such as ω-hydroxy fatty acids.Prepare and further enrich the current BVMO toolbox,laying a scientific foundation for the discovery of BVMO regioselectivity mechanism.This subject employed the reported monooxygenase PpBVMO from Pseudomonas putida KT2440 as a probe for genome mining,and used 10-ketostearic acid as a model substrate for functional screening,resulting in the identification of a new enzyme,GsB VMO from Gordonia sihwensis,with much better regioselectivity than PpBVMO.A study on the enzyme characterization of GsBVMO showed that the optimal pH for GsBVMO to catalyze the monooxygenation of 10-ketostearic acid was 8.0 and the optimal reaction temperature was 30℃,but the thermal stability of the enzyme was not ideal.Its half-life in 30℃ is only 2.5 h.The regioselectivity of the enzyme to the long-chain aliphatic keto acid 10-ketostearic acid derived from oleic acid was determined to be up to 97/3(normal ester/abnormal ester,molar ratio),higher than 74/26,indicating that GsBVMO is more suitable for the preparation of ω-hydroxy fatty acids from oleic acid,which can improve substrate utilization and reduce the formation of by-products.Substrate spectrum studies indicate that the enzyme prefers to catalyze long-chain aliphatic keto acids,and its catalytic activity for ketoester substrates is much higher than that of the corresponding keto acids.The enzyme prefers to catalyze linear ketones with medium chain length.The catalytic activity of this enzyme on cyclic ketones and aromatic ketones is generally not high,which indicates that this enzyme prefers to specifically catalyzes fatty ketones.The mutant GsBVMOC308L with better catalytic performance was obtained by protein engineering.Its pure enzyme specific activity reached 4.9 times that of the parent GsBVMOWT,and still maintained high regioselectivity of 97/3.The kinetic parameters of the mutant and the parental enzyme were determined,indicating that the catalytic efficiency constant(kcat/Km)of GsBVMOC308L for the model substrate 10-ketostearic acid was 484 s-1·mM-1,which was 8.9 times of GsBVMOWT.The reason is that the mutant has an 8.4-fold increase of kcat.Based on the study of enzymatic characterization,the reaction performance of GsBVMO catalyzing 10-ketostearic acid is studied.The reaction temperature,the pH of the reaction buffer,the amount of cofactor FAD,the form of catalyst and the way of adding the substrate is optimized.Finally,the complete conversion of 10 g/L(33 mM)substrate within 4.0 h was achieved,and the space-time yield reached 60.5 g·L-1·d-1,which was increased by 36 times.The molar ratio of the two ester acid products was 97/3(normal ester/abnormal ester).The high catalytic efficiency and regioselectivity of the mutant GsBVMOC308L indicates that it has the potential for participating in the preparation of medium-chain ω-hydroxy fatty acids from oils.