Study On Preparation Of Aromatic Aldehyde And Ketone Fragrances Compounds By Enzyme Cascade Catalysis

Flavors and fragrances are closely related to the improvement of people’s living standards and the development of food industry.Among them,aromatic aldehydes and ketones are important flavors and fragrances.With the enhancement of people’s awareness of product safety and the advocacy of health concepts,natural fragrances extracted from animal and plant raw materials by physical methods or synthesized by biological methods are more desired.Compared with traditional extraction methods and chemical synthesis methods for the preparation of aromatic aldehydes and ketones,biological enzyme catalysis method has the characteristics of desirable specificity,high catalytic efficiency and environmental friendliness.Moreover,the multi-step reactions could be carried out continuously in one reaction system without the need to separate intermediates in enzymatic cascade reactions,which have more application prospects.This study aims at the biocatalytic synthesis of aromatic aldehyde and ketone fragrance compounds.Cascade catalytic reaction systems of choline oxidase（An Ch Ox）-coupled unspecific peroxygenase（Aae UPO）and aryl-alcohol oxidases（Pe AAOx）-coupled unspecific peroxygenase（Aae UPO）were constructed.This study will provide a theoretical basis for the application of enzymatic cascade catalysis technology in the preparation of aromatic aldehyde and ketone fragrance compounds.The specific research contents and results are as follows:1.Preparation of key enzymes AnChOx and PeAAOx for the catalytic synthesis of aromatic aldehyde and ketone fragrance compounds.Heterologous expression of An Ch Ox and Pe AAOx in Escherichia coli was achieved.After purification by nickel column affinity chromatography and anion exchange chromatography,the effects of temperature and p H on the activity of Pe AAOx were investigated.The results showed that the yield of An Ch Ox was 80mg·L^-1,the protein concentration was 3.9 mg·L^-1,and the specific enzyme activity was 4.0U·mg^-1;the yield of Pe AAOx was 35 mg·L^-1,the protein concentration was 4.0 mg·L^-1,and the specific enzyme activity was 0.2 U·mg^-1.The optimum reaction temperature was 35℃,the residual enzyme activity was more than 94%after incubation at 30℃and 40℃for 2 h;the optimum reaction p H was 7.0,and the residual enzyme activity was more than 90%after incubation at p H 7.0 for 2 h.2.AnChOx-coupled AaeUPO cascade catalyzed preparation of aromatic ketone fragrance compounds from aromatic secondary alcohols.Using aromatic secondary alcohols as substrates,the effects of enzyme dosage,p H,temperature and other factors on the catalytic effect in the enzymatic cascade catalysis process were investigated,and the synergistic process of double-enzyme catalysis was explored.The feasibility of An Ch Ox-coupled Aae UPO cascade catalyzed by different aromatic secondary alcohols to prepare aromatic ketone fragrance compounds was verified.The results showed that:An Ch Ox was the key rate-limiting factor of the cascade catalytic process;under the conditions that the initial concentration of Aae UPO was 1.0μM,the initial concentration of An Ch Ox was 2.5μM,the p H of the reaction system was 8.0,the reaction temperature was 30°C,and the substrate concentration was 25.0m M,Aae UPO enzyme solution（the amount added was the same as the first addition,with a final concentration of 2.0μM）and An Ch Ox enzyme solution（the amount added was the same as the first addition,with a final concentration of 5.0μM）were added every 12 h,the product concentration was 17.0 m M after 24 h of reaction,and the conversion was 68.0%.In addition,the cascade catalytic reaction system was applied to the synthesis of five aromatic ketone fragrance compounds,and the conversion was between 8.0-68.0%.3.PeAAOx-coupled AaeUPO cascade catalyzed preparation of aromatic aldehyde fragrance compounds from aromatic primary alcohols.Using aromatic primary alcohols as substrates,the effects of enzyme dosage,p H,temperature and other factors on the catalytic effect of Pe AAOx-coupled Aae UPO cascade catalysis were investigated.The catalytic efficiency of this cascade catalysis was compared with the An Ch Ox-coupled Aae UPO reaction system and the Pe AAOx single enzyme catalyzed reaction system.The feasibility of Pe AAOx-coupled Aae UPO cascade catalyzed by different aromatic primary alcohols to prepare aromatic aldehyde fragrance compounds was verified.The results showed that:the concentration of Pe AAOx was 30.0μM,the concentration of Aae UPO was 2.0μM,the p H of the reaction system was 7.0,the reaction temperature was 40°C,and the substrate concentration was 50.0 m M after24 h,the product concentration was 42.0 m M,the conversion rate was up to 84.0%;the product concentration of the Pe AAOx-coupled Aae UPO reaction system was 1.45 times that of the An Ch Ox-coupled Aae UPO reaction system,and 1.24 times that of the Pe AAOx single-enzyme-catalyzed reaction system.In addition,the cascade catalytic reaction system was applied to the synthesis of six aromatic aldehyde fragrance compounds,and the conversion was between 10.1-84.1%.