Synthesis Of Raspberry Ketone By 4-hydroxybenzaldehyde Cascade Reaction

Raspberry ketone(RK)is the main aroma substance of raspberry fruit,which is widely used in perfume cosmetics and food additive industries.At present,raspberry ketone is mainly produced by chemical methods.Due to the low yield of microbial fermentation,the synthesis of RK by enzyme/whole-cell catalysis has attracted much attention.In the early stage,our laboratory realized the synthesis of RK from 4-Hydroxybenzylideneacetone(4-HBA)using the whole cell of Escherichia coli YB-9.On this basis,this paper explored the method of one-pot cascade and step-by-step cascade synthesis of raspberry ketone with 4-Hydroxybenzaldehyde(4-HBD)as the starting substrate.The specific research was as follows:(1)One-pot synthesis of RK with 4-HBD and acetone as substrates.Investigation of YB9 using its endogenous deoxyribonucleic acid aldolase(DERAEc)to catalyze the condensation of 4-HBD and acetone to generate the intermediate product 4-HBA,4-HBA was then reduced to RK using NADPH regeneration of YB-9,and it was found that YB-9 could synthesize a small amount of 4-HBA but no RK generated.Establishment of two-phase catalytic system composed of methyl tert-butyl ether(MTBE)and citric acid-sodium citrate buffer(SCB)and determined the volume fraction;engineered E.coli BL21(DE3)/pRSF-deoc(Z-1)overexpressing DERAEc was constructed,which was combined with YB-9 to synthesize RK from 4-HBD in doublebacteria one-pot catalysis.As a result,32 mmol·L-1 4-HBD and 320 mmo1·L-1 acetone were catalyzed for 24 h to obtain 0.11 mmol·L-1 RK.(2)One-pot synthesis of RK with 4-HBD and isopropanol as substrates.Engineered Escherichia coli co-expressing isopropanol dehydrogenase from Lactobacillus brevis and raspberry ketone synthase is constructed,which used isopropanol as the substrate to provide acetone and regenerated NADPH.As a result,32 mmol·L-1 4-HBD and 5%isopropanol reacted to synthesize 0.04 mmol·L-1 4-HBA,but no RK formation was detected.Analysis showed that RK could be converted by isopropanol dehydrogenase into another product.(3)Optimization of the parameters of 4-HBA synthesis from 4-HBD and acetone.Investigation of the reaction parameters of the first step in the 4-HBD cascade catalytic synthesis of RK.The results showed that the dosage of Z-1 was 4%(w/v),the concentration of 4-HBD was 120 mmol·L-1,the content of acetone was 80%,and the reaction temperature was 60℃,the reaction time was 48 h,the concentration of 4-HBA reached 111.36 mmol·L-1,and the yield was 92.80%.Carrageenan was used to prepare immobilized cells,and 120 mmol·L-14-HBD was used as the substrate to react for 36 h in each batch.The catalysis was repeated for 10 batches,and the molar conversion rate of 4-HBD remained above 90%.The average concentration of 4-HBA was 60.69 mmol·L-1.Further,by rationally modifying the organic solvent stability of wild-type DERAEc through the strategy of flexible site rigidification,a mutant DERAEc(S238D)was obtained,which was incubated in 60℃ and 80%acetone for 4 h,and the enzyme activity remained 58.04%.It was 3.24 times of the remaining enzyme activity of the wild type.(4)Optimization of the synthesis of RK from 4-HBA.Construction of E.coli BL21(DE3)/pRSF-ecidh-rirzsl(Z-4)overexpressing the isocitrate dehydrogenase.By coupling the NADPH regeneration system,61 mmol·L-1 4-HBA could be converted into 51.02 mmol·L-1 RK at the highest in 0.1 mol·L-1 pH 5.5 citric acid-sodium citrate buffer,with a yield of 83.63%.(5)Bibacterium catalyzed the synthesis of RK from 4-HBD in a step-by-step cascade,and the first step reaction conditions are:the content of Z-1/DERAEc(S238D)in whole cells was 4%(w/v)and 120 mmol·L-1 4-HBD was reacted in acetone at 60℃ for 36 h;in the second step,the reaction solution of the first step was decellularized and the acetone was recovered,citric acid-sodium citrate buffer and 4%(w/v)Z-4 were added for catalysis for 2 h to obtain a maximum of 50.00 mmol·L-1 RK,and the yield from 4-HBD to RK was 83.33%.