Study On The Acylation Reaction Of Tofu Fruit Catalyzed By Whole Cells In Non-aqueous Phase

As a natural small molecule glycoside compound,helicid has various biological activities,including sedation,acesodyne and anti-depression.However,its bioavailability is low due to the poor lipid solubility.Studies have found that the derivatives of helicid ester have better bioactivity and bioavailability than the parental compounds.The biosynthesis of glycoside derivatives has the advantages of mild reaction conditions,high regioselectivity,and environmental friendliness.Compared with pure enzyme catalysts,the use of microbial whole-cell catalysts avoids the tedious preparation process of pure enzyme and greatly reduces the cost of biocatalysis.Therefore,whole-cell catalysis has been concerned in the field of green catalysis.In this dissertation,the possibility of the acylation of helicid by whole-cell catalysts were investigated.A whole-cell catalyst capable of efficiently catalyzing the caproylation of helicid was screened and the key reaction conditions were studied.Furthermore,the effects of medium composition and the culture time on the biomass and catalytic activity of whole cell was investigated.Finally,the characteristics of the benzoylation of helicid catalyzed by whole-cell biocatalysts in ionic liquid?IL?-containing medium were studied.Among the selected microbial strains,Pseudomonas aeruginosa GDM 1.443 showed the ability to catalyze the caproylation of helicid efficiently.The optimal reaction solvent,substrate molar ratio,whole-cell dosage and reaction temperature were determined as acetone,30:1,40mg/m L and 40°C,respectively.Under these conditions,the initial reaction rate,conversion,and regioselectivity were 3.4 m M/h,99%,and>99%,respectively.In addition,a series of helicid esters were synthesized by P.aeruginosa cells.The effects of culture medium composition and culture time on the biomass and catalytic activity of the whole cells of P.aeruginosa were discussed.The results showed that the optimal medium composition was 0.1%soybean oil,0.2%tryptone,0.02%Mg SO₄·7H₂O,0.5%?NH₄?₂SO_4.The whole cells of P.aeruginosa obtained by culturing for 36 h had high biomass?0.72 g/L?and high catalytic activity?conversion rate 99%?.The biomass of whole cells of Pseudomonas aeruginosa reached 0.71 g/L after culturing for 24 h when the optimal medium was used for the amplification test of 5 L fermentation tank.In addition,the reactions of helicid with various aliphatic acyl donors had a higher initial reaction rate and substrate conversion rate when catalyzed by the whole-cell catalyst obtained from the optimized medium.The characteristics of the benzoylation of helicid catalyzed by whole-cell biocatalysts in IL-containing medium were studied.The results showed that adding proper amount ILs of[BMIM][PF₆],[HMIM][PF₆],[OMIM][PF₆],[BMIM][BF₄]and[BMIM][TF₂N]in the acetone reaction system could all effectively accelerate the benzoylation of helicid catalyzed by the whole cells of Aspergillus oryzae GDM 3.446.In acetone-[BMIM][PF₆]?5%,v/v?system,the initial reaction rate,conversion rate and regioselectivity were 7.3 m M/h,99%,and>99%.In addition,A.oryzae cells still retained original catalytic activity of 70%when used for 6 batches.This study not only opens up a novel and highly efficient approach for the acylation of helicid,but also establishes a new culture system for whole cells used to catalyze the synthesis of helicid derivatives.Meanwhile,the application scope of common microorganisms in the field of biocatalysis has been expanded.