Extraction Of Esterase From Pig Liver By Three-phase Partitioning And Its Applications In Preparation Of D-7-ACA And Ethyl Butyrate

Esterase is widely distributed in animals,plants,and microorganisms.It has wide substrate specificity and can catalyze hydrolysis and synthesis of a variety of compounds containing esters,amides,sulfones,or acetyl groups.Esterase has important applications in the field of biocatalysis,and this paper studies the extraction and initial studies on the enzymology of esterase from pig liver.The thesis studied its ability to catalyze the deacetylation of7-aminocephalosporanic acid to produce deacetyl-7-aminocephalosporanic acid.In addition,we explored the synthesis of ethyl butyrate by esterase in the organic phase.The main results of the thesis as follows:(1)Extraction of esterase from pig liver by three-phase partitioning has been established.The effects of different organic solvents,organic solvents dosage,ammonium sulfate concentration,and the pH of the system and extraction temperature on the three-phase extraction of pig liver esterase have been investigated.Based on the single factor experiments,a response surface method was used to optimize the best conditions.The optimal conditions were as follows:ammonium sulfate mass fraction 40%,the ratio of isopropanol to aqueous phase 1.5:1,pH 7.0,and temperature 25℃.Under these conditions,the enzyme activity recovery rate was 60.79%,the purification factor was 20.5,and the preservation of 75.1%of enzyme activity and the Km value of the maximum reaction rate was 2.01 mg/mL,corresponding to a maximum reaction rate of 15.758 mg/(mL·min).The preliminary studies on the enzymology of extracted pig liver esterase have shown that the optimal reaction temperature is 30℃and the optimal reaction pH is 7.0.The Michaelis constant was 2.01 mg/mL,corresponding to a maximum reaction rate of15.758 mg/(mL·min).(2)The deacetylation of 7-ACA to preparation D-7-ACA catalyzed by pig liver esterase was studied.The optimal preparation conditions were:pH 7.5,temperature 35℃,substrate concentration 2%,and the enzyme addition was30 U/mL,7-ACA conversion rate reached about 95%.Molecular docking simulation was performed to analyze the binding between pig liver esterase and 7-ACA.The binding free energy of the enzyme-7-ACA complex was calculated as-4.5 to-6.0 kcal/mol,which confirmed that the reaction could spontaneously occur.(3)Using chitosan as carrier material and glutaraldehyde as cross-linking agent,immobilized pig liver esterase microspheres were prepared.The enzymatic properties of the immobilized enzyme were investigated,and the results showed significant improvement in temperature stability and pH stability compared to the free enzyme.The immobilized enzyme was coupled with D311 anion exchange resin,which was used to establish a system for in situ separation and coupling between the immobilized enzyme and the deacetylation reaction.The system eliminated the inhibitory effect of the by-product acetic acid on the deacetylation reaction.After the reaction solution flow rate was 13 mL/min and the reaction time was 6 h,the conversion rate of7-ACA reached more than 86%.(4)Using macroporous resin D101 to adsorb pig liver esterase,a TA-Ti~Ⅳnano coating formed by the coordination reaction of tannic acid(TA)and(2-hydroxypropionic acid)diaminodium hydroxide(Ti-BALDH)was coated on its surface,enhancing the stability of immobilized pig liver esterase.The enzymatic properties of the TA-Ti~Ⅳ-capped PLE@Resins were shown to have improved temperature stability and pH stability are improved compared with PLE@Resins and Free PLE,and it has good tolerance to organic solvents.The immobilized enzyme was used to catalyze the synthesis of butyryl acetate from butyric acid and ethanol in n-hexane at a reaction temperature of 40℃,with a substrate concentration of 0.1 mol/L,a molecular sieve addition of 0.09 g/mL,and an immobilized enzyme addition of 0.2 g/mL,the conversion rate of butyryl acetate reached 84%after 30 h.After repeated use for 3 times,the esterification rate was still 54.21%in 30 h.