Research On The Enzymatic Properties And Hydrolysis Function Of Recombinant Ferulic Acid Esterase

Feruloyl esterases represent a diverse group of hydrolases catalyzing the cleavage andformation of ester bonds between plant cell wall polysaccharide and phenolic acid. They arewidely distributed in plants and microorganisms.Little of microbial feruloyl esterases havealso been discovered and overexpressed.Feruloyl esterase forms a part of the enzymecomplex that acts collectively and synergistically to completely hydrolyze xylan to itsmonomers.The enzyme has found potential uses in a wide variety of applications of interestto the agrifood and pharmaceutical industries.In this thesis，Ferulic acid methyl ester as substrate, Recombinant Ferulic acidesterase constructed was studied. First, the optimum reaction time of the RecombinantFerulic acid esterase, the optimum temperature, the optimum substrate concentration andthe optimal pH values were determined. The results showed that the optimum reactiontemperature was40°C, the optimum reaction time of50min, the optimum amount ofsubstrate is0.2mM, the optimum PH value of6.6. Studied about the influence of Organicsolvents, metal ions and surfactants on the enzyme activity of ferulic acid esterase. It wasshowed that metal ions, Ca2+, Na+, Mg2+, Cu2+, Fe2+, Zn2+and K+,inhibited the enzymeactivity of ferulic acid. The strongest inhibition was Fe2+, the inhibition rate was more than90%. Sopropyl alcohol, high concentrations of glycerol, Twen80and acetone has a certainActivation to ferulic acid esterase.High concentrations of glycerol was the highest Activator,up to about3.3times.Secondly, in compound enzyme hydrolysis reaction, FAE acted collaboratively withcellulose enzyme, showing good synergy. We had a response surface analysis with variablesof the FAE and cellulose enzyme addition amount, reaction temperature and reaction pHvalue. And then, we obtained that the optimal addition amount of cellulose enzyme was3uL，that the best FAE addition amount was400uL, and that the optimum temperature andpH was45℃and5.0respect. Through research of addition order, we discovered thathydrolysis efficiency increased by30%when FAE was added earlier than cellulose enzyme. The results of study on FAE synergistic action in hydrolysis reaction showed that FAEimproved the efficiency of the hydrolysis by8%.Finally, we established a screening method of FAE, and obtained a protein composed of152amino acids. And then we confirmed it was a kind of ferulic acid esters, molecularweight beyond16.72KD and its gene homology is higher than30%with other FAEs.Through the protein electrophoresis detection, we got its strip in the correspondingposition of, further proofing the above results. And this part of the work will continue after.