Dual Enzyme Immobilization And Its Catalytic Production Of Ferulic Acid In Deep Eutectic Solvents

Ferulic acid is a natural antioxidant with antioxidant and antithrombotic effects.It is widely used in food and medicine.Now,a large amount of waste water has been produced in the production process of ferulic acid leaching,which usually caused serious environmental pollution.Enzymatic hydrolysis method has the advantages of green environmental protection and less by-products,so it has attracted extensive attention of researchers.The combination of feruloyl esterase and xylanase can efficiently degrade DE starch wheat bran to produce ferulic acid,but the free enzyme is easy to inactivate and can not be recycled,which limits its industrial application.To solve these problems,this subject adopts the means of immobilized enzyme combined with solvent engineering(deep eutectic solvent instead of traditional buffer)to study the influence on enzymatic reaction.The main research contents and results are as follows:(1)Firstly,the effects of immobilized feruloyl esterase and xylanase and the combination of the two enzymes on their catalytic performance were studied.Feruloyl esterase and xylanase were immobilized on reversible soluble carrier Eudragit L-100,respectively.The conditions of immobilized enzyme were optimized.Under the optimal immobilization conditions,the immobilized enzyme showed better thermal stability and p H stability than the free enzyme.After incubating at 40℃ for 1 h,the immobilized ferulate esterase still had 50% activity remained and the immobilized xylanase still had about 90% remained.After incubation at p H 8.0 for 1 h,the activity of immobilized feruloyl esterase remained more than 50%,and that of immobilized xylanase was about50%.When the ratio of feruloyl esterase and xylanase was 1:5,the best compound ratio was reached.It was used to degrade DE starch wheat bran to produce ferulic acid,and showed good catalytic performance(after the free enzyme was compounded,the output of 6.6 U enzyme was about 6 μg ferulic acid,after the combination of immobilized enzyme,the output of 1.1 U enzyme is about 1 μg ferulic acid).(2)The effect of co-immobilization of feruloyl esterase and xylanase on their catalytic performance was investigated.Eudragit L-100 was used to co-immobilize feruloyl esterase and xylanase.The optimum reaction p H of feruloyl esterase was changed from 7.0 to 8.0,and the p H stability and thermal stability of feruloyl esterase were greatly improved.After incubation at 60 ℃ for 1 h,the co-immobilized feruloyl esterase still had 45% activity,the co-immobilized xylanase had more than 80% activity,After 1 h incubation at p H 8.0,co-immobilized feruloyl esterase still had more than90% activity and co-immobilized xylanase had more than 80% activity.The co-immobilised enzyme was used to break down wheat bran to produce ferulic acid,yielding 2.1 μg of ferulic acid at 0.66 U of enzyme addition and a reaction time of 12 h.The results showed that the co-immobilisation increased the efficiency of the dual enzyme synergistic ferulic acid production.The reusability of the co-immobilized enzyme was investigated and the activity of the co-immobilized enzyme was maintained at more than 60% after 5 cycles.(3)To optimise the effect of enzymatic reactions,the effect of deep eutectic solvents on the catalytic performance of the enzymes was investigated.The ability of the dual enzymes to decompose wheat bran to produce ferulic acid in five choline chloride-based deep eutectic solvents(DESs)was investigated.The results showed that the maximum rate of enzymatic reaction was achieved at 66.7% water content of choline-glycerol chloride,and under the same conditions,the enzymatic reaction with water as the medium was able to produce about 6.0 μg of ferulic acid,while in the chloride-glycerol(66.7% water content),it was able to produce about 35 μg of ferulic acid.The p H stability and thermal stability of feruloyl esterase in the deep eutectic solvent were improved,with more than 50% activity remaining after 1 h incubation at30°C and more than 75% activity remaining after 1 h incubation at p H 7.0.The results indicate that a suitable hydrogen bonding interaction between feruloyl esterase and the deep eutectic solvent molecules was formed,allowing the structural flexibility and rigidity of FAE to be balanced and maintained,which in turn had an effect on the stability of FAE.In conclusion,the method of immobilized enzymes combined with solvent engineering has greatly improved the activity and stability of the enzymes,and has enabled the reusability of the biological enzymes,resulting in significant savings in production costs.This study provides a theoretical basis for the development of a green and safe enzymatic reaction system.