Construction Of Multienzyme Hybrid Nanoflower Cascade Reaction System And Its Application In Catalytic Degradation Of Starch And Cellulose

Starch and cellulose are important components of biomass.These two materials have been widely used in energy,food,medicine and other industries.However,how to broaden the application of starch and improve the utilization rate of cellulose still faces many challenges.Therefore,it is of great significance to develop new methods to meet these challenges and make better use of starch and cellulose.As a kind of biological catalyst,enzyme has the characteristics of high catalytic efficiency,substrate specificity and mild reaction conditions.Multienzyme cascade reaction can realize one-step rapid conversion of reaction substrate to target product without separating intermediate products.However,due to the difficulty of preparing immobilized materials and the loss of enzyme activity during the immobilization process,the immobilized multienzyme cascade reaction still faces many problems.Enzyme hybrid nanoflowers have the advantages of high specific surface area,stable structure and simple preparation process,and are widely used in the enzyme immobilization field.Based on this,multienzyme hybrid nanoflower cascade reaction system was prepared in this study and applied to the catalytic conversion of starch and cellulose.The main research contents are as follows:?1?In this study,a novel multienzyme hybrid nanoflowers cascade reaction system?GA@GOx hybrid nanoflower?was constructed by compartmentalization method for the conversion of starch into gluconic acid in one pot.In the preparation process,the glucose oxidase?GOx?hybrid nanoflowers were first constructed by self-assembly method,and then,amylase?GA?was adsorbed on the surface of GOx hybrid nanoflowers by the coordination between GA and Cu²⁺.A series of immobilization parameters were studied to obtain the best catalytic activity of GA@GOx hybrid nanoflowers,including GA and GOx concentration,immobilization time and Cu²⁺concentration.The GA@GOx hybrid nanoflowers achieved the best catalytic activity when the GA and GOx concentrations were both 0.25 mg mL^-1,the immobilization time was 24 h,and the Cu²⁺concentration was 9 mM.Subsequently,the morphology,chemical composition and crystal structure of GA@GOx hybrid nanoflowers were studied in details.According to the kinetic parameters(K_m,V_max),the GA@GOx hybrid nanoflowers with specific spatial distribution of GA and GOx exhibited excellent substrate channeling and catalytic efficiency.The catalytic efficiency of GA@GOx hybrid nanoflowers was 1.5 times than that of the free multienzyme system.After 80 minutes,GA@GOx hybrid nanoflowers converted92.12%starch into gluconic acid in one pot.?2?The study first optimized the expression conditions of recombinant endoglucanase?EGLEH?and recombinant exoglucanase?CBHLEH?,and then,the ELP tag was utilized to separate and purify these two recombinant enzyme.Under the optimum conditions,the activity recovery and purification fold of EGLETH were81.56%and 18.16,while the enzyme activity recovery and purification fold of CBHLEH were 35.98%and 9.77,respectively.This study first coimmobilized EGLETH,CBHLEH and?-glucosidase?GLEH?by random co-immobilization method to form a novel multienzyme hybrid nanoflower cascade reaction system?EGLEH-CBHLEH-GLEH-NF?for the conversion of cellulose into glucose in one pot.The immobilization parameters were studied,including Cu²⁺concentration,immobilization time,immobilization temperature and total enzyme concentration.According to the reusability result,EGLEH-CBHLEH-GLEH-NF exhibited excellent performance for it possessed 61.59%of its original activity after eight cycles.Furthermore,comparedtothefreemultienzymesystem,EGLEH-CBHLEH-GLEH-NF also exhibited outstanding pH stability,thermal stability,storage stability,and catalytic efficiency.The kinetic parameters(K_m and V_max)of EGLEH-CBHLEH-GLEH-NF also indicated that the proximate enzyme in the multienzyme hybrid nanoflowers have better substrate affinity than that of free multienzyme system.