Multi-enzyme Co-immobilization For One-pot Synthesis Of Fructose 6-phosphate From Maltodextrin

In carbohydrate metabolism,fructose 6-phosphate(F6P)is an important intermediate in glycolysis,gluconeogenesis,pentose phosphate pathway and calvin cycle.It is also an important precursor for the synthesis of D-tagatose.D-tagatose is a natural rare hexose,which is used as a functional sweetener owing to its hypoglycemic and anticaries properties and favorable flavor.Currently,D-tagatose is produced predominantly from D-galactose and catalyzed by L-arabinose isomerase.However,it is not feasible for the large-scale production of D-tagatose because of high cost of the substrate D-galactose.A cost-effective synthetic pathway for D-tagatose production suggests the use of inexpensive maltodextrin.First,the maltodextrin is converted into F6P by enzymatic catalysis,and then,F6P is catalyzed by tagatose 1,6-bisphosphate aldolase(TBPA)to generate tagatose 6-phosphate.Finally,D-tagatose is synthesized by dephosphorylation of tagatose 6-phosphate.Therefore,there is an urgent need for a low cost and efficient enzymatic synthesis of F6P.To date,the synthesis of F6P is a complex multi-enzyme cascade reaction.However,due to the poor stability and reusability of free enzymes in this cascade reaction,F6P has not been effectively biotransformed.In this study,a multi-enzyme co-immobilization catalytic system was constructed to improve the stability of enzymes and convert the substrate maltodextrin into F6P in one-pot effectively.Methods and experiment results were as follows:(1)?-Glucan phosphorylase(?GP)and phosphoglucomutase(PGM)were simultaneous co-immobilizing on Duolite A568 exchange resin to produce glucose 6-phosphate(G6P).Under the optimum co-immobilization conditions,the highest enzyme loading was 12.38 mg/g resin,and the recovery rate of enzyme activity was 46.13%.Co-immobilized?GP&PGM retained63.4%of the initial activity after incubation at 75°C for 36 h,whereas free enzymes retained only 12%.(2)The enzyme loading and recovery rate of enzyme activity were relatively low when co-immobilized enzymes by the adsorption method,so the metal-organic framework was prepared and employed to co-immobilize?GP and PGM through nano-embedding method.When the ratio of Zn²⁺and 2-methylimidazole was 1:4,the enzyme loading was 18.69 mg/g ZIF-8.The simultaneous immobilization of?GP and PGM exhibited better storage stability than free enzymes.81.29%of the initial activity of?GP&PGM@ZIF-8 was maintained after storing at 4°C for 25 days.In contrast,the relative activity of free enzymes was dropped significantly below 50%.Furthermore,the conversion rates of the dual-enzyme co-immobilization and single-enzyme immobilization catalytic systems were 45.6%and 24.8%,respectively.(3)ZIF-8 was difficult to recover in the co-immobilization system,resulting in a waste of co-immobilized enzymes,so the resin Duolite A568 was again employed as a carrier to co-immobilize?GP,PGM and glucose 6-phosphate isomerase(PGI).To improve the enzyme loading and stability of enzymes under acidic condition,?-poly-lysine(EPL)was used to modify the free enzyme(?GP&PGM&PGI).Our results showed that the optimal temperature of co-immobilized enzyme was 70°C,which was 10°C higher than free enzyme.Furthermore,after conversion for 24 h,the final concentration of F6P produced by free enzymes and co-immobilized enzymes were 0.088 g/L and 0.257 g/L,respectively.These results suggested that co-immobilized?GP&PGM&PGI could act as a promising catalyst for one-pot production of F6P and this one-pot system of tri-enzymes co-immobilization is an effective way for bioconversion by bringing multi-enzyme together.Therefore,this study lays a theoretical foundation for the subsequent preparation of D-tagatose and provides ideas for the co-immobilization of multiple enzymes.