Multi-enzymatic Pathway In Vitro For Sorbitol Production From Starch:Construction And Optimization

Sorbitol is an important industrial raw material,which is widely used in,chemical,food and medicinal industries.The production of sorbitol by synthetic biology and optimized multi enzyme reaction system is a green preparation method,which can not only prevent the environmental pollution of the current preparation method,but also ensure the product purity.In this study,five continuous enzymatic reactions and NADH regeneration in vitro multi enzyme catalytic systems were constructed to convert maltodextrin（MD）into sorbitol.In particular,sorbitol 6-phosphate phosphatase with high specific catalytic performance for sorbitol 6-phosphate was evaluated and screened based on Rosetta ligand’s covalent docking method.The six enzymes in the multi enzyme catalytic pathway for sorbitol synthesis are α-Glucan phosphorylase（alpha glucan phosphorylase,αGP）,glucose phosphate mutase（PGM）,glucose 6-phosphate isomerase（PGI）,sorbitol 6-phosphate dehydrogenase（S6PDH）,sorbitol 6-phosphate phosphatase（s6pp）and formate dehydrogenase（FDH）.The expression of six enzymes in E.coli BL21（DE3）host was optimized and purified by nickel column to obtain pure enzymes and determine their catalytic activities.Through the preparation and optimization of the system,MD can be transformed into sorbitol through multi-stage in vitro multi enzyme system through one pot reaction.After optimization,the yield of 28 mmol/L maltodextrin to sorbitol was 27.6 mmol/L and the yield was 98.6%.Finally,in order to obtain a higher yield of sorbitol,the maltodextrin concentration was increased to 280 mmol/L.As a result,the yield of sorbitol was only 65.1mmol/L at 28 h.The results show that there is serious product inhibition in the transformation system,which is that sorbitol has a strong inhibitory effect on S6 PP and affects the production of sorbitol.In view of the inhibition of this product,protein engineering needs to be used to modify the enzyme and remove the inhibition in the future,so as to further improve the yield of sorbitol.In vitro multi enzyme catalytic system platform provides a biological refining technology for sorbitol production,which has predictable safety,environmental friendliness and sustainability,and represents a very potential alternative method for sorbitol production.