Application Of Acid Phosphatase From Shigella Flexneri In The Synthesis Of Rare Hexuloses

Rare hexuloses have physiological functions such as anti-tumor,anti-inflammatory,antihyperglycemic and immunosuppressive effects,and have broad application prospects in the food and pharmaceutical industries.Therefore,it is of great significance to establish an efficient and inexpensive synthetic system of rare hexuloses.In this study,the acid phosphatase from Shigella flexneri(Pho N-Sf)was applied to the synthesis system of rare hexuloses to provide a theoretical basis for the industrial production of rare hexuloses.The aim of this study was to establish a method for the synthesis of a series of rare hexuloses with glycerol as the only carbon source by multi-enzyme cascade reaction.Dihydroxyacetone phosphate(DHAP)-dependent aldolases catalyze the synthesis of hexulose-1-phosphate using DHAP as the donor and D-/L-glyceraldehyde(D-/L-GA)as the acceptor.First,to avoid directly using expensive and unstable DHAP,acid phosphatase Pho N-Sf and glycerol phosphate oxidase(GPO)were introduced into the cascade reaction system to synthesize DHAP and three DHAP-dependent aldolases: L-rhamnulose-1-phosphate aldolase(Rha D),L-fuculose-1-phosphate aldolase(Fuc A)and D-fructose-1,6-biophosphate aldolase(Fru A)were used to catalyze the condensation reaction between DHAP and added D-/L-GA to synthesize the hexuloses-1-phosphate.Meanwhile,hexulose-1-phosphate was dephosphorylated by Pho N-Sf to obtain the rare hexulose.Secondly,in order to solve the issue of additional GA,alditol oxidase(Ald O)and horse liver alcohol dehydrogenase(HLADH)were introduced into the cascade reaction to obtain D-GA and L-GA using glycerol as the substrate.As a result,a multienzyme cascade system was constructed for rare hexuloses synthesis with glycerol as the sole carbon source.Next,in order to avoid the complicated purification processes of enzymes,the enzymes were overexpressed in E.coli BL21(DE3)to establish a whole-cell catalytic system.Finally,enzymes were overexpressed in Clear Coli? BL21 to establish a safer whole-cell catalytic platform.The main research results of this study are as follows:(1)One-Pot Multienzyme system for rare hexuloses synthesis using glycerol: based on the property of Ald O to specifically oxidize glycerol to D-GA,Pho N-Sf,GPO,and Ald O were coupled with three aldolases to synthesize D-type rare hexuloses using glycerol as a substrate.The total concentration of D-allulose and D-sorbose was 14.18 g×L-1 using aldolase Rha D;The total concentration of D-allulose and D-sorbose produced was 10.10 g×L-1 using aldolase Fuc A.Based on the property of HLADH to specifically oxidize glycerol to L-GA,Pho N-Sf,GPO,HLADH,and NADH oxidase(NOX)were coupled with three aldolases to synthesize L-type rare hexuloses with glycerol as a substrate.The concentration of L-fructose was 10.70 g×L-1 using aldolase Rha D;The total concentration of L-tagatose and L-fructose was 8.12 g×L-1 using aldolase Fuc A;The concentration of L-sorbose was 9.64 g×L-1 using aldolase Fru A.(2)Whole cell multi-enzyme cascade reaction for rare hexuloses synthesis using glycerol: Pho N-Sf,Ald O,GPO and aldolase were overexpressed in BL21(DE3)and the total concentration of D-allulose and D-sorbose was 9.3 g×L-1 using aldolase Rha D;The total concentration of D-allulose and D-sorbose produced was 4.2 g×L-1 using aldolase Fuc A.To establish a safe and efficient biosynthetic platform,Pho N-Sf,GPO,Rha D and Ald O were overexpressed in Clear Coli? BL21 and the concentration of D-allulose and D-sorbose was 5.5 g×L-1 under optimized condition.