Study On The Production Of D-allulose By High-efficiency Secretion And Expression Of D-allulose 3-epimerase In Bacillus Subtilis

Rare sugar is a kind of monosaccharide and its derivatives with very little content in nature.It has been widely used in the field of food and pharmacy because of its unique physiological and biochemical characteristics.Among them,the sweetness of D-allulose is70%of sucrose,and the energy is almost zero.Moreover,it is safe and non-toxic.In addition,its unique physiological function also makes it have great potential in the fields of sweeteners and health products.However,there are few rare sugars in nature and it is difficult to extract them.Chemical synthesis has many adverse factors.Therefore,the synthesis of rare sugars by biotransformation has attracted extensive attention.According to the izumoring strategy,D-allulose 3-epimerase（DAE）catalyzes the formation of D-allulose by using cheap and easily available D-fructose as substrate.Therefore,D-allulose3-epimerase is the key enzyme for the synthesis of D-allulose.Most DAEs have the disadvantages of thermal stability,poor catalytic activity and low expression,but Sf DAE from Sinorhizobium fredii CCBAU 83666 has the advantages of good stability,high activity and high expression.In this study,the fructose kinase gene gmu E in B.subtilis genome and the genes fru A and lev G of fructose specific transporter elements IIabc and IId in PTS system were knocked out,and the chassis strainΔWB600without fructose consumption was constructed.Later,expression of Sf DAE in theΔWB600strain was analyzed and the reaction conditions of whole cell catalysis were optimized.Then,by screening the highly expressed single promoter,combining and optimizing the promoter group strategy,and screening the appropriate signal peptide of Tat pathway,the extracellular expression system of Sf DAE was constructed,and the recombinant B.subtilisΔWb600/p MA5-P_{Hpa II}-P_{amy E}-SP_{pho D}-sfdae was obtained which increased the expression of Sf DAE and made its extracellular enzyme activity reach 63.69±0.78U/m L.Then the fermentation parameters such as carbon source type and concentration,phosphate concentration and microelement concentration were optimized in shaking flask.Through large-scale production in the fermenter,the extracellular enzyme activity of the recombinant strain reached 175.64±4.296U/m L,and the high concentration fructose was catalyzed by the fermentation supernatant,reaching a conversion rate of more than 28%,which provided a theoretical basis for the production and application of Sf DAE in industry.