Cloning And Expression Of D-Tagatose 3-epimerases From Christensenella Minuta And Biosynthesis Of D-Allulose

The rapid changes in lifestyles of the people occurred with the global industrialization and economic development over the past decade.With the improvement of living standard,food availability has extended,which cause the significant negative effects in term of change in dietary pattern.As a result,it increases the diet-related chronic diseases such as diabetes,hypertension and hyperlipidemia caused by excessive intake of high-calorie and sugar diets.The traditional high-calorie sweeteners are gradually replaced by low-calorie functional sugar alcohols.As an important member of rare sugars,D-allulose,known as a calorie-free sweetener with70%sweeter and only 0.3%calorie of sucrose,also has hypoglycemic,hypolipidemic and neuroprotective effects.D-tagatose 3-epimerase family enzymes is the most effective biocatalyts for producing D-allulose.Although many different microbial sources of D-tagatose 3-epimerase family enzymes have been discovered,few of them can meet the industrial production conditions of acidic,thermally stable and high conversion rate.In addition,researches on the D-allulose biosynthesis method are incomplete and unsystematic.These make the failing of D-allulose's industrial production,resulting in high price,which limits its promotion and application in the market.In view of the above issues,discovery of novel D-tagatose 3-epimerase isomerase and development of an efficient process for D-allulose biosynthesis are expected to lay the foundation for the large-scale industrial production of D-allulose.The studies mainly include the following aspects:(1)A putative D-tagatose 3-epimerase isomerase of Christensenella minuta screened by bioinformatics techniques,and its Accession No.in NCBI is WP?066519968.1.The phylogenetic tree and multiple sequences comparison analysis revealed that the similarities between the putative protein and DTEase family enzymes are not high,ranging from 20%to 43%,but the amino acid residues of it are highly conserved with the active center,substrate binding site and metal ion binding site of DTEase family enzymes.Bioinformatics analysis showed that the protein was an unstable hydrophilic protein with a theoretical relative molecular weight of32,483.91Da.Three-dimensional modelling result showed that the putative protein monomer is a TIM barrel structure consisting of 13?-helices and 9?-folds.The codon optimized gene was synthesized to construct Escherichia coli Rosetta/p ANY1-Cm-dte.Then the enzymatic properties of purified recombinant DTE-CM were investigated.The results showed that this DTE-CM is strictly metal ion-dependent enzyme.The optimum metal ion is Ni²⁺,and the optimum concentration of Ni²⁺is 1 m M;The optimum p H 6.0 indicated this DTE-CM is an acidic enzyme;The optimum temperature of DTE-CM is 50?,and the stability is good at 50?,and low concentration of Ni²⁺can significantly improve its stability.The DTE-CM has a broad substrate spectrum with catalytic activities for D-tagatose,D-allulose,D-sorbose and D-fructose in decreasing turn,respectively.The K_mand K_cat/K_m for D-allulose are 53.8 m M and 124m M^-1min^-1,respectively.Finally,molecular docking of the enzyme was performed with four different substrate molecules,and the results indicated that the enzyme may be similar in function and catalytic mechanism with the DTEase family enzymes.(2)The induction expression conditions of the engineered bacteria were optimized,and the results showed that the optimal induction conditions were as follows:temperature 25°C,OD₆₀₀ 1.0,IPTG 1 m M,and 14 h.The enzymatic production of D-allulose was investigated.150.00 g/L of D-allulose was produced with 30.00%conversion ratio using 500 g/L D-fructose after 10 h.Using batch replenishment strategy,D-allulose production and conversion ratio reached to 185.00 g/L and 37.00%respectively after 4 time's replenishments.In addition,the conditions of whole-cell method for producing D-allulose were optimized.The conversion ratio was 18.5%under the optimal conditions(temperature 55°C,p H 6.5,Ni²⁺1 m M,whole-cell 50 g/L,D-fructose 300 g/L,and reaction time 30 h).Two whole cell batch replenishment strategies were explored,and the results showed that the reached towhen only D-fructose was added,the production and conversion ratio of D-allulose were 67.95 g/L and 22.65%respectively after 4 time's replenishments,and reached to 110.00 g/L and36.60%respectively after 6 time's replenishments of D-fructose and fresh whole cells.(3)In order to improve the whole cell recycling efficiency,D-allulose production by immobilized cells of sodium alginate glutaraldehyde(embedding-crosslinking method)was optimized.The optimal conditions were as follows:sodium alginate 2%,Ca Cl₂ 6%,cell 60 g/L,reactiontime 6 h,glutaraldehyde 0.02%,and crosslinking time4 h.Under the optimal conditions,the immobilized cells had good stability,and the relative enzyme activity remained 92%after 5 times'recycling.Furthermore,the production of D-allulose using immobilized cells with cheap substrate of fructose syrup was investigated.The result show that 42.86 g/L of D-allulose was produced after 48 h.