Targeted Mining And Characterization Of A Hyperthermostable Ketose 3-epimerase

D-Allulose is a hexoketose,an epimer corresponding to the C-3 position of D-fructose.Owing to its low energy and high sweetness,it can be used as a new sweetener in the food industry.The ketose 3-epimerase（KEase）can act on the C-3 position of D-fructose to produce D-allulose.The reported KEases generally suffer from poor thermal stability and are not suitable for industrial application.In this study,a hyperthermostable KEase from Labedella endophytica was screened through molecular dynamics simulation and gene mining techniques.It was classified as an L-ribulose 3-epimerase（LREase）based on bioinformatics analysis and identification of enzymatic properties.L.endophytica LREase has heat and acid resistance characteristics with high catalytic activity,making it an ideal enzyme for the industrial production of D-allulose.This study focuses on the following aspects.（1）Targeted mining of hyperthermostable KEase.Using the new enzyme mining tool Enzyme Miner,a primary screening library of hyperthermostable KEases was established;the hyperthermostable KEases were reselected using computational assistance,molecular dynamics simulations were carried out,and the RMSD values and number of hydrogen bonds of KEases from different microbial sources were used as indicators for comparison and establish a reselected library of hyperthermostable KEases;the thermodynamic properties of the enzymes were characterised and the final selection was made.By characterizing the thermodynamic properties of the enzymes,L.endophytica LREase with thermal potential was selected.Sequence alignment and phylogenetic tree analysis indicated that L.endophytica KEase was a potential LREase.（2）Optimization of heterologous expression and enzyme production conditions of L.endophytica LREase.A recombinant plasmid p ET22b（+）-Laen was constructed and transferred into E.coli BL21（DE3）to overexpress L.endophytica LREase.By optimizing the medium components and culture conditions,the final recombinant E.coli was induced to express L.endophytica LREase at a culture temperature of 20℃for 24 h.The enzyme activity reached 54.42±0.86 U/m L,which was 5.46 times higher than that of unoptimized culture condition.（3）Study on the enzymatic properties of L.endophytica LREase.L.endophytica LREase was successfully purified by Ni²⁺affinity chromatography.The enzymatic properties showed that the optimum reaction temperature was 80℃,the optimum p H was 6.0,the optimum metal ion was Ni²⁺,and the optimum substrate was L-ribulose.When D-fructose was used as the reaction substrate,the specific enzyme activity of L.endophytica LREase was 110.7±0.3U/mg,and the kinetic parameters k_cat,K_m and k_cat/K_m values were 4077.8 min^-1,49.6 m M and82.3 m M^-1min^-1,respectively.L.endophytica LREase showing strong catalytic activity for D-fructose.The thermodynamic properties of the enzyme showed that under the incubation of Co²⁺,the t_1/2 values of L.endophytica LREase were 37.7,9.0,and 4.6 h at 60,65,and 70°C,and the T_mvalue was 80.91°C,exhibiting an extremely strong thermal resistance characteristic.（4）Storage and production of L.endophytica LREase The storage t_1/2 of L.endophytica LREase were 28 and 16 d at 4 and 25℃in the presence of the stabilizer Co²⁺.At 70℃,p H 6.0,with 500 g/L D-fructose as the substrate,L.endophytica LREase could produce a synthetic154.2 g/L D-allulose with an equilibrium conversion rate of 30.8%.