Targeted Exploration And Characterization Of Cellobiose 2-epimerase

Epilactose and lactulose are non-natural oligosaccharides with many excellent physiological functions.They cannot be directly absorbed by the human body,so they will not raise blood sugar,which is consistent with the concept of modern healthy diet.Therefore,production of lactulose and epilactose shows a promising market prospect.Lactose is a natural carbohydrate that exists in the milk of most mammals.As a by-product of the cheese and casein manufacturing,lactose is mass-produced annually,which caused a significant environmental problem.Cellobiose 2-epimerase?CEase?can convert lactose to epilactose and lactulose,which can stably perform its catalytic function in complex milk systems.Molecular dynamics?MD?simulation technique was used to analyze the structural flexibility of CEase from new microbial sources in Uniprot database,and the root mean square deviation?RMSD?was obtained to predict its thermostability.It can be concluded from previous reports that CEase with higher thermostability may show better isomerization activity.The CEase with low thermostability can hardly catalyze isomerization,but they can maintain high epimerization activity at low temperature environment.The plasmid containing the genes of the CEase from Dictyoglomus thermophilum H-6-12?Dith-CE?and Treponema brennaborense DSM 12168?Trbr-CE?were transformed into Escherichia coli BL21?DE3??E.coli?for expression respectively.The maximum volumetric activity of the fermented broth of the recombinant E.coli with Dith-CE and Trbr-CE were11.9±0.8 U mL^-11 and 13.7±0.6 U mL^-1,respectively.The purified enzyme was obtained by fermentation and purification.Dith-CE shows a high thermostability which is consistant with the result from MD simulation.The optimum temperatures for epimerization and isomerization were 75?and 80?,respectively.The enzyme retained 75%of the residual enzyme activity after 12-h incubation at 75?.The optimum pH for the enzymatic reaction was 7.0,the measured specific activities of Dith-CE converting lactose to lactulose and epilactose were3.52±0.23 U mg^-1 and 160±6.5 U mg^-1,respectively.The Km and kcat values of Dith-CE for lactose epimerization were 523.2±9.98 mmol L^-1 and 441.4±15.4 s^-1,respectively.The Km and kcat values of Dith-CE for lactose isomerization were 235.2±11.2 mmol L^-1 and 3.98±0.3 s^-1,respectively.The enzymatic properties of Trbr-CE functioned optimally at 45°C and pH 7.0,The measured specific activities of Trbr-CE converting lactose to epilactose was 208.5±5.3 U mg^-1.The Km and kcat values of Trbr-CE for lactose epimerization were 57.8±1.94 mmol L^-1 and135.1±5.5 s^-1,respectively.Bacillus subtilis is a food safety grade industrial production microorganism.The plasmid pUB110 containing the genes of the Dith-CE and D-alanine racemase was transformed into Bacillus subtilis 1A751?dal^-?for expression.After 24-h fementation,the enzyme activity of the recombinant strain B.subtilis was more than 6 U mL^-1.Nutritional deficiency is used instead of antibiotics for screening,which avoids the harm to the human body and environment problems caused by the use of antibiotics.