Cloning, Expression, And Characterization Of Cellobiose 2-epimerases

Epilactose and lactulose are non-digestible disaccharides with good prebiotic properties. They are both valuable lactose derivatives and have promising physiological effects. Recently, a great deal of attention has been focused on their biological productions. Cellobiose 2-epimerase catalyzes the reversible epimerization between disaccharides including mannobiose, cellobiose lactose and epilactose. Some thermostable cellobiose 2-epimerases catalyze not only epimerization but also isomerization, through which epilactose and lactulose can be produced by lactose effectively. Thus, cellobiose 2-epimerase became a potential biocatalyst for industrial productions of epilactose and lactulose from the cheap material lactose with no co-substrates.In this study, two recombinant cellobiose 2-epimerases were cloned, expressed, purified characterized from two thermophilic bacteriums, the time courses of epilactose and lactulose large-scale productions by this two recombinant enzymes were studied.The genes encoding cellobiose 2-epimerases from Thermoanaerobacterium saccharolyticum JW/SL-YS485（protein ID: AFK87331.1） and Caldicellulosiruptor obsidiansis OB47（ADL43428.1） consisted of 1,176 bp and 1,170 bp nucleotides encode 392 and 390 residues. The genes were introduced into the p ET-22b（+） plasmids with the same restriction sites and transformed into E. coli BL21（DE3） for expression. The crude enzymes were purified by one-step nickel-affinity chromatography and affinity purifications were performed. The purified cellobiose 2-epimerases exhibited strong protein bands of approximately 47,000 and 43,500 on SDS-PAGE, which were similar to their calculated molecular masses. Cellobiose 2-epimerase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 showed maximal activity at p H 7.0 and 65°C. Cellobiose 2-epimerase from Caldicellulosiruptor obsidiansis OB47 had different optimum temperature for epimerization and isomerization reaction（70 and 75°C, respectively）, and its optimum p H was 7.5. This two enzymes exhibited considerable thermostability under incubation at less than 65°C and 75°C, respectively. Their Km, turnover numbers（kcat）, and catalytic efficiencies（kcat/Km） for lactose were 160.6 and 276.0 m M, 139.4 and 218.3 s^-1, 0.868 and 0.791 m M^-1 s^-1, respectively.At p H 7.0 and 60°C, 26% epilactose was produced from 200 g/L lactose by a 0.05 mg/m L cellobiose 2-epimerase from T. saccharolyticum after reaction for 4 h. Cellobiose 2-epimerase from C. obsidiansis exhibited activity of both epimerization reaction and isomerization reaction towards disaccharides, and produced 22 g/L epilactose and 108 g/L lactulsoe from 200 g/L lactose at 75°C after 4 h with a concentration of 1 mg/m L.