Immobilization Of Cellobiose 2-epimerase From Caldicellulosiruptor Saccharolyticus

Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase(Cs CE) catalyzes the single substrate lactose into lactulose and is the most efficient enzyme ever found for the enzymatic synthesis of lactulose. However, the utilization of Cs CE for industrial lactulose bioconversion which exhibits a tremendous advantage has not yet been fully studied. We endeavored to facilitate the enzymatic bioconversion of lactulose on an industrial scale by Cs CE in an immobilized form. In the following sections, we acquired the oppropriate immobilization parameters for permeabilized Cs CE cells, immobilized Cs CE cells, and immobilized Cs CE. The obtained three biocatalysts have been characterized and they used as the biocatalysts to produce lactulose have also been investigated.Firstly, ethanol was used as the effectiveness permeabilizing agent for Cs CE cells permeabilization after the high expression of Cs CE induced by lactose. The obtained permeabilized Cs CE cells were used as biocatalyst for maximum lactulose production. After incubated at Na2HPO4-Na H2PO4 buffer with 600 g/L lactose and 12.5 U/m L permeabilized Cs CE cells at 80 oC for 2 h, approximately 390.6 g/L lactulose was obtained with a conversion yield of 65.1%.Then, permeabilized Cs CE cells were immobilized via chitosan-tripolyphosphate(CS-TPP) flocculation and glutaraldehyde cross-linking. The retained activity of the immobilized Cs CE cells reached up to 90%. Compared to the free Cs CE, immobilized Cs CE cells have lower affinity to lactose but were significantly more active at all temperatures. Due to the excellent enhanced thermal and mechanical stability after immobilized, more than 90% of original activity was remained after 15 th cycle. The highest production of lactulose by immobilized Cs CE cells was 394.7 g/L with a conversion yield of 65.8% after being incubated at Na2HPO4–Na H2PO4 buffer for 2 h.Finally, thermal-purification of Cs CE was carried out. After incubated at 70 oC for 2 h, the activity recovery was 86.6% with a specific activity of 5.46 U/mg and a purifying fold of 6.14. Then Duolite A568 resin was used for the immobilization of thermal-purified Cs CE via simple adsorption and glutaraldehyde cross-linking. After immobilized, the optimal p H was shifted 0.5 units to a more weak acid value to 7.0 and the Michaelis constant Km was found to be increased 1.3-folds to 143.70±6.79 m M. The immobilized Cs CE exhibited excellent thermal and mechanical stability. The maximum production of lactulose by immobilized Cs CE was 309 g/L with a conversion yield of 51.6% under the condition of 600 g/L lactose, p H 7.0 and 70 oC for 2 h.