Puirifcation And Immobilization Of Cellobiose2-epimerase From Caldicellulosiruptor Saccharolyticus DSM8903

Lactulose has been considered as a prebiotic disaccharide with health promotingfunctional properties and pharmaceutical values. The commercial production of lactulose waschemical catalysis with a lot of disadvantages, such as complex side products, difficulties topurify lactulose and high-energy comsumption. Therefore, it has become a focus of currentresearch efforts to produce lactulose in a convenient and lowcost way, and a biocatalysismethod with the advantages of environmental-friendly production and easy purification hasbeen recently proposed. The encoding gene of cellobiose2-epimerase (CE, EC5.1.3.11) fromCaldicellulosiruptor saccharolyticus DSM8903was amplified by PCR and inserted into theplasmid pET-28a (+), and then transformed into the host E. coli BL21(DE3). To facilitate thelactulose production in biocatalysis methods, the subsequent studies of enzyme purification,the immobilization of CsCE on spore surface and magnetic particles were carried out, andthen the production of lactulose utilizing immobilized enzyme as biocatalyst was alsoinvestigated. The experimental results obtained were briefly described as follows:The recombinant enzyme was actively induced by isopropy-β-D-thiogalactoside (IPTG).In order to obtain high yield of Caldicellulosiruptor saccharolyticus cellobiose2-epimerase(CsCE), the optimum conditions for its production from the recombinant E. coli BL21(DE3)were carefully determined. Under the optimized induction conditions (time15h, IPTG0.5mmol/L, temperature28oC and OD600nm0.8), the highest recombinant CsCE yield wasobtained and the enzyme activity reached3.40U/ml.The enzyme was separated and purified to electrophoresis homogeneity utilized acombination of heat treatment and two chromatographic stages: Q ion-exchange and G-75gelfiltration. The apparent molecular weight estimated to be about47kDa by SDS-PAGE, whichwas consistent with molecular mass calculated from the amino acid sequence. The specificactivity of29.7U/mg of CsCE was28.3-fold from the enzymatic extracts. The enzymeexhibited maximal activity at80°C and pH7.5. Kinetic study indicated that the apparent Kmvalue for CsCE was84.6mM, when lactose was applied as substrate.Non-recombinant Bacillus subtilis spore was investigated as a novel support toimmobilize CsCE, as its hydrophobic and negatively charged surface layer could provide anappropriate platform for enzyme adsorption. The process of immobilization was optimized byinvestigating the pH values of reaction mixture, initial enzyme concentration andimmobilization temperature and time. The properties of immobilized CsCE and theinteractions between enzyme and spores were also investigated. It was observed that at theoptimal pH4.5, CsCE with a deduced isoelectric point of5.48has a net positive charge andcould be adsorbed onto the negatively charged surface of B. subtilis spore. Under the optimized conditions, the maximum adsorbed amount of CsCE was1.47mg/1011spores andthe measured activity was34.6U/1011spores, indicating the enzyme activity recovery was79.4%. The spore-immobilized CsCE presented a higher pH and thermal stability than freeenzyme. Total desorption of immobilized enzyme was only achieved under extremeconditions, indicating strong adsorption between CsCE and B. subtilis spores. Moreover，efficient binding between spore and enzyme may require a potent combination ofhydrophobic and electrostatic forces. B. subtilis spores could be regenerated and thespore-immobilized enzyme showed good reusability as it approximately retained70%of itsinitial activity after8recycles. The maximum production of lactulose by immobilized CsCEwas395g/l over the course of a4-h reaction. In consideration of the progresses above,Bacillus spore was proved to be a potential food-grade support for enzyme immobilization.Magnetic nonaparticles with superparamagnetism were prepared using the traditionalchemical co-precipitation. Then glycidyl methacrylate (GMA) and2-hydroxyethylmethacrylate (HEMA) were utilized as copolymering monomers on the basis of their reactivefunctional groups with potassium persulfate (KPS) presenting as initiator. The resultingFe3O4/P (GMA/HEMA) particles displayed a high immobilization capacity for CsCE and themaximum amount of the enzyme immobilized reached63.3mg/g with enzyme activityrecovery of65.1%. Immobilization did not significantly alter the biochemical properties ofCsCE. The maximum production of lactulose by immobilized CsCE was395g/l over thecourse of a4-h reaction, under the condition of700g/l lactose, pH7.5and80oC.