| Lactulose is a typical prebiotic disaccharide. Owing to its perceived health benefits,lactulose has been widely used in the fields of medical, food and animal feed. Commerciallyavailable lactulose is produced today mainly through the chemical isomerization of lactoseunder alkaline media. However, requirement of removal of catalysts and byproducts, andenergy requirement are the major drawbacks of such chemical process. In the last decade,enzymatic bioconversion of lactose into lactulose has been extensively studied to overcomethe above-mentioned problems. A study from our laboratory demonstrated that lactulose canbe successfully synthesized using immobilized β-galactosidase and immobilized glucoseisomerase dual-enzymatic system in organic-aqueous two-phase media. Under optimalconditions, the maximum lactulose concentration was approximately151g/L with aconversion yield of18.8%. Spore surface display technology, likely oriented immobilizationtechnology, enables the presentation of antigens or proteins of biological interest on thesurface of B. subtilis spores and has been successfully used for the production of vaccine andantibody. However, few studies have investigated the use of spore surface display system forthe development of whole-cell biocatalysts. In the case of immobilized lactulose-producingenzymes, very ofen, enzyme production was required and therefore the cost of the processwas increased. In considering both the disadvantages of the immobilized lactulose-producingenzymes and the excellent features of spore-surface display system, the primary aim of thepresent study was to display lactulose-producing enzymes on the spore surface. Theutilization of spore surface display system not only simplifies the procedure and also willfacilitate the lactulose production. Accordingly, an investigation of the display oflactulose-producing enzymes on the spore surface was carried out and the obtainedrecombinant spores were employed as whole cell biocatalyst for the lactulose production. Abriefly description of the obtained experimental results is given in the following sections.Firstly, three crust proteins CotX, CotY and CotZ were used as potential fusion partnersto target enhanced green fluorescence protein, encoded by the gene egfp, on the B. subtilisspores. Based on the results of the flourescence intensity experiment, the recombinant sporesof three engineered strains showed increased flouresence intensities than that of the wild typeB. subtilis168spores. To further confirm the possibility of CotX, CotY and CotZ acting asfusion partners, a tetrameric β-galactosidase was used as the second model protein. TheCotX-fused spore-displayed β-galactosidase exhibited an enzyme activity of0.39U/mg spores(dry weight), while the enzyme activities of CotY and CotZ-based recombinant protein were0.12and0.25U/mg spores. Data from the spore-displayed β-galactosidase assay showed thatthe display efficiency of β-galactosidase on the spore was in the order CotX>CotZ>CotY.An investigation into the utilization of CotX as a carrier for the display ofβ-galactosidase at the pore surface was performed. A combination of western blot,immunofluoresence microscope and flow cytometry was used to demonstrate that theCotX-linked fusion protein was successfully expressed on the B. subtilis spores. Further, theresulting spore-displayed β-galactosidase was able to hydrolyze ONPG, indicating that β-galactosidase was actively expressed on the spores. Next, the biochemical properties ofspore-displayed β-galactosidase were characterized in details. The optimum pH ofspore-displayed β-galactosidase was found to be6.0, decreased by a0.5pH unit as comparedto the recombinant enzyme. The spore-displayed β-galactosidase displayed its highest activityat75℃, a slightly higher than the purified recombinant enzyme (70℃). In additoin, the effectof the integrated and non-integrated expression models on the display efficiency ofβ-galactosidase on the spores was studied. It was found that the activities of spore-displayedβ-galactosidases obtained from the two different expression ways were determined to be0.42and1.34U/mg spores, respectively, suggesting that the use of non-integrated model is a betterchoice for the expression of β-galactosidase on the spores.To widen the application fields of spore-surface display systems, another CotX-basedspore display system that allows the expression of a thermostable glucose isomerase under thecontrol of the native CotX promoter PcotXwas constructed. Western blot was used to detect thepresence of the CotX-linked glucose isomerase fusion protein. Flow cyrometry showed that asignificant increase in flourescence intensity was observed for the recombinat spores ascompared to the wild type B. subtilis168spores. However, the recombinant spores exhibitedundetectalbe activity. These results evidenced that the CotX-based glucose isomerase fusionprotein was displayed, but not actively expressed on the spores. Further study is needed toreveal how the fusion protein folds. Besides this, the recombinant B. subtilis sporesexpressing β-galactosidase were used as a novel matrix for the immobilization of glucoseisomerase. The effcets of the pH value of buffer, the concentration of spore suspension andthe amount of the added enzyme on the adsorption efficiency of recombinant glucoseisomerase were optimized. Under optimal conditions, i.e., pH4.0sodium phosphate-citratebuffer,1×1012/mL spores concentration and2.0mg added enzyme amount, the activities ofspore-displayed β-galactosidase and adsorpted glucose isomerase was14.2and41.2U/mL.The obtained recombinant spores co-displaying β-galactosidase and glucose isomerase werefurther employed as a bi-enzyme system to enzymatically convert lactose into lactulose.Under optimal condition, i.e.,300g/L initial lactose concentration,65℃and pH7.5, thelactulose yield reached37.5g/L with a lactose conversion rate of12.5%.Finally, the CotX was used as a carrier to construct a spore-surface display system inwhich a new type lactulose-producing enzyme cellobiose2-epimerase was expressed on thespores. The resulting spore-displayed cellobiose2-epimerase was employed as a whole cellbiocatalyst to produce lactulose from lactose. Under optimal condition, i.e.,700g/L initiallactose concentration,80℃and pH8.0, the maximum lactulose yield was247.2g/L with alactose conversion rate of35.3%. The lactulose yield obtained in this study was much higherthan those of previous resulsts obtained from the dual-enzyme system in which consisted ofimmobilized β-galactosidase and immobilized glucose isomerase, and obtained from therecombinant spores co-diplaying β-galactosidase and glucose isomerase. |
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