Preparation and characterization of hybrid nanoflowers of cobalt phosphate and mutant cellobiose 2-epimerase

Abstract

Lactulose, a prebiotic, can be used as a functional food additive and a clinical drug for the treatment of hepatic encephalopathy, chronic constipation and other diseases. It has been widely used in food and medicine industry. Compared with chemical method of production, producing by enzymatic approach is not only energy-saving and eco-friendly, but also can reduce the generation of by-products. Therefore, this is a new trend of lactulose production. Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) is currently the most efficient enzyme for producing lactulose. E161D/N365P, a site-directed mutagenesis strain of CsCE, has better thermostability. However, the low reusability of free enzyme hinders its industrial application. This study was committed to the immobilization technology of E161D/N365P. EDNP@Co3(PO4)2 was prepared by organic-inorganic hybrid immobilization technology. Under the optimal synthesized condition, the activity recovery rate could reach 62.88%, and the encapsulation yield was 57.22%. Adding 0.8 mM Co2+ to the remaining solution which still contained residual enzyme after the first precipitation could further increase the total encapsulation yield to 65.82%, and at the same time, the nanoflower was structurally complete. Compared with the free enzyme, the thermostability of EDNP@Co3(PO4)2 was slightly improved below 60℃, and the optimum pH shifted to the acidic region, which could be beneficial to the clean production of lactulose. The nanoflower structure made EDNP@Co3(PO4)2 better at kinetic parameters. Its catalytic efficiency η was increased by 1.48 times in comparison with free enzyme, and after 8 cycles of reactions, the enzyme activity still remained over 70%. When producing lactulose with lactose as the substrate, the conversion rate of lactulose could reach 54.28% after 6 hours of reaction.