An engineered sialidase, Tr6, from Trypanosoma rangeli was used for biosynthetic production of 3'-sialyllactose, a human milk oligosaccharide case compound, from casein glycomacropeptide (CGMP) and lactose, components abundantly present in industrial dairy side streams. Four different enzyme reuse methods were compared to optimize the biocatalytic productivity, i.e. 3'-sialyllactose formation per amount of Tr6 employed: (i) His-tag immobilization on magnetic Cu2+-iminodiacetic acid-functionalized nanoparticles (MNPs), (ii) membrane immobilization, (iii) calcium alginate encapsulation of cross-linked Tr6, and (iv) Tr6 catalysis in a membrane reactor. Tr6 immobilized on MNPs gave a biocatalytic productivity of 84 mg 3'-sialyllactose/mg Tr6 after seven consecutive reaction runs. Calcium-alginate and membrane immobilization were inefficient. Using free Tr6 in a 10 kDa membrane reactor produced a 9-fold biocatalytic productivity increase compared to using free Tr6 in a batch reactor giving 306 mg 3'-sialyllactose/mg Tr6 after seven consecutive reaction runs. The 3'-sialyllactose yield on alpha-2,3-bound sialic acid in CGMP was 74%. Using circular dichroism, a temperature denaturation midpoint of Tr6, T-m, of 57.2 degrees C was determined. The thermal stability of free Tr6 was similarly high and the Tr6 was stable at the reaction temperature (25 degrees C) for at least 24 h. (C) 2013 Elsevier Inc. All rights reserved.