Journal article
Optimizing the biocatalytic productivity of an engineered sialidase from Trypanosoma rangeli for 3′-sialyllactose production
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 re-use 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 84mg 3′-sialyllactose/mg Tr6 after seven consecutive reaction runs. Calcium-alginate and membrane immobilization were inefficient. Using free Tr6 in a 10kDa membrane reactor produced a 9-fold biocatalytic productivity increase compared to using free Tr6 in a batch reactor giving 306mg 3′-sialyllactose/mg Tr6 after seven consecutive reaction runs.
The 3′-sialyllactose yield on α-2,3-bound sialic acid in CGMP was 74%. Using circular dichroism, a temperature denaturation midpoint of Tr6, Tm, of 57.2°C was determined. The thermal stability of free Tr6 was similarly high and the Tr6 was stable at the reaction temperature (25°C) for at least 24h.
Language: | English |
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Year: | 2014 |
Pages: | 85-93 |
ISSN: | 01410229 and 18790909 |
Types: | Journal article |
DOI: | 10.1016/j.enzmictec.2013.12.009 |
ORCIDs: | Zeuner, Birgitte , Mikkelsen, Jørn Dalgaard and Meyer, Anne S. |
3′-Sialyllactose Enzymatic membrane reactor Enzyme immobilization Thermal stability Trypanosoma rangeli trans-Sialidase
3'-sialyllactose Alginates Animals Biocatalysis Carbohydrate Conformation Carbohydrate Sequence Caseins Cattle Cellulose Cross-Linking Reagents Drug Compounding Enzymes, Immobilized Glucuronic Acid Hexuronic Acids Membranes, Artificial Molecular Sequence Data Mutagenesis, Site-Directed Nanoparticles Neuraminidase Oligosaccharides Peptide Fragments Pichia Protein Stability Protozoan Proteins Recombinant Fusion Proteins Temperature caseinomacropeptide