title: Optimizing the biocatalytic productivity of an engineered sialidase from Trypanosoma rangeli for 3'-sialyllactose production.
abstract: 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 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 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 24 h.
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Birgitte
authors.initials: B
authors.lastname: Zeuner
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Jianquan
authors.initials: J
authors.lastname: Luo
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Nyffenegger
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Ville
authors.initials: V
authors.lastname: Aumala
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Jørn Dalgaard
authors.initials: JD
authors.lastname: Mikkelsen
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs. Lyngby, Denmark. Electronic address: am@kt.dtu.dk.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
mesh_terms: D000464:Alginates; D000818:Animals; D055162:Biocatalysis; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002482:Cellulose; D003432:Cross-Linking Reagents; D004339:Drug Compounding; D004800:Enzymes, Immobilized; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D053758:Nanoparticles; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis*; D010446:Peptide Fragments / Q000378:metabolism; D010843:Pichia; D055550:Protein Stability; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D057509:Trypanosoma rangeli / Q000235:genetics / Q000378:metabolism*
chemical_list: C421467:3'-sialyllactose; D000464:Alginates; D002364:Caseins; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D006603:Hexuronic Acids; D008567:Membranes, Artificial; D009844:Oligosaccharides; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C065989:caseinomacropeptide; D020723:Glucuronic Acid; D002482:Cellulose; D009439:Neuraminidase
keywords: 3′-Sialyllactose; Enzymatic membrane reactor; Enzyme immobilization; Thermal stability; Trypanosoma rangeli; trans-Sialidase