title: Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase.
abstract: Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.
authors: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors: Jo
authors: J
authors: Tominaga
authors: Noriho
authors: N
authors: Kamiya
authors: Satoshi
authors: S
authors: Doi
authors: Hirofumi
authors: H
authors: Ichinose
authors: Tatsuo
authors: T
authors: Maruyama
authors: Masahiro
authors: M
authors: Goto
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D010455:Peptides; D011994:Recombinant Proteins; D011503:Transglutaminases; D000469:Alkaline Phosphatase