title: Functional immobilization of recombinant alkaline phosphatases bearing a glutamyl donor substrate peptide of microbial transglutaminase.
abstract: Covalent and site-specific protein immobilization catalyzed by microbial transglutaminase (MTG) was investigated using recombinant Escherichia coli alkaline phosphatase (AP) tagged with a glutamyl donor substrate peptide (MLAQGS) of MTG. A polystyrene surface physically coated with beta-casein or bovine serum albumin (BSA) was employed as an MTG-specific surface displaying reactive lysine residues. MTG-mediated protein immobilization through catalytic epsilon-(gamma-glutamyl)lysine bond formation between the peptide tag of recombinant APs and beta-casein- or BSA-coated surface was verified by the detection of AP activity on the surface. It was found that the length and the insertion position of the peptide tag did not significantly affect the efficacy of enzymatic immobilization of the recombinant APs. On the other hand, pH and ionic strength in the reaction media had crucial effects on the immobilization yields. Interestingly, the optimum pH range of MTG-mediated protein immobilization differed markedly from that for an MTG-catalyzed reaction in aqueous solution. The results suggest that the concentration of reactive species due to electrostatic interaction between the enzyme-substrate intermediate and the protein-adsorbed surface is a key factor governing MTG catalysis at a solid surface.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan; Center for Future Chemistry, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan. noritcm@mbox.nc.kyushu-u.ac.jp
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Satoshi
authors.initials: S
authors.lastname: Doi
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Ichinose
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005973:Glutamine / Q000737:chemistry*; D010455:Peptides; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry* / Q000235:genetics
chemical_list: D004800:Enzymes, Immobilized; D010455:Peptides; D011994:Recombinant Proteins; D005973:Glutamine; D011503:Transglutaminases; D000469:Alkaline Phosphatase