Functional immobilization of recombinant alkaline phosphatases bearing a glutamyl donor substrate peptide of microbial transglutaminase

Noriho Kamiya, Satoshi Doi, Yusuke Tanaka, Hirofumi Ichinose, Masahiro Goto

Research output: Contribution to journalArticle

17 Citations (Scopus)

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 β-casein or bovine serum albumin (BSA) was employed as an MTG-specific surface displaying reactive lysine residues. MTG-mediated protein immobilization through catalytic ε-(γ-glutamyl)lysine bond formation between the peptide tag of recombinant APs and β-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.

Original languageEnglish
Pages (from-to)195-199
Number of pages5
JournalJournal of Bioscience and Bioengineering
Volume104
Issue number3
DOIs
Publication statusPublished - Sep 1 2007

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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