Katsuya Seguro

Transglutaminase and its use for food processing

Some characteristics and applications of a transglutaminase, derived from a variant of Streptoverticillium mobaraense, namely microbial transglutaminase (MTGase), are considered. MTGase, mass-produced at low cost by fermentation, catalyses the crosslinking of most food proteins through the formation of an e-(γ-glutamyl)lysine bond, in the same way as well-known mammalian enzymes. However, MTGase is remarkable because it is calcium independent and its molecular weight is smaller than that of other known enzymes. The results of many studies suggest that MTGase, as well as other transglutaminases, has many potential applications in food processing and in other areas.

Overproduction of Microbial Transglutaminase in Escherichia coli, In Vitro Refolding, and Characterization of the Refolded Form

The Streptoverticillium transglutaminase (MTG) gene, synthesized previously for yeast expression, was modified and resynthesized for overexpession in E. coli. A high-level expression plasmid, pUCTRPMTG-02(+), was constructed. Furthermore, to eliminate the N-terminal methionine, pUCTRPMTGD2 was constructed. Cultivation of E. coli transformed with pUCTRPMTG-02(+) or pUCTRPMTGD2 yielded a large amount of MTG (200~300 mg/liter) as insoluble inclusion bodies. The N-terminal amino acid residue of the expressed protein was methionine or serine (the second amino acid residue of the mature MTG sequence), respectively. Transformed E. coli cells were disrupted, and collected pellets of inclusion bodies were solubilized with 8 M urea. Rapid dilution treatment of solubilized MTG restored the enzymatic activity. Refolded MTG, purified by ion-exchange chromatography, which had an N-terminal methionine or serine residue, showed activity equivalent to that of native MTG. These results indicated that recombinant MTG could be produced efficiently in E. coli.

In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence.

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.

Inhibition of passive cutaneous anaphylaxis by synthetic human immunoglobulin E peptide fragments

In an attempt to determine the regions responsible for type 1 immediate hypersensitivity, a total of 42 peptide fragments, which cover the CH3–CH4 domains in human immunoglobulin E (IgE), were chemically synthesized. Several peptide fragments located in the amino acid sequences Ala329—Thr357 and Arg119—Ala463, inhibited passive cutaneous anaphylaxis (PCA) in vivo. In order to pinpoint the sites responsible for the inhibition of the PCA reaction, various rragment peptides in these two regions were synthesized. As a result, residues Pro343—Leu348, Pro426—Thr433, and Ser456—Thr401 were suggested to be involved in type I immediate hypersensitivity.

Inhibition of passive sensitization of human peripheral basophils by synthetic human immunoglobulin E peptide fragments

To delineate the binding site in the human immunoglobulin E (IgE) molecule to the Fcϵ receptor on basophils and mast cells, we chemically synthesized a total of 71 peptide fragments within the sequence Ser300‐Lys547 in the human IgE molecule. The synthetic peptides were tested for their capacity to inhibit passive sensitization of human peripheral basophils with atopic patients serum containing the specific IgE against dust mites in vitro. It was found that a peptide fragment, Pro345‐Ile356, potently inhibited the passive sensitization. To clarify the minimal active core, various analogues, such as shortened, substituted (by Gly or Ala residue), omission and retro‐sequence peptides, were synthesized and assayed. The results suggested that the sequence Pro345‐Lys352 in the human IgE molecule would be an IgE binding site, and that a synthetic octapeptide, Pro345‐Phe‐Asp‐Leu‐Phe‐Ile‐Arg‐Lys352, inhibited the passive sensitization, probably by occupying the Fcϵ receptor sites on the cells.