Effect of enzymatic deamidation on the heat-induced conformational changes in whey protein isolate and its relation to gel properties.

The effect of protein-glutaminase (PG) on the heat-induced conformational changes in whey protein isolate (WPI) and its relation to gel properties was investigated. The structural properties of WPI treated with PG were examined by several analytical methods. The analysis of the fluorescence spectrum and the binding capacity of a fluorescent probe demonstrated that deamidation prevented the increase in the fluorescence intensity caused by subsequent heat treatment. Measurements of the molecular weight distribution of WPI showed that PG-treated WPI was not likely to polymerize even after heating. This is thought to be due to an increase in electrostatic repulsion between carboxylic acid groups and a decrease in the formation of disulfide bonds, which results in the decrease in heat-induced aggregation. The properties of heat-induced WPI gels were modified by deamidation. PG-treated WPI gels had a soft texture and a high water-holding capacity in the presence of salts.

Incorporation of 15N-labeled ammonia into glutamine amide groups by protein-glutaminase and analysis of the reactivity for α-lactalbumin.

Protein-glutaminase (PG) is an enzyme that catalyzes the deamidation of protein-bound glutamine residues. We found that an enzyme labeling technique (ELT), which is a stable isotope labeling method based on transglutaminase (TGase) reaction, is applicable for PG. PG catalyzed incorporation of (15)N-labeled ammonium ions into reactive glutamine amide groups in α-lactalbumin similarly to TGase and deamidated the most reactive glutamine amide group once labeled with (15)N. Furthermore, we investigated the effect of ammonium ions on the PG activity by peptide mapping, and more reactive glutamine residues were detected than were detected by the ELT in the presence of ammonium ions. This is probably because ammonium ions are competitive inhibitors, causing decreased reactivity for glutamine residues. We propose the reaction scheme of PG in the presence of the (15)N-labeled ammonium ions and show that the ELT method with PG is useful for evaluating the activity of PG.

Purification and characterization of an N-terminal acidic amino acid-specific aminopeptidase from soybean cotyledons (Glycine max).

A novel enzyme that catalyzes the efficient hydrolysis of Glu-Glu was isolated from soybean cotyledons by ammonium sulfate fractionation and successive column chromatographies of Q-sepharose, Phenyl sepharose, and Superdex 200. The apparent molecular mass of this enzyme was found to be 56 kDa and 510 kDa by SDS-polyacrylamide gel electrophoresis and Superdex 200 HR 10/30 column chromatography respectively. The enzyme had high activity against Glu-p-nitroanilide (pNA) and Asp-pNA, whereas Leu-pNA, Phe-pNA, Ala-pNA, and Pro-pNA were not hydrolyzed. The synthetic dipeptides Glu-Xxx and Asp-Xxx were hydrolyzed, but Xxx-Glu was not. The digestion of a Glu-rich oligopeptide, chromogranin A (Glu-Glu-Glu-Glu-Glu-Met-Ala-Val-Val-Pro-Gln-Gly-Leu-Phe-Arg-Gly-NH(2)) using this purified enzyme was also investigated. Glutamic acid residues were cleaved one by one from the N-terminus. These observations indicate that the enzyme removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. It is thought that it was an N-terminal acidic amino acid-specific aminopeptidase from a plant.

Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library.

Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.

Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626.

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, alpha-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM alpha-ketobutyrate, and 100 mM L-glutamate with a molar yield to alpha-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl(2) x 4H(2)O at 28 degrees C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and alpha-ketobutyrate was purified from A. simplex AKU 626. Mn(2+) and pyridoxal 5'-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.