Screening of novel DPP-IV inhibitory peptides derived from bovine milk proteins using a peptide array platform.

Dipeptidyl peptidase IV (DPP-IV) has become an important target in the prevention and treatment of diabetes. Although many DPP-IV inhibitory peptides have been identified by a general approach involving the repeated fractionation of food protein hydrolysates, the obtained results have been dependent on the content of each peptide and fractionation conditions. In the present study, a peptide array that provides comprehensive assays of peptide sequences was used to identify novel DPP-IV inhibitory peptides derived from bovine milk proteins; these peptides were then compared with those identified using the general approach. While the general approach identified only known peptides that were abundant in the hydrolysate, the peptide array-based approach identified 10 novel DPP-IV inhibitory peptides, all of which had proline at the second residue from the N-terminus. The proper or combined use of these two approaches, which have different advantages, will enable the efficient development of novel bioactive foods and drugs.

Peptide array-based inhibition ELISA for evaluating antigenicity in infant formulas.

With increased awareness among consumers regarding food safety and security, food allergen control has become an indispensable requirement in the food industry. Although several methods for detecting allergens in food products are available, highly sensitive techniques are required. In this study, we developed a technique named as peptide array-based inhibition enzyme-linked immunosorbent assay (ELISA), Pep-iEIA, for evaluating antigenicity and detecting cow's milk antigen in infant formula products, using a peptide array consisting of a series of overlapping peptides found in allergenic milk proteins. Pep-iEIA was used to examine five cow's milk-based infant formulas with different degrees of hydrolyzation, and the assay offered both more sensitive detection and detailed analysis of remaining antigenic peptides in allergen compared to conventional ELISA. The antigenicity level of the allergenic peptides identified using Pep-iEIA was confirmed by surface plasmon resonance assay. We believe that Pep-iEIA will be highly useful for antigenicity evaluation of dairy products consumed by infants and patients with cow's milk allergy.

Pep-MS assay: Protease hydrolysis assay system using photo-cleavable peptide array and mass spectrometer.

Food processing technology such as protein hydrolysis using proteases has been receiving a lot of attention, and it is important to accurately understand the cleavage specificity of each protease for selecting a protease suited to aims. Although numerous methods have been reported to reveal the substrate specificity of proteases, there is no method to evaluate simply, quickly, reasonably, and accurately. This study set out to devise Pep-MS assay, a novel assay system that can be used to comprehensively clarify positions at which proteases cleave, by combining a mass spectrometer and a photo-cleavable peptide array. First, we evaluated peptide array corresponding to the primary sequences of αS1-casein, αS2-casein and ß-casein with trypsin to verify the accuracy of the Pep-MS assay. The evaluation of cleavage positions by the trypsin protease reagent using the Pep-MS assay resulted in a matching rate of about 96.8% to rational cleavage positions. Next, we confirmed the cleavage positions in αS2-casein or ß-lactoglobulin by an industrial bacterial protease from Bacillus subtilis at some protease reaction temperatures or reaction times. The Pep-MS assay clarified the differences in the cleavage patterns due to the reaction temperature, and the change in the cleavage strength with the reaction time. Pep-MS assay is a promising method for evaluating the substrate specificity of proteases, which will be useful to find effective production conditions for functional peptide from foods and effective hydrolysis conditions for decreasing allergen of food proteins.

Effect of global transcriptional regulators related to carbohydrate metabolism on organic solvent tolerance in Escherichia coli.

The effect of global transcriptional regulators related to carbohydrate metabolism on organic solvent tolerance (OST) in Escherichia coli has been investigated. The OSTs of the E. coli K-12 BW25113 knockout mutants acrA, acrB, cra, crp, cyaA, fnr, and mlc were investigated on the basis of colony forming efficiency on an agar plate overlaid with organic solvents. The knockout mutants of the cyclic AMP receptor protein (Deltacrp) and adenylate cyclase (DeltacyaA) were found to increase their OSTs. However, their OSTs decreased to the level of the wild-type strain when the DeltacyaA cells were grown in the presence of exogenous cyclic AMP. These results indicate that the formation of the cAMP-Crp complex is related to OST. The microbial-adhesion-to-hydrocarbon test on the Deltacrp and DeltacyaA mutants revealed that these mutants bound less abundantly to the organic solvent phase. In the Deltacrp and DeltacyaA mutants, the expression levels of GadB and NuoG increased to the level of the wild type. The OSTs of DeltagadB and DeltanuoG mutants decreased, suggesting that the expressions of these proteins are involved in the increased OST in the Deltacrp and DeltacyaA mutants.

Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance.

Prefoldin is a jellyfish-shaped hexameric chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. In this work, we characterized the organic solvent tolerance of Escherichia coli cells that overexpress prefoldin and group II chaperonin from a hyperthermophilic archeaum, Pyrococcus horikoshii OT3. The colony-forming efficiency of E. coli cells overexpressing prefoldin increased by 1,000-fold and decreased the accumulation of intracellular organic solvent. The effect was impaired by deletions of the region responsible for the chaperone function of prefoldin. Therefore, we concluded that prefoldin endows E. coli cells by preventing accumulation of intracellular organic solvent through its molecular chaperone activity.

Increase of organic solvent tolerance by overexpression of manXYZ in Escherichia coli.

Transcriptional analysis has been investigated to detect the genes involved in organic solvent tolerance. A time course of gene expression profiles of Escherichia coli after exposure to organic solvents revealed that the expression levels of manX, manY, and manZ genes were strongly upregulated by 13.2-, 10.0-, and 7.0-folds, respectively, after 2 min and then decreased after 10 min. Organic solvent tolerance of E. coli was investigated by inducing overexpression of manX, manY, and manZ genes and manXYZ operon that encode a sugar transporter of the phosphotransferase system. Although the expression of manX, manY, and manZ alone was not effective, the organic solvent tolerance level was increased by the expression of manXYZ. The intracellular hexane level was kept lower in E. coli cells overexpressing manXYZ after 4 h of incubation with hexane.