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Molecular characterization of a novel aspartyl aminopeptidase that contributes to the increase in glutamic acid content in chicken meat during cooking.

ABSTRACT: The enzyme involved in the increase in glutamic acid content in chicken meat during cooking was identified and characterized. Chicken homogenate produced significantly more free glutamic acid and exhibited higher glutamyl p-nitroanilide (Glu-pNA) hydrolyzing activity than beef when heat cooked. Amino acid sequencing revealed the presence of aspartyl aminopeptidase (DNPEP) in chicken meat. Using RT-PCR, DNPEP gene expression was detected in chicken breast and thigh muscles, liver, and small intestine, together with various other peptidase genes. Full-length DNPEP cDNA was cloned, and recombinant chicken DNPEP (cDNPEP) was expressed in Escherichia coli. cDNPEP showed five-fold higher activity against Glu-pNA than against aspartyl-pNA, which represents a different substrate specificity than observed for recombinant bovine DNPEP (bDNPEP). The Km values of both DNPEPs with Glu p-NA substrates indicated a higher affinity of cDNPEP for glutamyl residues. This unique substrate specificity of cDNPEP contributes to efficient glutamic acid production in chickens.

SUBMITTER: Yuhara H

PROVIDER: S-EPMC8991601 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Molecular characterization of a novel aspartyl aminopeptidase that contributes to the increase in glutamic acid content in chicken meat during cooking.

Yuhara Hitomi H Ohtani Akira A Matano Mami M Kashiwagi Yutaka Y Maehashi Kenji K

Food chemistry. Molecular sciences 20210217

The enzyme involved in the increase in glutamic acid content in chicken meat during cooking was identified and characterized. Chicken homogenate produced significantly more free glutamic acid and exhibited higher glutamyl p-nitroanilide (Glu-pNA) hydrolyzing activity than beef when heat cooked. Amino acid sequencing revealed the presence of aspartyl aminopeptidase (DNPEP) in chicken meat. Using RT-PCR, DNPEP gene expression was detected in chicken breast and thigh muscles, l ...[more]

PMID: 35415631

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Project description:Chicken meat is rich in unsaturated fatty acids. Therefore, it is more susceptible to lipid oxidation and production of volatile organic compounds (VOC). In this study, we evaluated the fatty acids, antioxidants, and VOC profiles of raw and cooked meat samples derived from 4 strains of chicken differing in their growth rates, which were as follows: slow-growing (SG, Leghorn), medium-growing (MG, Hubbard and Naked Neck), and fast-growing (FG, Ross). The VOC profile of meat was measured using proton-transfer reaction-mass spectrometry (PTR-MS). The VOC were identified using PTR-time of flight-MS (PTR-ToF-MS). The data were analyzed using both univariate and multivariate models. Twenty main VOC were identified, which were classified into the following chemical categories: aldehydes, alkadienes, alkenes, furans, amides, alcohols, and other compounds. Our results revealed that the chicken genotype and the method of cooking strongly influenced the VOC profile of the meat. Identifying the relationships between these traits allowed us to highlight the trade-off of the main substrates such as n-3 and n-6 polyunsaturated fatty acids (PUFA), protective substances (antioxidants), and degradation products (VOC) of the poultry meat produced during cooking. The extent of VOC production and n-3 loss was found to be higher for the SG genotype. Reduction of n-6 was higher in MG, whereas small losses in antioxidants and PUFA were observed in the FG genotype, consequently, resulting in the lowest production of VOC. The SG and MG are genotypes more active from a kinetic point of view respect to the FG ones. For this reason, in the FG genotypes, the antioxidants are less involved in the oxidative stress induced by the movement; thus, they were available to protect the lipid of the meat during the cooking process. These results suggested that the use of SG and MG genotypes requires a specific dietary protocol (i.e., increasing the antioxidants content) to counteract the lipid oxidations in all the phases: in vivo, postmortem, and during/after cooking.

Project description:BackgroundDuring erythrocytic schizogony, Plasmodium falciparum interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the P. falciparum M18 aspartyl aminopeptidase (PfM18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) PfM18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.MethodsrPfM18AAP was produced as a hexahistidine-fusion protein in Escherichia coli and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin-PfM18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization.ResultsrPfM18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that rPfM18AAP aggregated into oligomers. An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C. The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with rPfM18AAP against spectrin and erythrocyte membrane proteins indicated that rPfM18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.ConclusionStudies characterizing rPfM18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, PfM18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.

Dataset Information

Molecular characterization of a novel aspartyl aminopeptidase that contributes to the increase in glutamic acid content in chicken meat during cooking.

Publications

Molecular characterization of a novel aspartyl aminopeptidase that contributes to the increase in glutamic acid content in chicken meat during cooking.

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