Single nucleotide polymorphisms in several porcine cathepsin genes are associated with growth, carcass, and production traits in Italian Large White pigs

To identify DNA markers associated with performance, carcass, and meat production traits including muscle postmortem cathepsin activity, sev- eral porcine genes encoding for lysosomal proteinases (cathepsin B, CTSB; cathepsin D, CTSD; cathepsin F, CTSF; cathepsin H, CTSH; cathepsin L, CTSL; and cathepsin Z, CTSZ) and for a cathepsin inhibitor (cys- tatin B) were investigated. Single nucleotide polymor- phisms were identified in CTSD, CTSH, CTSL, and CTSZ genes with a combination of in silico expressed sequence tag database mining and single-strand confor- mation polymorphism analysis. Sequencing and PCR- RFLP protocols were used to validate the identified polymorphisms. Allele frequencies at these loci were investigated in Italian Large White, Landrace, Duroc, Pietrain, Belgian Landrace, Hampshire, and Meishan breeds. Genotyping CTSD and CTSH markers made it possible to genetically map these genes to SSC 2 and 7, respectively. Markers in CTSD, CTSH, CTSL, and CTSZ genes, together with mutations we previously re- ported in cystatin B, CTSB, and CTSF genes, were genotyped in an Italian Large White sib-tested popu- lation (272 or 482 animals). For these animals, meat quality traits (cathepsin B activity, pH measured at 2 h postmortem, pH measured at 24 h postmortem, glyco- gen, lactate, and glycolytic potential of semimembrano- sus muscle) and EBV for ADG, lean cuts (LC), backfat thickness (BFT), ham weight (HW), and feed:gain ra- tio (FGR) were determined. Analyzed markers did not show any association with muscle cathepsin B activity. Thus, it could be possible that different genes, other than these investigated candidates, affect this trait, which is correlated with the excessive softness defect of dry-cured hams. The results of association analysis confirmed the effects we already reported in another study for CTSF on ADG (P = 0.008), LC (P = 0.001), and BFT (P = 0.02). Moreover, CTSD was associated with ADG, LC (P < 0.0001), BFT, HW, and FGR (P < 0.001); CTSH was associated with FGR (P = 0.026); and CTSZ was associated with ADG (P = 0.006), LC (P = 0.01), HW (P = 0.024), and FGR (P = 0.029). The biochemical and physiological functions of the lys- osomal proteinases, together with the results obtained in our investigation, suggest that the cathepsin gene family might play important roles affecting economic traits in pigs

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K(m) s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed