Association of natural resistance-associated macrophage protein 1 polymorphisms with Salmonella fecal shedding and hematological traits in pigs.

Background and Aim: Natural resistance-associated macrophage protein 1 encoding gene (Nramp1) plays a role in immune response and disease resistance. This study aimed to investigate the polymorphisms of Nramp1 intron 6 concerning Salmonella shedding and hematological traits in pigs. Materials and Methods: A total of 40 commercial pigs (three-way Large White x Landrace x Duroc cross) were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and analyze the relationship between the polymorphisms of the Nramp1 gene and Salmonella fecal shedding and hematological parameters. Results: Nramp1 was shown to be polymorphic in these pigs. The Nramp1 gene has two alleles (A and B) and two genotypes (AB and BB). The BB genotype had a higher frequency than the AB genotype. A significant relationship between the BB genotype and the number of Salmonella in feces compared to the AB genotype (p < 0.05) on 7 days post-inoculation (DPI) was revealed in the association analysis. The single-nucleotide polymorphism at intron 6 in the Nramp1 gene was linked to white and red blood cells 2 and 7 DPI (p < 0.05). Conclusion: The Nramp1 gene was suggested by these findings to be potentially used as a molecular marker for the genetic selection of disease susceptibility in pig breeding.

Structural insights into the unique polylactate-degrading mechanism of Thermobifida alba cutinase.

Cutinases are enzymes known to degrade polyester-type plastics. Est119, a plastic-degrading type of cutinase from Thermobifida alba AHK119 (herein called Ta_cut), shows a broad substrate specificity toward polyesters, and can degrade substrates including polylactic acid (PLA). However, the PLA-degrading mechanism of cutinases is still poorly understood. Here, we report the structure complexes of cutinase with ethyl lactate (EL), the constitutional unit. From this complex structure, the electron density maps clearly showed one lactate (LAC) and one EL occupying different positions in the active site cleft. The binding mode of EL is assumed to show a figure prior to reaction and LAC is an after-reaction product. These complex structures demonstrate the role of active site residues in the esterase reaction and substrate recognition. The complex structures were compared with other documented complex structures of cutinases and with the structure of PETase from Ideonella sakaiensis. The amino acid residues involved in substrate interaction are highly conserved among these enzymes. Thus, mapping the precise interactions in the Ta_cut and EL complex will pave the way for understanding the plastic-degrading mechanism of cutinases and suggest ways of creating more potent enzymes by structural protein engineering.

Identification and in vitro assessment of potential probiotic characteristics and antibacterial effects of Lactobacillus plantarum subsp. plantarum SKI19, a bacteriocinogenic strain isolated from Thai fermented pork sausage.

A total of 2257 lactic acid bacteria were preliminarily screened for antagonistic activity against Lactobacillus sakei subsp. sakei JCM 1157. Strain SKI19 was selected and identified at the subspecies level as Lactobacillus plantarum subsp. plantarum SKI19, using 16S rRNA gene sequence analysis combined with recA and dnaK genes' amplification. Antibacterial activity of SKI19 was completely lost after treatment of neutralized cell free culture supernatant with proteolytic enzymes, suggesting that SKI19 produced a bacteriocin-like substance that inhibited not only closely related species, but was also effective against Listeria monocytogenes DMST 17303. Viewed under scanning electron microscope, cell membranes of the indicator strain appeared to collapse after exposure to the bacteriocin-like substance. In vitro tests concerning probiotic properties, SKI19 survived under simulated gastrointestinal tract conditions, and adhesion of its cell surface to xylene and chloroform was 90.14 and 89.85%, respectively. Complete inhibition by SKI19 against pathogenic bacteria (Escherichia coli DMST 4212, L. monocytogenes DMST 17303, and Staphylococcus aureus DMST 8840) was observed in co-cultivation under anaerobic conditions. A safety assessment showed that SKI19 was susceptible to several antibiotics and had no haemolytic activity. PCR amplification of virulence factors with the specific primers for ace, asa1, cylLS , efaAfs , hyl, and gelE genes were negative for SKI19. Also, SKI19 did not harbor any hdc, tdc, odc or ldc genes involved in biogenic amine production. The results reveal that SKI19 has probiotic potential and antibacterial activity, and is safe for further application in certain food products.

Comparison of genetic structures and biochemical properties of tandem cutinase-type polyesterases from Thermobifida alba AHK119.

This study described the genetic map of tandem genes (est1 and est119) encoding cutinase-type polyesterases in Thermobifida alba AHK119 and comparison of wild type and mutant enzymes of Est1 and Est119. Two genes were independently and constitutively expressed. The activity of Est1 was higher by approximately 1.6-1.7-fold than that of Est119 towards p-nitrophenyl butyrate, although both enzymes shared 95% sequence identity and 98% similarity and possessed similar 3D structures except that several amino acids in the probable substrate-docking loops were different from each other. Calcium ion enhanced the activity and the thermostability of both enzymes. Based on conserved sequences among Thermobifida cutinases, valine, proline and lysine were introduced into Est1 at Ala68, Thr253 and Met256, respectively. Among wild and mutant enzymes of Est119 and Est1, Est1 (A68V/T253P) possessed three prolines in the substrate-docking loops and displayed the highest thermostability that spotlighted the important effect of proline numbers in the loops. Est1 (A68V/T253P) was stable for 1 h below 60°C and even at 65°C, more than 70% and 50% activities were maintained after 30 and 60 min, respectively. Est1 (A68V/T253P) degraded various aliphatic and aliphatic-co-aromatic polyesters and hydrophilized an amorphous PET film. The enzyme hydrolyzed a PET trimer model compound, indicating its specificity towards an ester bond between terephthalic acid and ethylene glycol.

Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119.

Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/ß-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second ß-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca(2+) and was stable up to 60 °C with 150 mM Ca(2+). Another identical gene was located in tandem in the upstream of est119.

Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119.

More than 100 bacterial strains were isolated from composted polyester films and categorized into two groups, Actinomycetes (four genera) and Bacillus (three genera). Of these isolates, Thermobifida alba strain AHK119 (AB298783) was shown to possess the ability to significantly degrade aliphatic-aromatic copolyester film as well as decreasing the polymer particle sizes when grown at 50 degrees C on LB medium supplemented with polymer particles, yielding terephthalic acid. The esterase gene (est119, 903 bp, encoding a signal peptide and a mature protein of 34 and 266 amino acids, respectively) was cloned from AHK119. The Est119 sequence contains a conserved lipase box (-G-X-S-X-G-) and a catalytic triad (Ser129, His207, and Asp175). Furthermore, Tyr59 and Met130 likely form an oxyanion hole. The recombinant enzyme was purified from cell-free extracts of Escherichia coli Rosetta-gami B (DE3) harboring pQE80L-est119. The enzyme is a monomeric protein of ca. 30 kDa, which is active from 20 degrees C to 75 degrees C (with an optimal range of 45 to 55 degrees C) and in a pH range of 5.5 to 7.0 (with an optimal pH of 6.0). Its preferred substrate among the p-nitrophenyl acyl esters (C2 to C8) is p-nitrophenyl hexanoate (C6), indicating that the enzyme is an esterase rather than a lipase.