Stabilization of Hydroxynitrile Lyases from Two Variants of Passion Fruit,Passiflora edulis Sims and Passiflora edulis Forma flavicarpa,by C-Terminal Truncation

Because the synthesis of chiral compounds generally requires a broad range of substrate specificity and stable enzymes, screening for better enzymes and/or improvement of enzyme properties through molecular approaches is necessary for sustainable industrial development. Herein, the discovery of unique hydroxynitrile lyases (HNLs) from two species of passion fruits, Passiflora edulis forma flavicarpa (yellow passion fruit, PeHNL-Ny) and Passiflora edulis Sims (purple passion fruit, PeHNL-Np), isolated and purified from passion fruit leaves is reported. These are the smallest HNLs (comprising 121 amino acids). Amino acid sequences of both enzymes are 99 % identical; there is a difference of one amino acid in a consensus sequence. PeHNL-Np has an Ala residue at position 107 and is nonglycosylated at Asn105. Because it was confirmed that natural and glycosylated PeHNL-Ny showed superior thermostability, pH stability, and organic tolerance to that of PeHNL-Np, it has been speculated that protein engineering around the only glycosylation site, Asn105, located at the C-terminal region of PeHNL-Ny, might contribute to the stabilization of PeHNL. Therefore, the focus is on improved stability of the nonglycosylated PeHNL by truncating its C-terminal region. The C-terminal-truncated PeHNLΔ₁₀₇ was obtained by truncating 15 amino acids from the C terminus followed by expression in Escherichia coli. PeHNLΔ₁₀₇ expressed in E. coli was not glycosylated, and showed improved thermostability, solvent stability, and reusability similar to that of the wild-type glycosylated form of PeHNL expressed in Pichia pastoris. These data reveal that the lack of the high-flexibility region at the C terminus of PeHNL might be a possible reason for improving the stability of PeHNL.     

Application of immunoproteomics to leptospirosis: towards clinical diagnostics and vaccine discovery

Each of the currently available methods for serodiagnosis of leptospirosis, including the microscopic agglutination test (MAT), has its own drawback(s) when used in clinical practice. A new diagnostic test is therefore required for an earlier and more accurate diagnosis of leptospirosis. We applied immunoproteomics to define potential immunogens from five serovars of Leptospira reference strains. A leptospiral whole cell lysate from each serovar was used as the antigen to react with IgG and IgM in the sera from four patients with a positive MAT. Sera from four non-leptospirosis patients with a negative MAT were pooled and used as the negative control. 2-D Western blot analysis showed that the degree of immunoreactivity corresponded with the MAT titers. No immunoreactive spots were detected when the pooled control sera were used. A total of 24 protein spots immunoreacted with IgM and/or IgG from patients with leptospirosis. These immunoreactive proteins were identified by MALDI-TOF MS and were classified into five groups, including flagellar proteins, chaperones/heat shock proteins, transport proteins, metabolic enzymes, and hypothetical proteins. More immunoreactive spots were detected with anti-human IgG in the sera of all patients and with all the serovars of leptospires used. Some of the identified proteins immunoreacted only with IgG, whereas the others were detectable with both IgM and IgG. Among the immunoreactive proteins identified, FlaB proteins (flagellin and flagellar core protein) have been shown to have a potential role in clinical diagnostics and vaccine development. These data underscore the significant impact of immunoproteomics in clinical applications.     

High efficacy bioconversion of starch to lactic acid using an amylolytic lactic acid bacterium isolated from Thai indigenous fermented rice noodles

Amylolytic lactic acid bacterium (ALAB) strain S21 was isolated from Thai indigenous fermented rice noodles and identified as Lactobacillus plantarum, based on 16S rDNA sequence and recA gene analysis. L. plantarum S21 exhibited a specific growth rate (μ) of 0.24 1/h in modified MRS broth containing 10 g/L of starch as the sole carbon source, and a high efficacy in producing lactic acid (9.41, 24.48, 41.84, 74.33, and 94.04 g/L from 10, 25, 50, 75, and 100 g/L of cassava starch, respectively), which are higher values than previously reported for ALAB. Crude amylase from L. plantarum S21 had broad pH stability (3.5–8.0), and hydrolyzed starch to maltose and glucose as the major and minor products. L. plantarum S21 should be considered useful for industrial bioconversion of starch to lactic acid.     

Texture and colour characteristics,and optimisation of sodium chloride,potassium chloride and glycine of reduced-sodium frankfurter

The three-components mixture design was applied to optimise a ratio of NaCl (0%–65%), KCl (35%–100%) and glycine (0%–20%) in reduced-sodium frankfurters. Fourteen frankfurters were analysed for texture and colour, and consumer (n = 100) acceptability. Results indicated that NaCl levels affected consumer acceptability of reduced-sodium frankfurters. Increasing NaCl generally increased texture hardness. Optimisation of a salt mixture was performed by superimposing contour plots of predicted acceptability scores (≥5.5 on a 9-points hedonic scale) of all sensory attributes and revealed the optimal salt mixture: 40.03%–63.66% NaCl, 35.00%–55.90% KCl and 0.00%–20.00% glycine. The optimal salt mixture contained 220–340 mg Na/100g frankfurter compared with 540 mg Na/100 g of the control formulation (100% NaCl). The mean overall liking score (5.9 vs. 5.9) of the optimal reduced-sodium frankfurter was not different from the control (100% NaCl). This optimal formulation had >25% sodium reduction and could be claimed as ‘reduced-sodium’ according to US Food and Drugs Administration regulation.     

Time influence on the interaction between Cyt2Aa2 and lipid/cholesterol bilayers

Protein–membrane interactions are still an important topic of investigation. One of the suitable experimental techniques used by the scientific community to address such question is atomic force microscopy. In a previous work, we have reported that the binding mechanism between the cytolytic and antimicrobial protein (Cyt2Aa2) and lipid/cholesterol bilayers was concentration‐dependent, leading to either the formation of holes in the bilayer or aggregates. Here we study such binding mechanism as a function of time at low protein concentrations (10 µg/mL). We demonstrate that although holes are formed during the first stages of the protein–lipid interaction, a reparation process due to molecular mobility in the bilayer leads to a homogenous and isotropic protein–lipid/cholesterol layer within 3 hr. The combination of imaging, force spectroscopy, and phase contrast delivered information about topography dynamics (molecular mobility), layer thickness, and mechanical properties of the protein–lipid/cholesterol system. These results highlight the importance of the observation time in (such type of) protein–lipid interactions (at low protein concentrations).     

Thermoelectricity of p-Fe2O3-SO3-SiO2-others and n-Fe2O3-SiO2-CuO-others

Thermoelectric minerals have been found at Loei Province, in the northeastern part of Thailand. Local mineral specimens were prepared in the powders and bulk solids form by crushing, calcination and annealing, pressure and sintering, cutting and polishing. Mineral samples were used to analyze the composition and phase, determine the thermoelectric property and efficiency, design and construct a thermoelectric generator. Chemical composition and phase identification of powder samples were analyzed by the x-ray fluorescence (XRF) and x-ray diffraction (XRD), respectively. XRF and XRD results indicated that the mineral samples comprised the SO₃-CaO-SiO₂-others, Fe₂O₃-SO₃-SiO₂-others, Fe₂O₃-SiO₂-others and Fe₂O₃-SiO₂-CuO-others. From the thermoelectric property and efficiency determinations, the p-SO₃-CaO-SiO₂-others, p-Fe₂O₃-SO₃-SiO₂-others, n-Fe₂O₃-SiO₂-others and n-Fe₂O₃-SiO₂-CuO-others bulks were found to exhibit the thermoelectric figure of merit in orders of 10^?14, 10^?11, 10^?14 and 10^?13 K^?1, respectively. A fabricated thermoelectric generator made from ten pairs of p-Fe₂O₃-SO₃-SiO₂-others and n-Fe₂O₃-SiO₂-CuO-others legs that can be provided the open circuit voltage and short circuit current up to 48.30 mV and 0.14 μA for a temperature difference of 39.80 K at room temperature, respectively. While the internal resistance decreased and reached a value of 665 kΩ.