Identification of dipeptidyl peptidase 3 as the Angiotensin-(1–7) degrading peptidase in human HK-2 renal epithelial cells

Angiotensin-(1–7) (Ang-(1–7)) is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic and pro-oxidant effects of the Ang II-AT₁ receptor axis. We previously identified a peptidase activity from sheep brain, proximal tubules and human HK-2 proximal tubule cells that metabolized Ang-(1–7); thus, the present study isolated and identified the Ang-(1–7) peptidase. Utilizing ion exchange and hydrophobic interaction chromatography, a single 80 kDa protein band on SDS-PAGE was purified from HK-2 cells. The 80 kDa band was excised, the tryptic digest peptides analyzed by LC–MS and a protein was identified as the enzyme dipeptidyl peptidase 3 (DPP 3, EC: 3.4.14.4). A human DPP 3 antibody identified a single 80 kDa band in the purified enzyme preparation identical to recombinant human DPP 3. Both the purified Ang-(1–7) peptidase and DPP 3 exhibited an identical hydrolysis profile of Ang-(1–7) and both activities were abolished by the metallopeptidase inhibitor JMV-390. DPP 3 sequentially hydrolyzed Ang-(1–7) to Ang-(3–7) and rapidly converted Ang-(3–7) to Ang-(5–7). Kinetic analysis revealed that Ang-(3–7) was hydrolyzed at a greater rate than Ang-(1–7) [17.9 vs. 5.5 nmol/min/μg protein], and the Km for Ang-(3–7) was lower than Ang-(1–7) [3 vs. 12 μM]. Finally, chronic treatment of the HK-2 cells with 20 nM JMV-390 reduced intracellular DPP 3 activity and tended to augment the cellular levels of Ang-(1–7). We conclude that DPP 3 may influence the cellular expression of Ang-(1–7) and potentially reflect a therapeutic target to augment the actions of the peptide.     

Superoxide dismutases from larvae of the camel tick Hyalomma dromedarii

Three superoxide dismutases (EC 1.15.1.1) (TLSOD1, TLSOD2 and TLSOD3) were purified from larvae of the camel tick Hyalomma dromedarii by ammonium sulfate precipitation, ion exchange and gel filtration columns. SDS-PAGE revealed that the subunit molecular masses of the SODs are 40 ± 2 kDa, 67 ± 1.5 kDa and 45 ± 2.6 kDa for TLSOD1, TLSOD2 and TLSOD3, respectively. TLSOD1 and TLSOD2 are monomeric proteins, while TLSOD3 isoenzyme exhibits dimeric structure with native molecular mass of 90 kDa. The pI values are estimated at pH 8.0, pH 7.2 and pH 6.6 for the three SODs which displayed pH optima at 7.6, 8.0 and 7.8, respectively. CuCl₂ and ZnCl₂ increase the activity of TLSOD2 and TLSOD3, while MnCl₂ increases the activity of TLSOD1. KCN inhibits the activity of TLSOD2 and TLSOD3, while a remarkable resistance of TLSOD1 isoenzyme was detected. TLSOD1 is suggested to be a manganese containing isoenzyme while TLSOD2 and TLSOD3 are suggested to be copper/zinc-containing isoenzymes. These results indicate the presence of three different forms of SODs in the larval stage of camel tick. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of non-traditional methods to control them.     

TGF-beta-induced early gene-1 overexpression promotes oxidative stress protection and actin cytoskeleton rearrangement in human skin fibroblasts

BackgroundTransforming growth factor beta inducible early gene-1 (TIEG-1), a member of the Krüppel-like factor, was identified as a primary response gene for TGF-β. The role of TIEG-1 in skin repair has been mainly addressed in vivo on TIEG-1 null mice model and the mechanism remains unexplored.MethodsWe investigated the modulation of TIEG-1 expression in normal human skin fibroblasts by either down-expressing or overexpressing the gene. We evaluated reactive oxygen species production and the cell viability of treated cells. The effect of TIEG-1 overexpression was monitored by wound healing assay and immunofluorescence staining of actin fibers organization and alpha-smooth muscle actin (α-SMA). Western blots were carried out to identify the level of expression or phosphorylation of key proteins such as cofilin, Rho GTPases, and p38 mitogen-activated protein kinase (p38 MAPK).ResultsTIEG-1 down-regulation had a deleterious effect on the cell viability. It was significantly reduced (65 ± 5%) and exposure to ultraviolet further increased this effect (47 ± 3%). By contrast, cells overexpressing TIEG-1 had a reduced reactive oxygen species production (75%) compared to control and mock-transfected cells. This overexpression also resulted in formation of actin stress fibers and increased α-SMA expression and an enhanced wound healing feature. RhoB GTPase was upregulated and phosphorylation of cofilin and p38 MAPK was observed.ConclusionTIEG-1 overexpression in normal human skin fibroblasts results in improved resistance to oxidative stress, myofibroblast-like conversion that involved RhoB signaling pathway with cofilin and p38 MAPK proteins activation.General significanceThis study enlightens the role of TIEG-1 role in skin biology.     

A blood stage fraction of Plasmodium berghei induces protective and long lasting immune response in BALB/c mice

Incorporation of the parasite's subcellular fractions in subunit vaccines can be a possible approach for formulation of vaccine against malaria. In this study, the immunogenicity and protective efficacy of 10,000 g fraction of blood stage Plasmodium berghei was evaluated in mouse model. This fraction induced higher levels of anti-parasite antibodies and provided complete and long lasting protection as compared to whole parasite antigens. Antiserum raised against it was immunoadsorbed on CNBr activated sepharose-4B to elute antigens from this fraction. Eluted antigens were characterized electrophoretically, and after lyophilization these were designated as ML-I (having 55, 64, 66, and 74 kDa proteins), ML-II (having 51, 64, 66, and 72 kDa proteins) and ML-III (having only 47 kDa protein) sub-fractions. Mice were immunized with these sub-fractions and immune responses induced by various immunization regimens were evaluated and compared with that of 10,000 g fraction. These sub-fractions imparted partial protection except ML-III, which was non-protective. 10,000 g fraction as a whole provided complete protection and generated significantly higher level of IL-2 and IFN-γ in immune mice. ML-I produced significant amount of IL-1 and IL-4 as compared to ML-II. Enhanced level of malaria-specific IgG1 was produced by ML-II, but IgG2a was significantly higher in ML-I immunized mice. Conclusively, this study identifies 10,000 g fraction as a promising blood stage vaccine candidate and suggests that a vaccine based upon multiple antigens may be more efficacious as compared to single antigen based formulations.     

Characterization of potential allergens in fenugreek (Trigonella foenum-graecum) using patient sera and MS-based proteomic analysis

BackgroundFenugreek is a legume plant used as an ingredient of curry spice. Incidents of IgE-mediated food allergy to fenugreek have been reported. Coincidence with allergy to peanut, a major food allergen, seems to be common suggesting a rather high rate of cross-reactivity.ObjectiveCharacterization of fenugreek allergens using patient sera and mass spectrometry-based proteomic analysis.MethodsAllergenic fenugreek proteins were detected by immunoblotting, using sera from 13 patients with specific IgE to peanut and fenugreek. IgE-binding proteins were analyzed by peptide mass fingerprinting and peptide sequencing.ResultsA fenugreek protein quintet in the range from 50 kDa to 66 kDa showed high IgE-affinity, the protein at 50 kDa reaching the strongest signals in all patients. Proteomic analyses allowed the classification of several fenugreek proteins to a number of allergen families. Fenugreek 7S-vicilin and 11S-legumin were partly sequenced and revealed considerable homologies to peanut Ara h 1 and Ara h 3, respectively. The presence of a fenugreek 2S albumin and pathogenesis-related (PR-10) plant pollen protein was assumed by database searching results.ConclusionIn this study, individual fenugreek proteins were characterised for the first time. Observed homologies to major peanut allergens provide a molecular explanation for clinical cross-reactivity.     

Complexity of the heat-soluble LEA proteome in Artemia species

The brine shrimp Artemia is a well known stress tolerant invertebrate found on most continents. Under certain conditions females produce cysts (encysted gastrulae) that enter diapause, a state of obligate dormancy. During developmental formation of diapause embryos several different types of stress proteins accumulate in large amounts, including the late embryogenesis abundant (LEA) proteins. In this study we used a combination of heterologous group 3 LEA antibodies to demonstrate that the heat-soluble proteome of the cysts contains up to 12 distinct putative group 3 LEA proteins that complement the group 1 LEA proteins found previously. Most antibody-positive, heat-soluble proteins were larger than 50 kDa although antibody positive proteins of 20–38 kDa were also detected. Both nuclei and mitochondria had distinct complements of the putative group 3 LEA proteins. A few small group 3 LEA proteins were induced by cycles of hydration–dehydration along with one protein of about 62 kDa. The expression of group 3 LEA proteins, unlike members of group 1, was not restricted to encysted diapause embryos. Three to five putative group 3 LEA proteins were expressed in gravid females and in larvae. Cysts of different species from various geographic locations had distinct complements of group 3 LEA proteins suggesting rapid evolution of the LEA proteins or differences in the type of group 3 Lea genes expressed. Our results demonstrate the potential importance of group 3 LEA proteins in embryos and other life cycle stages of this animal extremophile.     

Effect of molecular size and modification pattern on the internalization of water soluble β-(1 → 3)-(1 → 4)-glucan by primary murine macrophages

It has been shown that β-(1 → 3)-(1 → 4)-glucans (BG34) from barley and oats can trigger recognition and internalization by murine and human macrophages. Increasing evidence has suggested that macrophage recognition and internalization of BG34 are dramatically affected by the purity of BG34, the molecular weight and chemical modification. In this study, we investigated the structural features of BG34 for macrophage recognition and internalization. We prepared homogeneous BG34s of 10 kDa (BG34-10), 200 kDa (BG34-200) and 500 kDa (BG34-500) with high purity, and then introduced green fluorescence FITC to the reducing ends (Re) or main chain (Mc). The results of size exclusion chromatography, ¹³C NMR, fluorescence microscopy, FACS analyses and MTS assay demonstrated that non-toxic BG34 of 10 kDa (BG34-10) effectively trigger macrophage internalization. The internalization was adversely affected by modifying the main chain of BG34-10 but not the reducing end. Studies using blocking antibodies on several CD11b⁺ and CD11b^? cells suggested that CD11b may play an important role in mediating macrophage internalization of BG34-10. Quantitative RT-PCR and intracellular cytokine stain revealed that macrophages generate increased level of CD11b and TNF-α in response to BG34-10. This study for the first time demonstrated the molecular size (10 kDa) and pattern of modification (reducing end modification) for BG34-10 to mediate macrophage internalization. Since BG34 is water soluble, biocompatible and biodegradable FDA-approved material, this mechanism of BG34-10 can be used to design drug delivery system targeting macrophages.     

IL-22 activates oxidant signaling in pulmonary vascular smooth muscle cells

Reactive oxygen species (ROS) mediate cell-signaling processes in response to various ligands and play important roles in the pathogenesis of cardiovascular diseases. The present study reports that interleukin-22 (IL-22) elicits signal transduction in vascular smooth muscle cells (SMCs) through a ROS-dependent mechanism. We find that pulmonary artery SMCs express IL-22 receptor alpha 1 and that IL-22 activates STAT3 through this receptor. IL-22-induced signaling is found to be mediated by NADPH oxidase, as indicated by the observations that the inhibition and siRNA knock-down of this enzyme inhibit IL-22 signaling. IL-22 triggers the oxidative modifications of proteins through protein carbonylation and protein glutathionylation. Mass spectrometry identified some proteins that are carbonylated in response to IL-22 stimulation, including α-enolase, heat shock cognate 71 kDa protein, mitochondrial 60 kDa heat shock protein, and cytoplasmic 2 actin and determined that α-tubulin is glutathionylated. Protein glutathionylation and STAT3 phosphorylation are enhanced by the siRNA knock-down of glutaredoxin, while IL-22-mediated STAT3 phosphorylation is suppressed by knocking down thioredoxin interacting protein, an inhibitor of thioredoxin. IL-22 is also found to promote the growth of SMCs via NADPH oxidase. In rats, pulmonary hypertension is found to be associated with increased smooth muscle IL-22 expression. These results show that IL-22 promotes the growth of pulmonary vascular SMCs via a signaling mechanism that involves NADPH oxidase-dependent oxidation.     

Identification of target proteins of clinical immunity to Plasmodium falciparum in a region of low malaria transmission

The target molecules of antibodies against falciparum malaria remain largely unknown. Recently we have identified multiple proteins as targets of immunity against Plasmodium falciparum using African serum samples. To investigate whether potential targets of clinical immunity differ with transmission intensity, we assessed immune responses in residents of low malaria transmission region in Thailand. Malaria asymptomatic volunteers (Asy: n = 19) and symptomatic patients (Sym: n = 21) were enrolled into the study. Serum immunoreactivity to 186 wheat germ cell-free system (WGCFS)-synthesized recombinant P. falciparum asexual-blood stage proteins were determined by AlphaScreen, and subsequently compared between the study groups. Forty proteins were determined as immunoreactive with antibody responses to 35 proteins being higher in Asy group than in Sym group. Among the 35 proteins, antibodies to MSP3, MSPDBL1, RH2b, and MSP7 were significantly higher in Asy than Sym (unadjusted p < 0.005) suggesting these antigens may have a protective role in clinical malaria. MSP3 reactivity remained significantly different between Asy and Sym groups even after multiple comparison adjustments (adjusted p = 0.033). Interestingly, while our two preceding studies using African sera were conducted differently (e.g., cross-sectional vs. longitudinal design, observed clinical manifestation vs. functional activity), those studies similarly identified MSP3 and MSPDBL1 as potential targets of protective immunity. This study further provides a strong rationale for the application of WGCFS-based immunoprofiling to malaria vaccine candidate and biomarker discovery even in low or reduced malaria transmission settings.     

Levan-type FOS production using a Bacillus licheniformis endolevanase

In the present work we describe an enzymatic production method to obtain β2-6 fructose oligosaccharides (levan-type FOS) through a sequential reaction in which a bacterial endolevanase is applied to levan produced from sucrose by bacterial levansucrases. A putative gene encoding an endolevanase, designated as LevBl, was identified through a bioinformatics search, isolated from a strain of Bacillus licheniformis IBt1 from our own collection and expressed in Escherichia coli. LevB1 showed a specific activity of 1.8 U/mg protein at 35 °C in 50 mM phosphate buffer pH 6.0. A first order kinetic behavior was found when up to 150 g/L of low molecular weight levan (8.3 kDa) was used as the substrate. The product profile was determined by HPAEC-PAD and consisted of levan-type FOS with a polymerization degree between 2 and 8, with levanbiose as the major product after long reaction times. Yields of 97% of levan-type FOS were obtained when 1.0 U/mL of LevB1 reacted with 100 g/L of levan produced by the levansucrase from Bacillus subtilis. Finally, it was observed that levan-type FOS are efficiently fermented by probiotic lactic acid bacteria.     

Bio-reduction of hexachloroplatinic acid to platinum nanoparticles employing Acinetobacter calcoaceticus

Acinetobacter calcoaceticus PUCM 1011 efficiently synthesized platinum nanoparticles (PtNP) of size 2–3 nm intracellularly when challenged with hexachloroplatinic acid. Salt concentration (1 mM), temperature (30 °C), pH (7) and incubation period (72 h) influenced the efficiency of monodisperse cuboidal PtNP synthesis. Resolution of ordered lattice fringes with “d” value of 0.23 nm corresponding to (1 1 1) plane and EDAX confirmed presence of metallic platinum. AFM, TEM and HR-TEM confirmed synthesis of PtNP and its effect on cell viability. Total cell protein profile for 120 h with an interval of 24 h after PtNP synthesis revealed prominent four protein bands (97, 66, 43 and 29 kDa) when compared to control. Combinations of three proteins initiated PtNP synthesis within 4 h in range of 1–4 nm and few in picometers under HR-TEM. This is the first report of PtNP synthesis employing whole cell and total cell protein of A. calcoaceticus.     

A novel thermostable GH5_7 β-mannanase from Bacillus pumilus GBSW19 and its application in manno-oligosaccharides (MOS) production

A novel thermostable mannanase from a newly isolated Bacillus pumilus GBSW19 has been identified, expressed, purified and characterized. The enzyme shows a structure comprising a 28 amino acid signal peptide, a glycoside hydrolase family 5 (GH5) catalytic domain and no carbohydrate-binding module. The recombinant mannanase has molecular weight of 45 kDa with an optimal pH around 6.5 and is stable in the range from pH 5–11. Meanwhile, the optimal temperature is around 65 °C, and it retains 50% relative activity at 60 °C for 12 h. In addition, the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents. Bpman5 can efficiently convert locus bean gum to mainly M2, M3 and M5, and hydrolyze manno-oligosaccharides with a minimum DP of 3. Further exploration of the optimum condition using HPLC to prepare oligosaccharides from locust bean gum was obtained as 10 mg/ml locust bean gum incubated with 10 U/mg enzyme at 50 °C for 24 h. By using this enzyme, locust bean gum can be utilized to generate high value-added oligosaccharides with a DP of 2–6.     

Multi-chaperone-peptide-rich mixture from colo-carcinoma cells elicits potent anticancer immunity

Background: Chaperones play an important role in inducing anti-cancer immunity. To explore the probability of using chaperone-peptide-rich complexes extracted from colo-carcinoma cells as anti-cancer vaccine, we extracted and prepared chaperone-peptide-rich complexes from CT26 cells, which were subsequently investigated on anti-cancer efficacy. Methods: The crude extracts of the CT26 cells treated with heat and Trichosanthin were precipitated with salt and dialyzed to remove proteins below 50 kDa and above 300 kDa in molecular weight; the proteins with the molecular weights in 70 kDa, 90 kDa, 95 kDa, 110 kDa and 170 kDa were collected through gel filtration and SDS-PAGE. After confirmation, the purified proteins were used to determine their effects on lymphocyte proliferation, the activities of NK and CTL, tumor suppression and the tumor-bearing mouse survival. Results: The majority of the chaperone-peptides of anti-cancer immunity in CT26 cells, including HSP70-antigen peptide, HSP90-antigen peptide, gp96-antigen peptide, HSP-110 antigen peptide, HSP170-antigen peptide, was satisfactorily extracted that the multi-chaperone-peptide-rich mixtures were obtained. All the mixtures prepared could elicit lymphocyte proliferation, enhance the activities of CTL and NK, reinforce the tumor suppression and prolong the mouse survival. Conclusions: The multi-chaperone-peptide-rich mixtures could be prepared via dialysis and gel filtration combining with SDS-PAGE. Both the heat stress and Trichosanthin could induce and increase the mixtures, of which that treated by 42 °C heat and Trichosanthin was found to possess the strongest anti-cancer efficacy.     

Glycosylated Alpha-1-acid glycoprotein 1 as a potential lung cancer serum biomarker

Presently existing screening approaches for lung cancer are not being proving sufficient and sensitive, so a study was conducted to identify disease related biomarker proteins for diagnostic applications. A total of 100 lung cancer patients (88 non-small cell lung cancer and 12 small cell lung cancer) and 50 healthy controls were included in this study. Serum samples of patients and healthy controls were subjected to a series of proteomic approaches and as a result of two dimensional gel electrophoresis, a ∼43 kDa protein was found to be differentially expressed compared to healthy controls. Quantitative profiling of two dimensional gels by Dymension software analysis displayed 3.58 fold increased expression of ∼43 kDa protein in squamous cell carcinoma and 2.92 fold in case of adenocarcinoma. Mass spectrometric analysis resulted in identification of 8 differentially expressed proteins, out of which human Alpha-1-acid glycoprotein 1 was targeted for further validations. This candidate protein exhibited N-linked glycosylation at five amino acid residues; 33, 56, 72, 93, and 103 with significant score of 0.66, 0.78, 0.78, 0.53 and 0.66, respectively. Sandwich ELISA quantified high serum levels of Alpha-1-acid glycoprotein 1 in squamous cell carcinoma (2.93 g/l ± 1.22) and adenocarcinoma (2.39 g/l ± 1.13) when compared with healthy controls (0.83 g/l ± 0.21). One-way ANOVA analysis predicted highly significant variation of Alpha-1-acid glycoprotein 1, among all the study types (F-value 65.37, p-value 0.000). This study may prove as a non-invasive, cost effective and sensitive scheme for diagnosis of lung cancer, by passing the expensive and painful screening procedures.     

Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria

An extracellular protease from a newly isolated seawater haloalkaliphilic bacterium, haloalkaliphilic bacteria Ve₂-20-9₁ [HM047794], was purified and characterized. The enzyme is a monomer with a 37.2 kDa estimated molecular weight. It catalyzed reactions in the pH range 8–11 and performed optimally at pH 10. While maximal activity occurred at 50 °C, the temperature profile shifted from 50 to 80 °C in 1–3 M NaCl. The enzyme's thermal stability was probed using circular dichroism (CD) spectroscopy with NaCl at 50 and 70 °C. The changes in the enzyme's secondary structure were also analyzed using Fourier transform infrared spectroscopy (FTIR). The N-terminal amino acid sequence GKDGPPGLCGFFGCI exhibited low homology with other bacterial proteases, which highlights the enzyme's novelty. The enzyme was labile in anionic surfactant (1% w/v SDS) but showed stability in non-ionic surfactants (Tween 20, Tween 80 and Triton X-100 all 1% v/v), commercial detergents, and oxidizing and reducing agents. The enzyme's excellent stability in commercial detergents highlights its potential as a detergent additive.     

Gastroprotective and mucosa homeostatic activities of coconut milk and water on experimentally induced gastropathies in male wistar rats

In this biphasic study, 45 male wistar rats were divided into 9 groups. In Phase 1, Group 1 was treated with normal saline and served as the overall control, group 2 was treated with 95% Ethanol and represents the ulcer control, groups 3 and 4 received coconut water (CW; 4 ml/100 g BWt) and milk (CM; 4 ml/100 g BWt) for 4weeks while group 5 received Omeprazole (Omep; 20 mg/kg BWt) during terminal week. 95% Ethanol-induced ulceration followed the treatments in all except group 1. In the second phase, Group 1 was the overall control, group 2 served as ulcer control by receiving acetic acid only, group 3 received coconut milk, and group 4 received omep. CM and omep were administered post-ulcer induction for 3 and 6 days twice daily. Blood collection after 1hour was through cardiac puncture for haemocytometry, and gastric tissues harvested for histopathological investigations. Results showed significantly reduced ulcer score and gastric lesion index in Omep, CW and CM groups compared to ulcer control. WBC, neutrophil, lymphocyte counts in Omep, CW and CM groups were significantly reduced compared to ulcer and overall control groups. C-reactive protein was significantly reduced in CM compared to control. Neutrophil Infiltration score reduced while mucus cell density increased significantly in Omep; CM compared to control. EGFR and CD 31 assessment revealed significantly higher expressions in coconut-milk group compared to the ulcer control. We conclude that the protective effects of coconut (water and milk) is expressed by inflammation suppression, upregulation of mucus cell population and catalyses mucosa homeostasis via angiogenesis and mucosal cell proliferation following mucosa. erosion.     

Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miamiensis avidus

The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30 kDa; serotype II, 38 kDa; serotype III, 34 kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores—< 51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.     

Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes,antioxidants, and N-acetyl chitooligosaccharides

A chitinase (CHT), a chitosanase (CHS) and a protease (PRO) were purified from the culture supernatant of Serratia sp. TKU020 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHT, CHS and PRO determined by SDS-PAGE were approximately 65 kDa, 55 kDa and 55 kDa, respectively. CHT and CHS were inhibited by Mn²⁺, EDTA and PRO was inhibited by Mg²⁺, EDTA. The antioxidant activity of TKU020 culture supernatant was 78% (DPPH scavenging ability). N-Acetylglucosamine (GlcNAc) and N-acetyl chitobiose (GlcNAc)₂ were also produced from the culture supernatant by using TKU020 strain fermentation. The maximum production of GlcNAc and (GlcNAc)₂ was 1.3 mg/mL and 2.7 mg/mL, respectively, after 4 days of fermentation. With this method, we have shown that squid pen wastes can be utilized and it is effective in the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides, facilitating its potential use in industrial applications and functional foods.     

In-depth exploration of Hevea brasiliensis latex proteome and “hidden allergens” via combinatorial peptide ligand libraries

The proteome of Hevea brasiliensis latex has been explored in depth via combinatorial peptide ligand libraries. A total of 300 unique gene products have been identified in this latex, whose proteome has been largely unknown up to the present. In search for unknown allergens, control latex and eluates from the ligand libraries have been fractionated by two-dimensional mapping, blotted and confronted with sera of 18 patients. In addition to the already known and named Hevea major allergens, we have unambiguously detected several others like, for instance: heat shock protein (81 kDa), proteasome subunit (30 kDa), protease inhibitor (8 kDa), hevamine A (43 kDa) and glyceraldehyde-3-phosphate dehydrogenase (37 kDa). Gene Ontology analysis of analyzed fractions has shown that major functions are substantially unchanged after sample treatment, while novel biological functions appeared that were undetectable in the crude sample.