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Abstract The endoxylanase XT6 produced by Geobacillus stearothermophilus is a desirable candidate for industrial applications. In this study, the gene encoding XT6 was cloned using the pET-28a expression vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant XT6 production was improved by optimizing cell lysis (sonication, chemical, and enzymatic lysis) and expression conditions. Sonication in a 0.05 M sodium phosphate (pH 6.0) buffer resulted in the highest xylanase activity (16.48 U/ml). Screening and optimization of induction conditions using the Plackett–Burman Design and Box-Behnken Design (BBD) approaches revealed that cell density pre-induction (OD600 nm), post-induction incubation time, and IPTG concentration significantly (p

Abstract Cellulase-producing microorganisms produce low titres of β-glucosidases with low tolerance to glucose. This study aimed to improve production, purify, and characterize a β-glucosidase from a newly isolated Neofusicoccum parvum strain F7. β-Glucosidase production was significantly enhanced by a sequential statistical modelling approach from 1.5-fold in Plackett–Burman design to 2.5 U/ml in the Box–Behnken design compared to the preliminary one variable at a time experiments (1.6 U/ml). The optimal conditions for enzyme production by BBD were 12 days of fermentation at 20 °C, 175 rpm, 0.5% glycerol and 1.5% casein in pH 6.0 buffer. Three β-glucosidase isoforms referred to as Bgl1, Bgl2, Bgl3 were purified and characterized from the optimized crude extract displaying IC50 values of 2.6, 22.6 and 319.5 mM for glucose, respectively. Bgl3 with a molecular mass of approximately 65 kDa demonstrated the highest tolerance to glucose among the isoforms. The optimum activity and stability for Bgl3 was observed at pH 4.0 in 50 mM sodium acetate buffer with 80% β-glucosidase residual activity retained for three hours. This isoform also retained 60% residual activity at 65 °C for one hour which was then reduced to 40% which remained stable for another 90 min. The β-glucosidase activity of Bgl3 was not enhanced after the addition of metal ions in assay buffers. The K m and v max for 4-nitrophenyl-β-d-glucopyranoside were 1.18 mM and 28.08 µmol/min, respectively indicating high affinity for the substrate. The ability to withstand the presence of glucose in conjunction with its thermophilic nature indicates promise for this enzyme in industrial application.

Abstract Xylanases are hydrolytic enzymes with a wide range of applications in several industries such as biofuels, paper and pulp, food, and feed. The objective of this study was to optimize the culture conditions and medium components for maximal xylanase production from a newly isolated Trichoderma harzianum strain using the Plackett–Burman Design (PBD) and Box Behnken Design (BBD) experimental strategies. Xylanase production was enhanced 4.16-fold to 153.80 U/ml by BBD compared to a preliminary one-factor-at-a-time (OFAT) activity of 37.01 U/ml and 2.24-fold compared to the PBD (68.70 U/ml). The optimal conditions for xylanase production were: 6 days of fermentation, incubation temperature of 70 °C, pH 5.0, agitation of 160 rpm, and 1.2% wheat bran and ammonium sulphate. The experimental design effectively provided conditions for the production of an acidic-thermostable enzyme with exciting potential for application in animal feed improvement. The acidic-thermostable xylanase was purified from the submerged culture and SDS-PAGE analysis revealed a molecular weight of 72 kDa. This protein had maximum xylanolytic activity at pH 6.0 and 65 °C and was stable for 4 h retaining > 70% activity and exhibited substrate specificity for beechwood xylan with a K m of 5.56 mg/ml and V max of 1052.63 µmol/min/mg. Enzyme activity was enhanced by Fe2+, Mg2+, and Zn2+. There was an absence of strong inhibitors of xylanase activity. Overall, these characteristics indicate the potential for at least two industrial applications.

Abstract Dendritic cells (DCs) are the antigen-presenting cells that initiate and direct adaptive immune responses, and thus are critically important in vaccine design. Although DC-targeting vaccines have attracted attention, relevant studies on chicken are rare. A high diversity T7 phage display nanobody library was constructed for bio-panning of intact chicken bone marrow DCs to find DC-specific binding nanobodies. After three rounds of screening, 46 unique sequence phage clones were identified from 125 randomly selected phage clones. Several DC-binding phage clones were selected using the specificity assay. Phage-54, -74, -16 and -121 bound not only with chicken DCs, but also with duck and goose DCs. In vitro, confocal microscopy observation demonstrated that phage-54 and phage-74 efficiently adsorbed onto DCs within 15 min compared to T7-wt. The pull-down assay, however, did not detect any of the previously reported proteins for chicken DCs that could have interacted with the nanobodies displayed on phage-54 and phage-74. Nonetheless, Specified pathogen-free chickens immunized with phage-54 and phage-74 displayed higher levels of anti-p10 antibody than the T7-wt, indicating enhanced antibody production by nanobody mediated-DC targeting. Therefore, this study identified two avian (chicken, duck and goose) DC-specific binding nanobodies, which may be used for the development of DC-targeting vaccines.

Abstract Fungal endophytes have the capacity to biosynthesize secondary metabolites that are produced by their host plants. In this study, a dilactone terpenoid of C16 architecture was isolated from the fungal endophytes of Kigelia africana, in our attempt to identify anti-Pseudomonas aeruginosa metabolites. Thirty-eight fungal isolates were cultured for biomolecule production over a period of thirty days. Extracts from three (ZF 34, ZF 52 and ZF 91) of the fungi showed good anti-P. aeruginosa activity, with ZF 52 presenting the best MIC of 19.53 µg/mL and was accordingly subjected to chromatographic separation. Based on nuclear magnetic resonance (NMR) spectroscopy, high resolution mass spectrometry and single crystal X-ray diffraction (XRD) analyses, the isolated compound was identified as a C16-terpene dilactone, with a structure consistent with that of the known diterpene, CJ-14445. The isolated dilactone showed anti-P. aeruginosa activity with MIC of 0.61 µg/mL, signifying the antibacterial potential of the biomolecule. The bioactive fungal isolate (ZF 52) was identified as Neofusicoccum luteum based on genomic DNA sequencing. This is the first report of the endophyte N. luteum from K. africana and the first reported occurrence of CJ-14445 in the fungus.