Biotechnology

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Bioaccumulation of heavy metals together with medicinal properties of Pleurotus spp cultivated on agro-industrial substrates supplemented with wheat bran and maize flour.
(2023) Mkhize, Senzosenkosi Surprise.; Pooe, Ofentse Jacob.; Simelane, Mthokozisi Blessing Cedric.
Over the years, mushrooms have been used as a source of food and as medicinal therapeutics, with numerous biological properties such as; antimicrobial, anticancer, hepatoprotective, and antidiabetic. Identifying optimum mushroom growing conditions and substrates may improve mushroom productivity, quality, safety, and subsequent biological properties of P. ostreatus mushrooms. Therefore, the current study sought to investigate the effects of supplementing the mushroom-growing substrates on the biological properties of mushrooms. The study also evaluated the ability of Pleurotus ostreatus to accumulate heavy metals from locally available mushroom-growing substrates. Our observations indicated that the P. ostreatus mushroom potentially absorbed heavy metals from all the growing substrates, indicating its potential for bioremediation. The absorption of heavy metal by P. ostreatus was not influenced by the type of substrates used to cultivate the mushroom. The addition of supplements significantly improved the mushroom yield, and biological properties of P. ostreatus. The P. ostreatus mushroom extracts showed significant radical scavenging activity against DPPH and ABTS. Significant antimicrobial activities against Staphylococcus aureus and Escherichia coli were observed. Finally, the study investigated the potential of biosynthesis of zinc oxide nanoparticles (ZnONps) using Pleurotus ostreatus mushroom as the capping and reducing agent. The synthesized ZnONps were stable and proved to have antioxidant and antimicrobial activity against Pseudomonas aeruginosa, E. coli, Klebsiella pneumonia, and Enterococcus faecalis. Finally, the findings suggest that edible P. ostreatus mushrooms grown from supplemented substrates can potentially be used for green synthesis of ZnONPs, and also as an alternative source for antioxidant and antimicrobial products.
Designing T-cell epitope-based vaccine against Eimeria infection in chicken using immunoinformatics approach.
(2021) Madlala, Thabile.; Adeleke, Matthew Adekunle.; Okpeku, Moses.
Chicken coccidiosis is the most significant ubiquitous, intestinal parasitic disease known to infect chickens globally. It is recognised for incurring significant production loss to the poultry industry, caused by single or multiple Eimeria spp. infections which threaten chicken welfare and productivity. The emergence of drug resistance in parasites and pathogenicity reversion has put pressure on the poultry industry to reduce chemoprophylactic drugs and live vaccines as preventive measures against coccidiosis. Recombinant DNA vaccines have shown promising results as an alternative option, but complete protection has not been reported highlighting the need for the design of new vaccine against this disease. In this study, Eimeria antigens Immune Mapped Protein-1(IMP1) and Microneme Protein-2(MIC2) were explored using reverse vaccinology and immunoinformatics tool to predict and design potential multiepitope vaccine candidate against coccidiosis. A total of 28 and 19 antigenic T-cell epitopes were predicted and used to construct two multiepitope vaccines with 610 and 512 amino acids for IMP1 and MIC2, respectively. The produced vaccines exhibited favoured characteristics for an ideal vaccine candidate; they were antigenic (Vaxijen score of 0.5989 and 0.5103), immunogenic (scores: 10.15 and 9 419), thermostable (instability index <40 ), and non-allergic. The presence of IFN-gamma and IL-4 inducing epitopes in the constructed vaccine enables vaccine to trigger a cellular and humoral response within the host. Molecular docking of designed vaccines with toll-like receptors (TLR4 and TLR5) to determine vaccine interaction and stability was confirmed by molecular dynamics simulation root-mean square deviation (RMDS) and root-mean-square fluctuation (RMSF) analysis. The designed vaccines induced immune response through production of cytokines and antibodies associated with tertiary response. When exposed to online immune simulation C-ImmSim, both vaccines produced potent immune response through production of IgG, Tc and Th cell and memory Bcells. The constructed multiepitope vaccine in this present study is highly promising and as such further experimental work should be done to confirm its suitability against chicken coccidiosis.
Diversity of Eimeria tenella apical membrane antigen-1 from chickens in Mpumalanga province and its in silico epitope prediction as a vaccine candidate.
(2021) Tenza, Petronella Nokukhanya.; Adeleke, Matthew Adekunle.; Fatoba, Abiodun Joseph.
Coccidiosis has been a significant challenge in the poultry industry. There is a high request for the modification of a cost-effective immunizing agent to curtail this disease. Apical membrane antigen 1 (AMA1) has been reported as a protective antigen in sub-unit vaccine development against several apicomplexan parasites such as Plasmodium falciparum, Eimeria tenella and Eimeria maxima. However, knowledge of genetic diversity in this vaccine candidate is imperative. Also, to minimize the cost and time involved in producing a vaccine, computational vaccine design has received much attention through the immunoinformatics method. Therefore, screening for the potential vaccine epitopes in AMA1 that can induce cellular and humoral immune response through the immunoinformatics technique looks promising. This investigation aimed to detect the level of genetic diversity amid Eimeria tenella Apical Membrane Antigen 1(EtAMA1) in selected farms in Mpumalanga province and predict vaccine epitopes from this antigen. Four hundred fresh faecal samples were collected from 10 selected broiler chicken farms in Mpumalanga. The samples were screened for Eimeria oocyst using a compound microscope, and samples containing oocyst were further screened for E. tenella using molecular methods. AMA1 (n=103) was amplified from positive samples for E. tenella, and resulted amplicons were sent to Inqaba Biotec for sequencing and analyzed using MEGA6.06 and DnaSP programs. The results revealed low levels of genetic diversity among Mpumalanga EtAMA1 sequences which were measured by nucleotide diversity (0.0007) diversity, haplotype diversity (0.113) and haplotype number (3). Correspondingly, the haplotype network revealed 4 haplotypes, 3 of which consist of samples from Mpumalanga. Identification of immunogenic B- and T-cell epitopes from EtAMA1 sequences was further carried out and were used to construct a multiepitope vaccine (MEV) using immunoinformatics approaches. The constructed MEV is 311 amino acids long. It was constructed by linking 6 B-cell, 3 CD8+ epitopes and 6 CD4+ epitopes with appropriate adjuvant and linkers. Both adjuvant and linkers were used to increase the immunogenicity of the MEV. The designed MEV was highly antigenic and non-allergenic. The results showed a strong binding affinity of MEV with TLR4. These results suggest that the predicted vaccine could be a significant vaccine candidate against chicken coccidiosis through further experimental validation is still necessary.
Epigenetic priming and in Vitro mutagenesis in sugarcane (Saccharum Spp. Hybrids) for resistance to Fusarium Species and Aldana Saccharina (Lepidoptera: Pyralidae).
(2022) Govender, Eshani.; Watt, Maria Paula Mousaco Deoliveira.; Snyman, Sandy Jane.; Rutherford, Richard Stuart.
In the South African sugar industry, there have been substantial economic losses of R1 billion/annum caused by the indigenous pyralid borer, Eldana saccharina (Lepidoptera: Pyralidae). To develop control measures for E. saccharina in sugarcane, it is important to understand the interactions between the stalk borer and Fusarium spp. In previous studies, in vitro assays have shown that Fusarium strains may be antagonistic (e.g., F. sacchari PNG40) or beneficial (e.g., F. pseudonyamai SC17). F. pseudonyamai SC17 is a potential endophytic indicator of E. saccharina infestation, as the association between borer infestation and infection by the fungus causes Fusarium stalk rot in sugarcane. Studies have reported that the presence of endophytic fungi may have several benefits to the host plant, e.g., the production of phytohormones such as indole-3-acetic acid (IAA), which promotes plant development. The study aimed to: 1) choose a suitable resistance priming agent between hexanoic acid (Hx) and cis-jasmone (CJ); 2) determine an appropriate culture filtrate (CF) concentration for in vitro screening of calli and plantlets for tolerance to F. pseudonygamai; 3) develop a protocol (epigenetic priming and mutagenesis) to generate mutants: primed only (0.6 mM Hx), a combination of priming and mutagenic agents (100 μM 5-AzaC + 16 mM EMS-induced); 4) screen for indole-3-acetic acid production by F. pseudonygamai; and 5) characterise in vitro selected mutants for E. saccharina and F. pseudonygamai resistance by comparing the levels of resistance between unprimed, primed, and primed + mutagenic plantlets through ex vitro screening. When cis-jasmone (CJ) and hexanoic acid (Hx) were investigated for their effect on priming for pathogen resistance, 0.6 mM Hx was selected as the optimum priming agent concentration for both the callus and plantlet regeneration stages. At the highest CF concentration (100 ppm) at the embryo germination stage, the number of plantlets was greatly reduced to 58 and 98 plantlets/0.1 g of callus, for cultivars 88H0019 and N41 respectively, compared to more than 600 plantlets/0.1 g of callus in the no CF control. Unexpectedly, in the plantlet regeneration stage all the tested CF concentrations had a significant positive effect on the percentage of plantlets that re-rooted compared with the control. Both cultivars showed a 95 - 100 % rooting ability of the plantlets, which was significantly higher than the percentage of plantlets that rooted in the embryo germination media (EGM1) containing no CF (60 - 70 %) (p < 0.001). Likewise, all the concentrations of the CF had a positive effect on the root length of plantlets, with 1500 ppm CF resulting in the highest root length of 31.5 mm ± 4.3 for 88H0019 and 34.05 mm ± 3.9 for N41. Hence, F. pseudonygamai SC17 could not be used as an in vitro selection agent in a root re-growth assay. Due to the enhanced effect of F. pseudonygamai SC17 CF on root growth, the fungal isolate’s potential to produce indole acetic-3-acid (IAA) was assessed. F. pseudonygamai produced the highest IAA concentration (743.1 nM) in the presence of L-tryptophan than in the treatment without L-tryptophan (457.2 nM). This suggests that the observed enhanced root growth may be due in part to the production of auxin (IAA) in the F. pseudonygamai SC17 CF. Acclimatised in vitro plantlets (8-9 months old) were inoculated only with F. pseudonygamai SC17 or dual inoculated: firstly, with F. pseudonygamai SC17, then 1-2 2nd instar E. saccharina larvae that were placed into the leaf whorls 2 weeks later. To confirm tolerance of the putative mutants, fungal isolations were performed on the stem sections above the inoculation lesion from symptomatic and asymptomatic plants. The results revealed that the putative mutant plants that were primed with Hx only and treated with a combination of mutagens (EMS and 5-AzaC) and priming agent exhibited a significant decrease in lesion severity as compared with controls. For both treatments, a mild lesion severity rating was recorded for plants inoculated with only SC17 for cultivars N41 and 88H0019. For the plants that were dual inoculated there was a significant difference in the lesion severity ratings between the two treatments (p < 0.001). The lesion severity rating was moderate for cultivar 88H0019 (primed with Hx) and mild for cultivar N41 (primed with Hx). Plants from the combined treatment for both cultivars resulted in a mild lesion severity rating. This protocol could be valuable in generating commercially important cultivars that are tolerant and resistant to F. pseudonygamai SC17 and possibly other sugarcane pathogens. Planting resistant cultivars is recommended as an economical and the best method for controlling diseases and pests. This approach used in this study will have the least impact on the environment and increase yields without the need for expensive chemical applications and labour.
Optimization of antifungal production by Bacillus species in the presence of nanoparticle supplementation and preliminary scale-up.
(2023) Nzimande, Sikhulile Nosimo.; Gueguim Kana, Evariste Bosco.; Ramchuran, Santosh Omrajah.; Sanusi, Adeyemi Isaac.
The intensive agricultural practices used to meet global crop production demands have resulted in the rigorous use of chemical pesticides. These ultimately compromise crop production as well as the environment. In order to alleviate this, cheaper and environmentally friendly, biocontrol agents have been considered as an alternative to chemical pesticides. Biosurfactants are a promising alternative to chemical pesticides due to their higher biodegradability, lower toxicity, and environmental friendliness. Amongst the many bacterial and fungal biosurfactant producers, biosurfactants from Bacillus species show promise as biocontrol agents. These biosurfactants are known for their wide biotechnological use in agricultural, industrial, and medicinal fields. However, large scale production is still faced with challenges such as low yields and high production cost thus raising the need for modelling, optimization, catalytic and scale up investigations. Hence, a study was undertaken with the aim of enhancing biosurfactant production through process modelling and optimization with subsequent assessment of the scale up potential of the optimized process. A Response Surface Methodology (RSM) using box Behnken design was used to investigate the optimal process conditions for improved biosurfactant production from B. subtilis BS20. The investigated process parameters included glucose concentration (10 – 30 g/L), incubation temperature (25 – 45℃) and incubation time (24 – 96 h). The developed model gave a high coefficient of determination (R2 ) = 0.86, p-value of 0.0279 and F-value of 4.62 for the modelled biosurfactant production. Optimized process conditions of 11.5 g/L glucose concentration, 24 h incubation time and 41o C for incubation temperature were obtained and produced a maximal antifungal activity of 68 mm. Moreover, supplementary inclusion of seven (7) different nanoparticles as a biocatalyst in the cultivation of B. subtilis BS20 was carried out using the optimal process condition to further improve antifungal (biosurfactant) production. The inclusion of nanoparticles favored increased biomass yield, but biosurfactant with high antifungal activity was not obtained. Moreover, when it comes to commercializing new bioprocess and bioproduct developments, bio – process scale-up in the biotechnology industry is an essential stage. This study therefore evaluated the scale up of biosurfactant production based on constant power consumption, Reynold number and impeller tip speed. The stirrer speed (n), impeller diameter (di), number of impellers (N), power number (Np), broth density (⍴), working volume and geometric factor (fc) were correlated with impeller tip speed (Vtip), Reynolds number (𝑅𝑅𝑅𝑅) and power consumption rate (P/V) to obtain the most suitable criterion for biosurfactant production in a 10 L bioreactor. Implementing constant Vtip value from the 1 L scale: 93 rpm, Reynold number (Re) 5.9E - 04, Power (P) 0.32 W, Power to Volume ratio (P/VL) 160 W/m3 , circulation time (tc) 5.2 s and shear stress (γ) 15.5 S-1, at 41 °C, gave the highest antifungal activity of 65 mm zone of inhibition in the 10 L scale bioreactor. The antifungal activity obtained for constant Vtip were comparable to those obtained at 1L bioreactors (57 mm), this showed that the bioprocess dynamics for achieving high antifungal activity are available, further paving the way for feasible commercialization strategies. This study has elucidated the optimum process conditions for B. subtilis BS20 metabolism for improved biosurfactant production resulting in significant antifungal activity. Furthermore, findings showed that the inclusion of nanoparticles biocatalyst to the process enhanced biomass yields. Process scale up provided preliminary data for large scale production of biosurfactant production from B. subtilis BS20.
Screening, optimization of process parameters and scale-up of native and recombinant thermophilic Xylanases and their application in chicken feed hydrolysis.
(2023) Dhaver, Priyashini.; Govinden, Roshini.; Sithole, Bruce.; Pletschke, Brett.
Lignocellulosic biomass is a renewable raw material that has gained industrial interest due to its abundance, low cost, and potential to mitigate greenhouse gas emissions. Biomass is treated with various microbial enzymes to produce desired products under ideal conditions. Thermophilic microorganisms are excellent sources of thermostable enzymes that can tolerate extreme conditions. Optimized xylanases can be produced through genetic engineering, and recombinant DNA techniques. The biotechnological potential of xylanases from thermophilic microorganisms is discussed and the ways they are being optimized and expressed for industrial applications. Monogastric animal farming relies on grain feedstocks with non-starch polysaccharides (NSP’s) and anti-nutritive factors that cause adverse effects like increased digesta viscosity and nutrient inaccessibility which leads to reduced feed conversion, energy metabolism, and growth. Exogenous enzymes have been used to reduce viscosity and increase nutrient absorption in poultry and pigs. Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest due to their ability to modulate the composition of the gut microbiota. This study aimed to isolate and screen potential xylanolytic fungi from soil and tree bark samples in South Africa and to determine their growth conditions for maximum xylanase production. The highest xylanase activity was produced by Trichoderma harzianum. The enzyme with a molecular weight of 72 kDa retained >70% activity after 4 h at pH 6.0 and 70°C. The study also identified multiple isoforms of xylanase, which could be beneficial for animal feed and biofuel industries. The xylanase was purified from the submerged culture and displayed maximum xylanolytic activity at pH 6.0 and 65°C and the enzyme was activated by Fe2+, Mg2+, and Zn2+. Enzyme production was then optimized for maximal xylanase production strain using the Plackett-Burman Design (PBD) and Box Behnken Design (BBD), screening, and optimization design strategies, respectively. Xylanase production was enhanced to 153.80 U/ml by BBD representing a 3.99-fold increase and a 2.24- fold increase, respectively compared to the preliminary one-factor-at-a-time (OFAT) activity of 68.7 U/ml. The experimental design effectively provided conditions for the production of an acidic enzyme based on pH and incubation time. This is an exciting prospect for the application of enzymes in animal feed improvement (pH 5.0). The Geobacillus stearothermophilus glycoside hydrolase family 10 xylanase, endoxylanase XT6, is a promising candidate for industrial application. This gene was cloned and expressed and the xylanase was applied as an additive to locally produced chicken feeds. Optimization of cell lysis and expression conditions led to enhanced recombinant XT6 xylanase production. The recombinant XT6 xylanase was purified using cobalt chromatography, resulting in a 43 kDa protein with 15.69-fold purity. Cellulose, hemicelluloses, and lignin are the primary sources of fermentable sugars in lignocellulosic feedstock. Carbohydrate-active enzymes which can help release functional compounds from the carbohydrate matrix, such as phenolics are used to modify polysaccharides for industrial purposes. However, it should be noted that the primary action of carbohydrate enzymes like cellulases or xylanases is specific to the carbohydrate structure and may involve the hydrolysis of polysaccharides like xylan or other complex carbohydrate-lignin compounds,rather than being directly responsible for the release of phenolic compounds. Corn, with its antioxidant potential, is used in animal feed production, thus improving its quality for animal feed supplementation is crucial. The crude and purified T. harzianum xylanases as well as the recombinant XT6 xylanase were applied to locally produced chicken feeds in an experimental BBD design to optimize hydrolysis to monosaccharides and XOS. The Response Surface Methodology (RSM) results showed that higher (8.05 U/ml) levels of reducing sugars were produced for the crude T. harzianum xylanase and starter feed, than on the grower feed (3.11 U/ml). Treatment with the purified T. harzianum and recombinant XT6 xylanases produced lower levels of reducing sugars with similar levels for both feed types of starter feed (2.81 U/ml) and (2.98 U/ml), compared to the grower feed (2.41 U/ml) and (2.62 U/ml), respectively. Profiling of the hydrolysis products by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed that the chicken feed enzymatic hydrolysates contained a range of monosaccharides (mannose, glucose and galactose) and XOS, with xylobiose being the predominant XOS. Toxicity studies showed that the higher dilutions of the feed enzymatichydrolysates were not toxic to HEK293 cells. Therefore, the T. harzianum and recombinant XT6 xylanase are appropriate for application in the feed industry to produce XOS. These results are promising for future studies and application in the poultry feed industry as additives. The novelty of this study was the identification and characterization of a thermostable xylanase from a South African T. harzianum isolate, the application of experimental design to optimize its production and as well as that of a recombinant XT6 xylanase. The recombinant XT6 xylanase exhibited high yields in bioreactor production with activity superior to that of a commercial xylanase preparation, further emphasizing its potential for commercialization through scalingup techniques and its industrial application. Moreover, the enzymes investigated in this researchhold promise for the production of prebiotics in animal feed applications.

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