Biotechnology

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Production and characterization of DNA ligases isolated from Kogelberg Biosphere metagenomics library.
(2021) Zuma, Lindiwe Khumbuzile.; Pooe, Ofentse Jacob.
Microbial enzymes have been described as an underutilized source of novel enzymes with potential economic advantages. Recently discovered enzymes such as DNA ligase from metagenomic studies, have been shown to achieve great potential in transforming the reagent market specifically in the African continent. Reagent proteins are frequently utilized in the research field widely and are prone to protein degradation and shelf-life reduction. Hence, this study sought to improve biological activity, shelf life and stability of the two DNA ligases identified from Kogelberg Biosphere metagenomics library. Two recombinant DNA ligases expression studies were done using E.coli BL21 and purification studies were done subsequently using affinity chromatography. Both recombinant DNA ligases (Ligsv081 & LigpET30) were successfully expressed and purified as homogenous proteins. In this study two approaches were used to enhance the biological DNA ligases, the first approach used was PEGylation. The purified proteins were conjugated to PEG using site-specific PEGylation and non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze the secondary structure of the PEG conjugated DNA ligases. Thermal stability assays were then employed to assess protein stability in the conjugation with PEG. Site-specific PEGylation enhanced ligase activity and reduced the formation of protein aggregates. The second approach involved DNA ligase co-expression in the presence of PfHsp70 or chimeric transcription factor, respectively. Protein co-expression and co-purification assays were conducted. The co-expression and co-purification assays of both proteins with chimeric transcription factor (cTF) were successful, followed by co-expression and co-purification of LigpET30-PfHsp70. Ligation assays were conducted to assess bioactivity of proteins. All DNA ligase complexes were functional and their melting point was increased. Taken together, site-specific PEGylation and protein co-expression with PfHsp70 potentially extended the shelf-life and stability of the proteins. PEGylation strategies and co-expression strategies can potentially be used to enhance reagents in diagnostic and therapeutic tools in molecular biology field.
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.
The isolation and characterisation of proteases from Euphorbia tirucalli, E. triangularis, and Carica papaya latex.
(2023) Boodhoo, Akira.; Coetzer, Theresa Helen Taillefer.
Plant proteases play an important role in the food and industrial sectors from meat tenderisers to milk clotting agents and even anti-parasitic agents. Proteases have been identified in plant latex, but many proteases have not been isolated and characterised. This research aimed to isolate and characterise proteases from the latex of Euphorbia tirucalli, E. triangularis, and Carica papaya. Three-phase partitioning (TPP) of E. tirucalli plant latex revealed the presence of two active proteases on gelatin-containing zymograms, that were subsequently separated by size exclusion chromatography. These proteases were classified as a 75 kDa serine protease (E. tiru SP), inhibited by PSMF, and SBTI and a 37 kDa cysteine protease (E. tiruCP), inhibited by E-64. Analysis of E. triangularis latex by TPP and p-aminobenzamidine affinity chromatography showed the presence of three serine proteases inhibited by PMSF and SBTI, E. laris SP1 (>97.4 kDa), E. laris SP2 (68 kDa) and E. laris SP3 (38 kDa). These proteases showed stability in constant ionic strength buffers from pH 4 to 9, both with and without the presence of the reducing agent cysteine. Papain was isolated from the latex of Carica papaya for use as a control protease in zymograms on account of the anomalous behaviour of commercial papain preparations on these gels. Papain was isolated by two methods: ammonium sulfate precipitation followed by TPP and CM-cellulose cation exchange chromatography. The isolated papain was detected by rabbit anti-papain antibodies in a dot blot and western blot and showed inhibition by E-64. The latex from all three plant species showed milk clotting activity with higher activity in the presence of calcium chloride. These findings suggest that isolated plant latex proteases from the Euphorbia species can be used in the food industry as milk clotting agents. Further characterisation of these isolated proteases should identify further uses in biotechnology.

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