Enzymes play a powerful role as catalysts with high specificity and activity under mild environmental conditions. Significant hurdles, such as reduced solubility, reduced shelf-life, aggregate formation, and toxicity, are still ongoing struggles that scientists come across when purifying recombinant proteins. Over the past three decades, PEGylation techniques have been utilized to significantly overcome low solubility; increased protein stability, shelf-life, and bioactivity; and prevented protein aggregate formation. This review seeks to highlight the impact of PEG-based formulations that are significantly utilized to obtain favourable protein physiochemical properties. The authors further discuss other techniques that can be employed such as coexpression studies and nanotechnology-based skills to obtaining favourable protein physiochemical properties.
The sugar cane tops were obtained from farms around the northern part of KwaZulu-Natal province, South Africa (UVS farm at longitudes 28°42'24.9"S 31°54'09.0"E). The bagasse substrate, WB supplement and test mushrooms (P. ostreatus) were all obtained at the South African Department of Agriculture and Rural Development. The mushroom strain (P. ostreatus) was pre-cultured on potato dextrose agar and incubated in the dark environment at 25 ºC and then maintained as working spawn cultures at 4 ºC.The modified method outlined by Crisan and Sands (1978) was utilized for spawn preparation, whereby 1 kg of sorghum grains were soaked overnight in 1.5 l ABSTRACT Background: Pleurotus ostreatus, is one of the most cultivated mushrooms with great economic and medicinal value that can be easily grown on various bio-waste substrates. However, biosafety evaluations on these mushrooms are rarely conducted. Thus, we sought to evaluate the concentration or presence of Heavy metals in P. ostreatus mushrooms cultivated on agro-bio-waste products. Furthermore, the effect of adding agro wastes on wheat bran (WB) cultivated mushrooms was evaluated. Methods: Mushrooms grown in sugar cane tops and bagasse were supplemented with varying levels of WB. Atomic absorption spectrophotometer was applied to evaluate the concentration of heavy metals in the substrates and within mushrooms. Furthermore, DPPH free radical scavenging activity was used to determine antioxidant activity of mushroom extracts. Results: The transfer factor analysis (TF) showed that mushrooms have an affinity to absorb Zn, Cd, Cu and Cr from all tested substrates during cultivation (TF>1). The addition of WB supplement into substrates resulted into significant increase in mushroom yield. However, the increased addition of WB, inversely affected the DPPH scavenging activity of the P.ostreatus methanolic extracts. Conclusion: The bioabsorption of heavy metals by P. ostreatus is depended on the metal type. Based on these findings, mushrooms grown on these agro-waste appear to be safe and potent scavenging ability against free radicals.
Background: Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are routinely used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5’-phosphorylated end to a 3’-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source. Objective: The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases Method: We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins were conjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected various PEGylation conditions. Results: In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-fold for plasmid ligations and reduced the formation of protein aggregates. Conclusion: Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.
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