Directed evolution for soluble and active periplasmic expression of bovine enterokinase in Escherichia coli

Bovine enterokinase light chain (EKL) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EKL gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by kcat/Km, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased kcat and Tm values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site

Non-mulberry Silk Fibroin Biomaterial for Corneal Regeneration

Successful repair of a damaged corneal surface is a great challenge and may require the use of a scaffold that supports cell growth and differentiation. Amniotic membrane is currently used for this purpose, in spite of its limitations. A thin transparent silk fibroin film from non-mulberry Antheraea mylitta (Am) has been developed which offers to be a promising alternative. The silk scaffolds provide sufficient rigidity for easy handling, the scaffolds support the sprouting, migration, attachment and growth of epithelial cells and keratocytes from rat corneal explants; the cells form a cell sheet, preserve their phenotypes, express cytokeratin3 and vimentin respectively. The films also support growth of limbal stem cell evidenced by expression of ABCG2. The cell growth on the silk film and the amniotic membrane is comparable. The implanted film within the rabbit cornea remains transparent, stable. The clinical examination as well as histology shows absence of any inflammatory response or neovascularization. The corneal surface integrity is maintained; tear formation, intraocular pressure and electroretinography of implanted eyes show no adverse changes. The silk fibroin film from nonmulberry silk worms may be a worthy candidate for use as a corneal scaffold

Micropatterned silk-fibroin/eumelanin composite films for bioelectronic applications

There has been growing interest in the use of natural bionanomaterials and nanostructured systems for diverse biomedical applications. Such materials can confer unique functional properties as well as address concerns pertaining to sustainability in production. In this work, we propose the biofabrication of micropatterned silk fibroin/eumelanin composite thin films to be used in electroactive and bioactive applications in bioelectronics and biomedical engineering. Eumelanin is the most common form of melanin, naturally derived from the ink of cuttlefish, having antioxidant and electroactive properties. Another natural biomaterial, the protein silk fibroin, is modified with photoreactive chemical groups, which allows the formation of electroactive eumelanin thin films with different microstructures. The silk fibroin/eumelanin composites are fabricated to obtain thin films as well as electroactive microstructures using UV curing. Here, we report for the first time the preparation, characterization, and physical, electrochemical, and biological properties of these natural silk fibroin/eumelanin composite films. Higher concentrations of eumelanin incorporated into the films exhibit a higher charge storage capacity and good electroactivity even after 100 redox cycles. In addition, the microscale structure and the cellular activity of the fibroin/eumelanin films are assessed for understanding of the biological properties of the composite. The developed micropatterned fibroin/eumelanin films can be applied as natural electroactive substrates for bioapplications (e.g., bioelectronics, sensing, and theranostics) because of their biocompatible properties.The authors acknowledge the FRONTHERA project (Frontiers of technology for theranostics of cancer, metabolic and neurodegenerative diseases) n degrees NORTE-01-0145-FEDER0000232, the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement n degrees 668983. FoReCaST (Forefront Research in 3D Disease Cancer Models as in vitro Screening Technologies), and FCT grants POCI-01-0145-FEDER-031590, PD/BD/150546/2019 and PTDC/BTM-ORG/28168/2017. VKY acknowledges support from the National Science Foundation (CBET1704435)

Utilizing Reduced Graphene Oxide-Iron Nanoparticles Composite to Enhance and Accelerate the Removal of Methyl Blue Organic Dye in Wastewater

In this work, a nano-composite is used to remove dye from wastewater of different industries. For this purpose, thesynthesis of a magnetic 1:1 composite made of iron nanoparticles (NPs) using reduced graphene oxide is a novel techniqueand tested for Methyl Blue (MB) dye adsorption from aqueous solution. In this study Fe nanoparticles in reduced Graphenecomposite (FGOC) has been prepared using Graphene Oxide (GO). X-ray diffraction, FTIR spectroscopy and Ramanspectroscopy, are used to identify the structures. Many methods have been developed for MB removal in wastewater. One ofthe most popular methods is adsorption because it is simple and high-efficiency, and the adsorbent is crucial. It reached amaximum MB adsorption at pH 7. The kinetic study indicated that the adsorption of MB process was fitted well to thequasi-first-order and quasi-second-order kinetic models. The isotherm study revealed that the MB adsorption process obeyedthe Langmuir and Freundlich adsorption Isotherms models. The GO adding content and absorption conditions on the methylblue removal efficiencies were investigated. This adsorbent is easily recovered by an external magnetic field from thetreated wastewater and has high reusability

Utilizing Reduced Graphene Oxide-Iron Nanoparticles Composite to Enhance and Accelerate the Removal of Methyl Blue Organic Dye in Wastewater

914-921In this work, a nano-composite is used to remove dye from wastewater of different industries. For this purpose, the synthesis of a magnetic 1:1 composite made of iron nanoparticles (NPs) using reduced graphene oxide is a novel technique and tested for Methyl Blue (MB) dye adsorption from aqueous solution. In this study Fe nanoparticles in reduced Graphene composite (FGOC) has been prepared using Graphene Oxide (GO). X-ray diffraction, FTIR spectroscopy and Raman spectroscopy, are used to identify the structures. Many methods have been developed for MB removal in wastewater. One of the most popular methods is adsorption because it is simple and high-efficiency, and the adsorbent is crucial. It reached a maximum MB adsorption at pH 7. The kinetic study indicated that the adsorption of MB process was fitted well to the quasi-first-order and quasi-second-order kinetic models. The isotherm study revealed that the MB adsorption process obeyed the Langmuir and Freundlich adsorption Isotherms models. The GO adding content and absorption conditions on the methyl blue removal efficiencies were investigated. This adsorbent is easily recovered by an external magnetic field from the treated wastewater and has high reusability