Pepsin immobilization: Influence of carbon support functionalization.

Among the matrices for enzyme immobilization, activated carbon has been standing out in immobilization processes due to its properties and to its characteristics that provide superficial modification by inserting new functional groups capable of binding the enzymes forming covalent bonds. In this study the effect of different modification methods of activated carbon (functionalization with genipin, metallization, metallization in the presence of chelating agent, and functionalization with glutaraldehyde) on efficiency of pepsin immobilization was evaluated. The effect of immobilization pH and the reaction medium on hydrolysis activity of bovine casein was also evaluated. The functionalization of activated carbon using iron ions allowed an immobilization capacity of 98.93 mg·g-1, with immobilization efficiency greater than 99%, and enzyme activity of 2.30 U, which was higher than the other modifications, and closer to the enzyme in the native form activity (3.32 U). In general, the carbon surface modifications were responsible for forming more stable bonds between support and enzyme, improving its proteolytic activity (from 1.84 to 2.30 U) when compared to traditional immobilization methods by adsorption and covalent binding using glutaraldehyde (from 1.04 to 1.1 U).

Oriented immobilization of antibodies onto sensing platforms - A critical review.

The immunosensor has been proven a versatile tool to detect various analytes, such as food contaminants, pathogenic bacteria, antibiotics and biomarkers related to cancer. To fabricate robust and reproducible immunosensors with high sensitivity, the covalent immobilization of immunoglobulins (IgGs) in a site-specific manner contributes to better performance. Instead of the random IgG orientations result from the direct yet non-selective immobilization techniques, this review for the first time introduces the advances of stepwise yet site-selective conjugation strategies to give better biosensing efficiency. Noncovalently adsorbing IgGs is the first but decisive step to interact specifically with the Fc fragment, then following covalent conjugate can fix this uniform and antigens-favorable orientation irreversibly. In this review, we first categorized this stepwise strategy into two parts based on the different noncovalent interactions, namely adhesive layer-mediated interaction onto homofunctional support and layer-free interaction onto heterofunctional support (which displays several different functionalities on its surface that are capable to interact with IgGs). Further, the influence of ligands characteristics (synthesis strategies, spacer requirements and matrices selection) on the heterofunctional support has also been discussed. Finally, conclusions and future perspectives for the real-world application of stepwise covalent conjugation are discussed. This review provides more insights into the fabrication of high-efficiency immunosensor, and special attention has been devoted to the well-orientation of full-length IgGs onto the sensing platform.

Papain immobilization on heterofunctional membrane bacterial cellulose as a potential strategy for the debridement of skin wounds.

We combined the chemical and physical methods of papain immobilization through the aldehyde groups available on oxidized bacterial cellulose (OxBC) to provide high proteolytic activity for future applications as bioactive dressing. Bacterial cellulose (BC) was obtained by the fermentation of Komagataeibacter hansenii in Hestrin-Schramm medium for 5 days, followed by purification and oxidation using NaIO4. Surface response methodology was used to optimize papain immobilization (2%, w/v) for 24 h. The independent variables: pH (3-7) and temperature (5 to 45 °C) were investigated. The mathematically validated optimal conditions of 45 °C and pH 7 had a statistical effect on the immobilization yield (IY) of papain in OxBC (52.9%). These ideal conditions were also used for papain immobilization in BC (unoxidized). The IY of 9.1% was lower than that of OxBC. OxBC-Papain and BC-Papain were investigated using thermal analysis, confocal microscopy, and diffusion testing. The OxBC support exhibited a more interactive chemical structure than the BC support, and was capable of immobilizing papain by covalent bonds (-C-NHR) and adsorption (ion exchange), with 93.3% recovered activity, 49.4% immobilization efficiency, and better thermal stability. Papain immobilized to OxBC by adsorption displayed 53% widespread papain activity. The results indicate the potential of prolonged bioactivity in debrided chronic wounds.

Different strategies to co-immobilize dextransucrase and dextranase onto agarose based supports: Operational stability study.

Co-immobilization is a groundbreaking technique for enzymatic catalysis, sometimes strategic, as for dextransucrase and dextranase. In this approach, dextranase hydrolytic action removes the dextran layer that covers dextransucrase reactive groups, improving the immobilization. Another advantage is the synergic effect of the two enzymes towards prebiotic oligosaccharides production. Thus, both enzymes were co-immobilized onto the heterobifunctional support Amino-Epoxy-Glyoxyl-Agarose (AMEG) and the ion exchanger support monoaminoethyl-N-ethyl-agarose (Manae) at pH 5.2 and 10, followed or not by glutaraldehyde treatment. This work is the first attempt to immobilize dextransucrase under alkaline conditions. The immobilized dextransucrase on AMEG support at pH 10 (12.78 ± 0.70 U/g) presents a similar activity of the biocatalyst produced at pH 5.2 (14.95 ± 0.82 U/g). The activity of dextranase immobilized onto Manae was 5-fold higher than the obtained onto AMEG support. However, the operational stability test showed that the biocatalyst produced on AMEG at pH 5.2 kept >60% of both enzyme activities for five batches. The glutaraldehyde treatment was not worthwhile to improve the operational stability of this biocatalyst.