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In this study, trypsin from the porcine pancreas was immobilized on a heterofunctional support prepared by activating chitosan (Chit) hydrogel with glutaraldehyde (GA), then functionalizing it with glycine (Chit–GA–Gly). The catalytic performance of the immobilized trypsin in the hydrolysis reactions was compared with the catalytic performance of the immobilized enzyme on glutaraldehyde-activated chitosan (Chit–GA) and chitosan hydrogel (Chit). The maximum concentration of immobilized protein on Chit–GA–Gly was approximately 16 mg·g−1 at pH 9.0 (5 mmol·L−1 buffer sodium carbonate) at 25 °C from an offered protein loading of 20 mg·g−1. This biocatalyst exhibited maximum specific activity (SA) of 33.1 ± 0.2 nmol·min−1·mg−1 for benzoyl-DL-arginine-p-nitroanilide (BAPNA) hydrolysis, twice as high as the enzyme immobilized on the classic Chit–GA support (SA values ranging between 6.7 ± 0.1 nmol·min−1·mg−1 and 8.1 ± 0.1 nmol·min−1·mg−1). The Elovich kinetic model was used to describe the adsorption process using low (3 mg·g−1) and high (20 mg·g−1) initial protein loadings. The optimum temperature for BAPNA hydrolysis catalyzed by the immobilized trypsin (60 °C) was 10 °C higher than that of its soluble form. Additionally, the immobilized enzyme was 16 to 20 times more stable than its soluble form at 50–55 °C. Thermodynamic studies were conducted to elucidate the kinetics of the thermal inactivation process of soluble and immobilized forms. Complete hydrolysis of bovine serum albumin (BSA) at 37 °C was achieved after 2 h using a soluble enzyme, while for its immobilized form, the hydrolysis yield was 47%. Reuse tests revealed that this biocatalyst retained 37% of its original activity after 10 successive hydrolysis batches. Based on these results, this support could be used as an interesting alternative for producing heterogeneous biocatalysts with high catalytic activity and thermal stability when producing protein hydrolysates. [ABSTRACT FROM AUTHOR]

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). [Display omitted] • Activated carbon structure has been modified through the insertion of reactive groups. • Pepsin immobilization on heterofunctional supports. • New modifications to activated carbon improve its immobilization efficiency. • Genipin can be used as a natural substitute for glutaraldehyde. • Modifications with metal ions presented the highest activity for the derivatives. [ABSTRACT FROM AUTHOR]

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 NaIO 4. 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. Unlabelled Image • The optimum papain immobilization condition at 2% (w/v) was at 45 °C and pH 7. • Yield immobilization, activity recovered and immobilized were assessed by SRM. • OxBC wet membrane was a heterofunctional support, immobilizing papain chemically and physically. • The association of immobilization mechanisms was confirmed by confocal microscopy. • OxBC membrane immobilized with papain is a strategy to obtain a bioactive dressing. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

There has been increasing interest in the development of solvent systems for efficient and less energy intensive CO 2 capture and desorption. In this work, we have developed a hybrid solvent system (30 wt.% MEA + 7.5 wt% PZ + 15wt% K 2 CO 3 ) containing a thermo-stable carbonic anhydrase (CA) and Zn complex (Zn-Im) together on a hetero-functional support (HFS CaCO 3 /H 3 BO 3 /SiO 2 ) (termed as CA/Zn-Im: HFS). The hybrid solvent containing CA/Zn-Im: HFS exhibit excellent gravimetric CO 2 uptake (21.65 wt%) over the neat hybrid solvent (13.75 wt%). Gravimetric and pressure drop experiments confirmed that HFS and Zn-Im plays key role in improving the CO 2 capture activities of CA in hybrid solvent. Low temperature desorption (90 °C) suggested that the hybrid solvent containing CA/Zn-Im: HFS has 1.57 times higher desorption kinetics compared to the neat hybrid solvent. Moreover, CA was highly stable in the hybrid solvent medium and could be reused for 15 consecutive cycles with retention of 90% of its initial activity. The development in various key aspects of CO 2 capture suggested that the hybrid solvent with CA/Zn-Im: HFS is an efficient solvent system to improve conventional amine based scrubbing technology. [ABSTRACT FROM AUTHOR]

Octyl-agarose is a support that permits the one step immobilization, stabilization and purification of lipases. However, the enzyme may be released from the support under drastic conditions. This paper describes a new heterofunctional support, octyl agarose beads activated with divinyl sulfone, that has proved to be useful to produce very stable and active biocatalysts of lipases from Candida rugosa (CRL), Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL), able to work under any reaction conditions without risking enzyme desorption. The three enzymes failed in immobilization on glyoxyl-octyl supports for different reasons. The immobilization at pH 5 permitted to keep the good properties of octyl agarose. Further incubation at pH 8 permitted to establish at least one covalent enzyme-support bond per enzyme molecule (preventing the risk of enzyme desorption), avoiding the inactivation produced at pH 10, and the final result is that all three new biocatalysts are more active than the octyl-glyoxyl counterparts and much more stable (e.g., 20 using CRL). The end of the enzyme-support reaction was achieved via blocking the vinylsulfone groups with different nucleophiles (cationic, anionic, hydrophobic, etc). This not only determined the final enzyme stability, but also the activity, selectivity and even specificity of the different immobilized preparations. [ABSTRACT FROM AUTHOR]

Peroxidases from Pleurotus eryngii have been investigated for their ability to degrade recalcitrant, phenolic pollutants. The use of crude enzymatic extracts can reduce the high costs associated with enzyme purification, and enzyme immobilization can enhance enzyme stability and recovery. The present study tests the effectiveness of various conditions for crude enzyme stabilization in polyethylene glycol and glycine solutions, and immobilization on monofunctional and heterofunctional agarose solid supports. Glycine at 0.5 M at 4 °C and pH 4 was most effective stabilization agent for the crude enzymatic extracts, and enzyme immobilization efficiency was greatest for heterofunctional supports. MANA-glyoxyl heterofunctional supports were demonstrated to have the greatest enhancement of decolorization (1.3-fold) and velocity of substrate consumption (fivefold). Therefore, the application of crude enzymatic extracts to industrial processes, such as dye decolorization, represents a cost-effective alternative to purified enzymes. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Stepwise immobilization promotes uniform orientation of bound IgGs onto the immunosensor. • Heterofunctional matrices are more cost-effective immuno-platform with great orientation controllability. • Exploitation of site-oriented full-length IgGs provide more option for immunosensing. [ABSTRACT FROM AUTHOR]