Design and characterization of alcalase-chitosan conjugates as potential biocatalysts.

In this study, alcalase (protease from Bacillus licheniformis) immobilization by adsorption, enzyme crosslinking and covalent enzyme binding to activated chitosan microbeads were examined. The biocatalysts highest activity was obtained by covalent immobilization of alcalase onto a solid support. The alcalase covalent immobilization onto different types of chitosan beads obtained by inverse emulsion technique and electrostatic extrusion was studied. Parameters examined under different conditions were beads diameter, enzyme loading, enzyme capacity yield, and biocatalyst activity. The highest activity and enzyme loading of 23.6 IU/mg protein and 340.2 mg/g, respectively, were achieved by the enzyme immobilized onto chitosan microbeads obtained by the electrostatic extrusion technique. FT-IR analysis was used to confirm formation of alcalase-chitosan conjugates. The activity of optimally produced alcalase-chitosan microbeads was then verified in the industrially feasible reaction systems of egg white and soy protein hydrolysis. The high degree of hydrolysis of 29.85 ± 0.967% after 180 min and five successive reuses obtained under real conditions (50 °C, pH 8) verified the covalently bound alcalase to chitosan beads a promising candidate for use in industrial egg white protein hydrolysis process.

Immobilization of horseradish peroxidase onto kaolin.

Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer-Emmett-Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of H2O2) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi-Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes.

Immobilization of horseradish peroxidase onto Purolite® A109 and its anthraquinone dye biodegradation and detoxification potential.

Horseradish peroxidase (HRP) is a highly specific enzyme with great potential for use in the decolorization of synthetic dyes. A comprehensive study of HRP immobilization using various techniques such as adsorption and covalent immobilization on the novel carrier Purolite® A109 with a special focus on enzymatic decolorization and toxicity of artificially colored wastewater. The immobilized preparations with an activity of 156.21 ± 1.41 U g-1 and 85.71 ± 1.62 U g-1 after the HRP adsorption and covalent immobilization, respectively, were obtained. Stability and reusability of the immobilized preparations were also evaluated. A noteworthy decolorization level (~90%) with immobilized HRP was achieved. Phytotoxicity testing using Mung bean seeds and acute toxicity assay with Artemia salina has confirmed the applicability of the obtained immobilized preparation in industrial wastewater plants for the treatment of colored wastewater.

Influence of ultrasound probe treatment time and protease type on functional and physicochemical characteristics of egg white protein hydrolysates.

The objective of this study was to discover the relationship between the ultrasound probe treatment (UPT) on egg white proteins (EWPs) before EWPs hydrolysis by different proteases, and the functional properties of the obtained hydrolysates. To fulfill this goal, the protein solubility, foaming, and emulsifying properties were studied as a function of the UPT time and then related to the surface characteristics and structural properties. The changes in the hydrolysates microstructures and macromolecular conformation, induced by the UPT, were followed using scanning electron microscope analyzis (SEM) and Fourier transforms infrared spectroscopy (FTIR). The results showed that UPT influenced (P < 0.05) the proteolysis of egg white proteins for all examined treatment times. Alcalase hydrolysates (AHs) and papain hydrolysates (PHs) were found to have a higher solubility, as a consequence of their relatively higher foaming, and emulsifying properties compared to the untreated hydrolysates. The changes in surface hydrophobicity, sulfhydryl content and surface charge of AHs and PHs indicated unfolding of EWPs affected by ultrasound. SEM analyzis showed that UPT destroyed the microstructures of AHs and PHs, while FTIR spectra indicated remarkable changes in the macromolecular conformation of AHs and PHs after UPT. This study revealed that by combining ultrasound pre-hydrolysis treatment under controlled conditions with thoughtful proteases selection, hydrolysates with improved functional properties could be produced, enhancing utilization of EWPs in food products.

Covalent Immobilization of Enzymes on Eupergit® Supports: Effect of the Immobilization Protocol.

A selection of best combination of adequate immobilization support and efficient immobilization method is still a key requirement for successful application of immobilized enzymes on an industrial level. Eupergit® supports exhibit good mechanical and chemical properties and allow establishment of satisfactory hydrodynamic regime in enzyme reactors. This is advantageous for their wide application in enzyme immobilization after finding the most favorable immobilization method. Methods for enzyme immobilization that have been previously reported as efficient considering the obtained activity of immobilized enzyme are presented: direct binding to polymers via their epoxy groups, binding to polymers via a spacer made from ethylene diamine/glutaraldehyde, and coupling the periodate-oxidized sugar moieties of the enzymes to the polymer beads. The modification of the conventionally immobilized enzyme with ethylenediamine via the carbodiimide route seems to be a powerful tool to improve its stability and catalytic activity.

Ultrasound Pretreatment as an Useful Tool to Enhance Egg White Protein Hydrolysis: Kinetics, Reaction Model, and Thermodinamics.

The impact of ultrasound waves generated by probe-type sonicator and ultrasound cleaning bath on egg white protein susceptibility to hydrolysis by alcalase compared to both thermal pretreatment and conventional enzymatic hydrolysis was quantitatively investigated. A series of hydrolytic reactions was carried out in a stirred tank reactor at different substrate concentrations, enzyme concentrations, and temperatures using untreated, and pretreated egg white proteins (EWPs). The kinetic model based on substrate inhibition and second-order enzyme deactivation successfully predicts the experimental behavior providing an effective tool for comparison and optimization. The ultrasound pretreatments appear to greatly improve the enzymatic hydrolysis of EWPs under different conditions when compare to other methods. The apparent reaction rate constants for proteolysis (k2 ) are 0.009, 0.011, 0.053, and 0.045 min-1 for untreated EWPs, and those pretreated with heat, probe-type sonicator, and ultrasound cleaning bath technologies, respectively. The ultrasound pretreatment also decreases hydrolysis activation (Ea ) and enzyme deactivation (Ed ) energy, enthalpy (ΔH), and entropy (ΔS) of activation and for the probe-type sonication this decrease is 61.7%, 61.6%, 63.6%, and 32.2%, respectively, but ultrasound has little change in Gibbs free energy value in the temperature range of 318 to 338 K. The content of sulfhydryl groups and ζ potential show a significant increase (P < 0.05) for both applied ultrasound pretreatments and the reduction of particle size distribution are achieved, providing some evidence that the ultrasound causes EWP structural changes affecting the proteolysis rate.