Two-way reaction of versatile peroxidase with artificial lignin enhances low-molecular weight fractions.

In recent years, versatile peroxidase (VP) has emerged as a promising enzyme for biotechnological applications, as it can oxidize lignin without the external mediators. To gain insights into the breakdown process of artificial lignin by VP, reaction between the two was studied. Degradation products were fractionated using ultrafiltration and analyzed by RP- high performance liquid chromatography with mass detection (HPLC-MS) chromatography. Four fractions were obtained based on their molecular sizes: >10, 3-10, 1-3, and <1 kDa. Interestingly, while VP did not significantly alter the yields of these fractions, the chromatograms revealed the presence of oligomers with different molecular weights (MWs) resulting from the enzymatic activity. The VP exhibits a dual role in its enzymatic activity: both degrading and synthesizing these oligomers. This was confirmed by principal component analysis (PCA). The positive correlations were found between certain oligomers (D1 and D2, D5 and D6, as well as between D7, D10, T2, and T4), suggesting their simultaneous degradation. On the other hand, a negative correlation was found between the monomer and some oligomers (D7, D10, T2, and T4), indicating the decomposition of these oligomers into monomers. These findings shed light on the intricate interplay between VP and artificial lignin, offering valuable insights for potential applications in lignin valorization.

Xylanase Production by Solid-State Fermentation for the Extraction of Xylooligosaccharides from Soybean Hulls§.

Research background: The development of a novel process for the production of xylooligosaccharides (XOS) based on the 4R concept is made possible by the integration of numerous techniques, especially enzymatic modification together with the physical pretreatment of renewable materials. This study aims to integrate the use of agricultural wastes for the production of xylanase by a new strain of Penicillium sp. and value-added products, XOS. Experimental approach: For the production of xylanase, a solid-state fermentation was performed using wheat bran as substrate. To obtain the most active crude extract of xylanase, the time frame of cultivation was first adjusted. Then, the downstream process for xylanase purification was developed by combining different membrane separation units with size exclusion chromatography. Further characterisation included determination of the optimal pH and temperature, determination of the molecular mass of the purified xylanase and analysis of kinetic parameters. Subsequently, the hydrolytic ability of the partially purified xylanase in the hydrolysis of alkali-extracted hemicellulose from soybean hulls was investigated. Results and conclusions: Our results show that Penicillium rubens produced extracellular xylanase at a yield of 21 U/g during solid-state fermentation. Using two ultrafiltration membranes of 10 and 3 kDa in combination with size exclusion chromatography, a yield of 49 % and 13-fold purification of xylanase was achieved. The purified xylanase (35 kDa) cleaved linear bonds ß-(1→4) in beechwood xylan at a maximum rate of 0.64 µmol/(min·mg) and a Michaelis constant of 44 mg/mL. At pH=6 and 45 °C, the purified xylanase showed its maximum activity. The xylanase produced showed a high ability to hydrolyse the hemicellulose fraction isolated from soybean hulls, as confirmed by thin-layer chromatography. In the hydrothermally pretreated hemicellulose hydrolysate, the content of XOS with different degrees of polymerisation was detected, while in the non-pretreated hemicellulose hydrolysate, the content of xylotriose and glucose was confirmed. Novelty and scientific contribution: Future research focusing on the creation of new enzymatic pathways for use in processes to convert renewable materials into value-added products can draw on our findings.

Impact of Different Enzymatic Processes on Antioxidant, Nutritional and Functional Properties of Soy Protein Hydrolysates Incorporated into Novel Cookies.

Soy protein concentrate (SPC) was hydrolyzed using several commercial food-grade proteases (Alcalase, Neutrase, papain, Everlase, Umamizyme, Flavourzyme) and their combination to obtain promising ingredients in the manufacture of functional bakery products. In all cases, the hydrolysis caused nutritional, sensory, and rheological changes in SPC, as well as protein structural changes like increased surface hydrophobicity and content of exposed SH groups with the magnitude of these changes depending on enzyme specificity. The hydrolysis with the combination of Neutrase and Flavourzyme (NeuFlav) increased essential amino acid content by 9.8% and that of Lys by 32.6% compared to SPC. This hydrolysate showed also significant antioxidant activities including ABTS and superoxide anion scavenging activity and metal-chelating ability. The addition of all hydrolysates in wheat flour decreased water adsorption and increased development time to some extent due to gluten network weakening, but also decreased the rate of starch retrogradation, contributing to the increase of the shelf-life of bakery products. The NeuFlav tasted less bitter than other hydrolysates, while E-nose provided a discrimination index of 93 between control and hydrolysates. It appeared that the addition of the NeuFlav hydrolysate in a cookie formulation improved protein content and nutritional quality and directed to its higher general consumer acceptability than cookies formulated with only wheat flour.

Synergistic Effect of Enzyme Hydrolysis and Microwave Reactor Pretreatment as an Efficient Procedure for Gluten Content Reduction.

In this study, we assessed the effects of microwave irradiation of wheat gluten proteins as a pretreatment performed in a microwave reactor that could accurately control process parameters as a function of power and temperature, as well as comparing it with conventional heat treatment. The aim was to identify suitable combinations of partial enzymatic hydrolysis and microwave pretreatment parameters to produce gluten hydrolysates with reduced allergenicity and conserved techno-functional features for food application. FTIR analysis, and total and reactive SH group contents confirmed that the microwave-controlled heating can significantly change the secondary structure and conformation of gluten protein. The microwave treatment had the largest effect at 200 W and 100 °C, at which the content of gluten has been reduced by about 2.5-fold. The microwave pretreatment also accelerated the enzymatic hydrolysis of gluten, changing the kinetic profile. The apparent hydrolysis rate constants (k2) were 1.00, 3.68, 3.48, 4.64 and 4.17 min-1 for untreated gluten, and those pretreated with microwave power of 200, 400, 600 and 800 W, respectively. Compared to the heat treatment, it appeared that microwave specific non-thermal effects had a significant influence on the gluten structure and allergenicity and, in combination with the enzymatic hydrolysis, ultimately yielded protein hydrolysates with enhanced antioxidant and functional properties.

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.

Enzyme production by solid-state fermentation on soybean meal: A comparative study of conventional and ultrasound-assisted extraction methods.

Protease, cellulase, and α-amylase producing Bacillus subtilis strain was cultivated by solid-state fermentation technique using soybean meal as a substrate. The aim of the present study was to establish a highly efficient enzymes' extraction method as a first stage in downstream processing. The conventional extraction procedure was optimized by determining pH, stirring rate, solid/liquid ratio, and time of extraction on enzymes' recoveries from fermented soybean meal. Yields of leached enzymes were compared to the amounts of enzymes that are achieved with ultrasound-assisted extraction (UAE). UAE was established to be superior method for obtaining higher yields of proteases (up to 330 IU) and α-amylases (825 IU), under significantly shorter extraction time and gaining more concentrated product. However, the obtained model predicts that conventional process led to a product with a higher cellulolytic activity (≥7.5 IU).

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.

Production of enzymes by a newly isolated Bacillus sp. TMF-1 in solid state fermentation on agricultural by-products: The evaluation of substrate pretreatment methods.

Study on potential of different agro-industrial waste residues for supporting the growth of newly isolated Bacillus sp. TMF-1 strain under solid-state fermentation (SSF) was conducted aiming to produce several industrially valuable enzymes. Since the feasibility of the initial study was confirmed, further objectives included evaluation of several pretreatments of the studied agricultural by-products (soybean meal, sunflower meal, maize bran, maize pericarp, olive oil cake and wheat bran) on the microbial productivity as means of enhancing the yields of produced proteases, α-amylases, cellulases and pectinases. Among acid/alkaline treatment, ultrasound and microwave assisted methods, chemical treatments superiorly affected most of the studied substrates. Highest yields of produced proteases (50.5IUg-1) and α-amylases (50.75IUg-1) were achieved on alkaline treated corn pericarp. Alkaline treatment also promoted the secretion of cellulases on maize bran (1.19IUg-1). Highest yield of pectinases was obtained on untreated soybean meal (64.90IUg-1).

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.

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.

Decolorization of anthraquinonic dyes from textile effluent using horseradish peroxidase: optimization and kinetic study.

Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP) in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C) and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C). The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes.