Complete wastewater discoloration by a novel peroxidase source with promising bioxidative properties

BACKGROUND Our study aimed to characterize and prospect immobilization strategies for a novel fungal peroxidase - POD (EC 1.11.1.7) and to insert it in the context of pollutant remediation, since these compounds pose risks to human and environmental health. The enzymatic extract was obtained by submerged fermentation of the fungus Trichoderma koningiopsis in an alternative substrate, consisting of fresh microalgal biomass. The immobilization efficiency was evaluated by monitoring the residual activity (RA) and the discoloration potential (DP) of a synthetic dye solution. Concomitantly, the catalytic properties of free POD were explored, and the most promising storage strategy to maintain the enzymatic activity was studied. RESULTS The novel guaiacol peroxidase expressed specific activity of up to 7801 U mg−1 in the free form, showing stability when subjected to up to 80°C in a pH range between 4.0-8.0. Furthermore, the bioproduct immobilized on magnetic nanoparticles expressed up to 689% RA and 100% DP. An increase in the RA of the enzyme, both in free and immobilized form, was also observed after storage for up to 8 months. The synthesized magnetic nanozymes showed good reusability, maintaining 13546 U mg−1 after ten cycles and removing 94% of color in a second batch. Toxicological evaluation with Allium cepa indicated that the enzymatic process of color removal with immobilized POD was essential to eliminate genotoxic effects. CONCLUSION T. koningiopsis peroxidase production and immobilization presented in our work are promising for the enzyme market and for the wastewater treatment technologies, considering its high bioxidative potential.

Antioxidant activity of Gryllus assimilis meal hydrolysed by Aspergillus oryzae proteases

The consumption of insects is an alternative protein source of a high nutritional value, in comparison to other traditional animal and vegetable proteins, and is suitable for food and as animal feed. This work aimed to study the antioxidant activity of cricket meal (Gryllus assimilis) partially hydrolysed by proteases from a Aspergillus oryzae, which was cultivated in the cricket meal. The cricket meal showed great potential for obtaining protease from A. oryzae, with an average enzymatic activity of 112±8 U/g of dry substrate after 96 h of fermentation. The enzymatic extract applied to the cricket meal increased its antioxidant properties, increasing the reduction of the 2,2-diphenyl-1-picrylhydrazyl radical by 2 times, compared to non-hydrolysed cricket meal. These initial results are promising, demonstrating the benefits of the partial hydrolysis of cricket meal.

Application of Crude Enzymatic Extract In Mushroom Processing To Recover High Value Products

In the present study, a fungal strain was isolated from mushroom waste dump-site and was described based on the morphological and molecular characteristics. The crude enzymatic extract was prepared by fermenting pineapple peels using the newly isolated fungal strain under solid-state condition. The enzymatic saccharification conditions of mushroom were optimized using the central composite design based on the response surface methodology. The isolate had black colony color, conidial head biseriate and small conidia which are synonymous with Aspergillus niger. The phylogenetic analysis using the rDNA ITS sequencing further revealed that the isolate was identical (≥99%) to A. niger. The crude extract displayed CMCase, Fpase and xylanase activities of 20.73U/mL, 34.57U/mL and 118.03U/mL respectively. The saccharification using the crude extract at optimal conditions of pH 6.5, temperature 50oC, enzyme loading of 5% (v/v) and time of 12h achieved maximum glucose yield of 1.639 mg mL-1 which is 1.1 folds higher than the predicted value. This study demonstrated the potential use of crude enzymatic extract from the newly isolated A. niger as a viable and efficient low-cost approach to mushroom processing using enzymes.

Proteolytic Analysis of Different Tuna (Thunnus albacares) Viscera for Obtaining Protein Hydrolysates

This project aims to obtain and characterize protein hydrolysates from the solid residues (dark muscle) and effluents (cooking water) generated from the processing of yellowfin tuna (Thunnus albacares) and evaluate their potential to be applied as organic fertilizer in farming. From the studied viscera (stomach, pyloric blind, liver, pancreas, and intestine), it was found that the pyloric blind of the tuna digestive system represents an adequate source of alkaline proteases that act on the dark muscle of tuna and cooking water, at pH optimum of 10.5 and temperature of 50 °C. The results of SDS-PAGE electrophoresis and enzymatic inhibition indicate that the proteolytic activity exhibited by the enzymatic extract of the pyloric blind of tuna is mainly due to serine protease enzymes, especially trypsin type, and it showed a proteolytic activity similar to that of a commercial protease coming from Bacillus sp. It is possible to use these enzymes in processes that require pH values between 8 and 11 and mild temperatures conditions (30-50°C). The cooking water showed a low yield and high ash content, which may decrease the protein hydrolysates' nutritional quality and functional properties.

Purification, Characterization and N-terminal Protein Sequencing of the Enzyme Dextransucrase Produced by Leuconostoc mesenteroides

Background: The wide use of dextran in many different applications, makes its industrial production a challenge and, hence, to obtain a control branched structure of this enzyme research is in progress. Objectives: In the present paper, the enzyme dextransucrase, produced by cultivation of the bacterium Leuconostoc mesenteroides CMG713, was purified and characterized. Methods: The produced dextransucrase was partially purified by PEG400 obtaining a purification factor of 29.4-fold and an overall yield of 18.3% from the initial crude enzymatic extract. Results: The partially purified dextransucrase had a specific activity of 24.0 U/mg and presented a molecular weight of about 200 kDa. In addition, the produced dextransucrase was stable at 30ºC and pH 5.5 for 3 days and led to a highly soluble dextran with wide potential industrial applications. The current study has successfully partial purification, characterization and conformation of dextransucrase produced by fermentation of the bacterium Leuconostoc mesenteroides CMG713.

Complete wastewater discoloration by a novel peroxidase source with promising bioxidative properties

The occurrence of micropollutants in aqueous matrices has become a global concern and a challenge for wastewater treatment plants. Monitoring their toxicity has shown that these compounds, even at low concentrations, pose risks to human and environmental health. Therefore, our study aimed to prospect immobilization strategies for non-commercial oxidoreductase enzymes and insert them in the context of pollutant remediation. The enzymatic extract was obtained by submerged fermentation of the fungus Trichoderma koningiopsis in an alternative substrate, consisting of fresh microalgal biomass from the phycoremediation process. The immobilization efficiency of peroxidase (POD) was evaluated by monitoring the residual activity (RA) and the discoloration potential (DP) of a synthetic dye solution. Concomitantly, the catalytic properties of free POD were explored, and the most promising storage strategy to maintain the enzymatic activity was studied. Guaiacol peroxidase from T. koningiopsis expressed specific activity of up to 7801.1 U mg− 1 in the free form, showing stability when subjected to up 80°C in a pH range between 4.0–8.0. Furthermore, the bioproduct immobilized on magnetic nanoparticles expressed up to 688% RA and 100% DP. An increase in the RA of the enzyme, both in free and immobilized form, was also observe after storage for up to 8 months. The synthesized magnetic nanozymes showed good reusability, maintaining 13546.4 U mg− 1 after ten cycles and removing 93% of color in a second batch. Toxicological evaluation with Allium cepa indicated that the enzymatic process of color removal with immobilized POD, despite maintaining unwanted cytotoxic effects, was essential to eliminate genotoxic effects. In this sense, the immobilization processes of T. koningiopsis peroxidase presented in our work are promising for the enzyme market and for the wastewater treatment sector.

Production and biochemical characterization of xylanases synthesized by the thermophilic fungus Rasamsonia emersonii S10 by solid-state cultivation

The xylanolytic enzyme complex hydrolyzes xylan, and these enzymes have various industrial applications. The goal of this work was to characterize the endoxylanases produced by the thermophilic fungus Rasamsonia emersonii in solid-state cultivation. Tests were carried out to evaluate the effects of pH, temperature, glycerol and phenolic compounds on enzyme activity. Thermal denaturation of one isolated enzyme was evaluated. The crude extract from R. emersonii was applied to breakdown pretreated sugarcane bagasse, by quantifying the release of xylose and glucose. The optimum pH value for the crude enzymatic extract was 5.5, and 80 �C was the optimum temperature. Regarding the stability of the crude extract, the highest values occurred between the pH ranges from 4 to 5.5. Several phenolic compounds were tested, showing an increase in enzymatic activity on the crude extract, except for tannic acid. Zymography displayed four corresponding endoxylanase bands, which were isolated by extraction from a polyacrylamide gel. The thermodynamic parameters of isolated Xylanase C were evaluated, showing a half-life greater than 6 h at 80 �C (optimum temperature), in addition to high melting temperature (93.3 �C) and structural resistance to thermal denaturation. Pretreated sugarcane bagasse breakdown by the crude enzymatic extract from R. emersonii has good hemicellulose conversion to xylose.