Evaluation of structural quality of the enzyme Azoreductase (2HPV).
Azo dyes are frequently used at an industrial level to restore the color of raw materials once it has faded away, make an original color more vibrant or with the purpose of giving a material a different color that is considered more attractive. These processes however, have a negative impact on the environment, evidenced in colored wastewater that is subsequently dumped into water bodies, causing disruptions in the natural balance of ecosystems and deteriorating human health. Traditional strategies for the treatment of effluents contaminated with azo dyes are limited to physical and chemical processes that have a high energy and economic cost. For these reasons, current challenges are focused on the use of microorganisms capable of transforming dyes into less toxic products. This chapter will present a description of the main characteristics of azo dyes and the different methods used for their treatment, with special emphasis on the benefits associated with biological treatment. Likewise, it will provide relevant information about Enterococcus and show its potential to degrade azo dyes.
- azo dyes
Annually, more than a million tons of synthetic dyes are produced around the world for use in the leather, textile, pharmaceutical, food, cosmetic, paint, plastic and paper industries , of which, at least 60% represent azo dyes . In addition to being recalcitrant towards various degradation processes , azo dyes produce dangerous chemical substances such as aromatic amines, known for their toxic, allergenic, carcinogenic and mutagenic effect on living organisms .
The impact of azo dyes on the environment is related to the enormous amounts of hazardous waste associated with industrial processes, which in most cases, is then released directly to water bodies without proper treatment. A further aggravating factor is that due to the inability of at least 35% of azo dyes to adhere to substrates, heavy metals have been incorporated during the dyeing process, and these act as mordants, favoring the fixation of the dye .
Colorants associated with metals such as copper, cobalt and especially chromium, are difficult to degrade and represent an important source of environmental contamination due to their increased presence in organic load. They generate adverse and irreversible eco-toxicological effects, bioaccumulation phenomena and biomagnification in flora and aquatic fauna and alteration of biogeochemical cycles .
This powerful metal-dye complex has carcinogenic and mutagenic properties for humans exposed to effluents contaminated with dyes. It can lead to skin cancer due to photosensitization, photodynamic damage, allergic contact dermatitis, renal, reproductive, hepatic, cerebral dysfunction, irritation of the respiratory tract and asthma .
Traditionally, physicochemical methods have been used to treat effluents contaminated with azo dyes, but their high economic and energy cost and the environmental effects associated with their use have changed the focus, in recent years, on the use of microorganisms. These are successful biological alternatives due to their survival properties, adaptability, enzymatic activity and chemical structure. Additionally, hybrid technologies have been developed, which are able to take the best of each technology and surpass the limitations of current conventional treatments .
The environmental importance of
While international organizations consider
As a contribution to this discussion and element for further research, this article presents a review of the potential of
1.1 The potential of bacteria for the degradation of azo dyes
For the degradation of azo dyes, bacteria have an efficient enzymatic system that allows them to carry out a series of catabolic activities, with azoreductase and laccase enzymes being responsible for the transfer of electrons to the azo bond of the dye and the production of aromatic amines .
The mechanism of degradation by azoreductase enzymes consists of two phases. The first, called the reducing phase, begins with the cleavage of the azo bond (-N=N-) by catalyzed reduction of the enzyme under anaerobic/anoxic or microaerophilic conditions, where NADH molecules, derived from carbohydrate metabolism are used as electron donors . In the second phase, as a result of this division, relatively simple intermediate aromatic amines are generated, which are deaminated or dehydrogenated by bacteria through aerobic processes under aerobic conditions, which leads to complete degradation of azo dyes .
Laccases, on the other hand, are copper oxidases that degrade dyes in the presence of oxygen through mechanisms that involve direct or indirect oxidation using redox mediators to accelerate the reaction. This involves the removal of a hydrogen atom from the hydroxyl and amino groups, replacing it with phenolic substrates and aromatic amines .
Bacterial action in the degradation of azo dyes is increased due to their ability to act through consortiums or synergistic associations that act as biological inducers. The union of the catabolic functions of each microorganism makes them even more useful alternatives to improve the discoloration rate of effluents contaminated with dyes, as they have greater resistance to abiotic conditions and lower rates of enzyme inactivation, especially in large-scale operations .
1.2 Enterococcus, potentially degrading bacteria of the complex azoic dyes - heavy metals
One of the bacteria identified as an effective biological alternative for the removal of metal-dye synergy is
For a long time, the environmental importance of
The ability of
The ATCC 6569
The binding affinity of
In relation to metal removal,
Taking as reference the results of the Environmental Quality Program of Tourist Beaches , samples of water and sand were taken at Bocagrande beach in Cartagena Colombia, taking as reference points the areas with the highest concentration of users and suspected of contamination from a source point of marine water dumping.
To search for
The samples were processed through the membrane filtration method. In the case of the sand samples, 10 g of these were diluted in 90 mL of deionized water, the supernatant being considered as a filterable material. Filters were transferred to Slanetz & Bartley agar and incubated for 48 hours at 35 ± 0.5° C . After the incubation period, the colony count was performed and the results were reported in CFU/100 ml of sample. To confirm the identification of
Biochemical tests were carried out for the confirmation of the genus
For the identification of microorganisms by MALDI-TOF Mass Spectrometry, it was necessary to extract ribosomal proteins, using the formic acid extraction method. The analysis of the mass spectra was performed using a Microflex LT mass spectrometer, using the MALDI Biotyper 3.4 software package from Bruker Daltonik . The interpretation of the results was based on accepting scores between 2.0 and 1.7 for the identification of genus and species. Scores below 1.7 were considered unreliable.
Advanced proteomic computational techniques were used; Their advantages lie not only in the speed and economic cost of the process, but also in the effectiveness for the structural and functional analysis of the azoreductase enzymes present in
3. Results and discussion
Of the 54 samples analyzed by the membrane filtration method, in 36 samples (64.86%) colony growth was obtained on Slanetz and Bartley agar, presumptive of the genus
This is due to the ability of microorganisms to adhere to particulate materials, from which they obtain protection against predation and adverse environmental conditions such as: solar radiation, pH, temperature or bioavailability of nutrients. At the same time, this provides them with a food source that allows them to survive for longer periods, favoring their multiplication .
Confirmatory biochemical tests were performed to the 36 samples in which growth of presumptive colonies of the genus
Another feature that characterizes
Regarding the tests for the determination of species, the enzymatic activity of gelatinase was not expressed in any of the 36 strains identified as presumptive for
On the other hand, the ability to reduce potassium tellurite is one of the tests that allows differentiation of
Taking into account the discrepancy in the results obtained, high-precision confirmatory tests were performed using matrix-assisted laser ionization mass spectrometry or MALDI TOF. Unique mass peaks are considered specific biomarkers for each genus and species. In species discrimination, MALDI-TOF MS allowed the identification of 12 strains belonging to three different species of
The in-silico analysis showed a low amount of cysteine residues and a high amount of aliphatic amino acids in the primary structure, which indicated that Azoreductase (2HPV) is some intracellular proteins. The hydrophobicity condition of cysteine suggests that the enzyme is nonpolar and hydrophilic in nature. The presence of a high percentage of α helices indicates that Azoreductase (2HPV) is considered thermostable, as shown in Figure 3.
More than 90% of the amino acids were located in the allowed region of the Ramachandran graph, which indicates their stability in nature. The results obtained by SAVES showed that the enzyme have stable crystallography and the SWISS-MODEL QMEAN, ANOLEA and ERRAT analyzes confirmed their good quality. The structural analysis established that Azoreductase (2HPV) have better thermal stability and a superior quality model than other enzymes degrading dyes such as peroxidases and laccases , as shown in Table 1.
|Enzymes||3D-1D score (%)||ERRAT quality factor||QMEAN Z-score||AA in FR of Ramamchandran plot (%)|
The STRING analysis (protein –protein) identified that the proteins that interact with the azoreductase studied had an unknown 3D structure. However, the formation of interconnection networks was evidenced, possibly due to the interaction with bacteria that is genetically similar, which is especially favorable in dye degradation processes using bacterial consortia. At an industrial level, this improves the discoloration rate of effluents contaminated with dyes, as it has greater resistance to abiotic conditions and lower rates of enzyme inactivation, especially in large-scale operations .
The Bocagrande beach in Cartagena, Colombia is one of the most visited Colombian destinations by locals, as well as national and international tourists. Its high number of users throughout the year, the dumping of domestic waste generated by tourist activity, as well as other drainage carried by rain, are all considered triggers of pollution in this ecosystem.
Matrix-assisted laser ionization mass spectrometry or MALDI TOF identified other species apart from
According to the World Health Organization guideline values for recreational marine waters at risk of transmitting gastrointestinal diseases (EGI) and acute febrile respiratory disease (ERFA), the results of this study indicate that Bocagrande’s beaches are in category A; This means that the concentration of
The current biotechnological challenges lead to the development of solutions that guarantee the quality of our ecosystems and the health of human beings exposed to environmental imbalance. In relation to the problems associated with the use of dyes in different industrial processes, there have been many technological strategies developed to reduce the polluting load in industrial effluents and in receiving water bodies.
Dye removal strategies have evolved over the years. This happened due to the development of new physical and chemical methods, which progressed towards the use of environmentally friendly and cost effective biological solutions for the industry. These biological solutions have used plants, algae and other microbial biomasses as an alternative for dye removal. However, bacteria are the most robust microorganisms that, due to their structure and genome, become potential degraders of recalcitrant contaminants such as azo dyes.
The competitive advantages of bacteria are, among others, their short life cycle, their ability to adapt, and their metabolic action; they are able to degrade and detoxify the secondary metabolites produced in the discoloration process. These properties prevail in bacterial communities present in marine ecosystems, considering that these are capable of removing, in monoculture or in consortium, individual colorants, mixtures of colorants and the metal-colorant complex. Their use, although underexploited, becomes relevant with the advent of emerging technologies connected with nanotechnology, alternative energy, circular economy and environmental sustainability.
The mechanisms involved in the simultaneous removal of dyes and the metal-dye complex, the enzyme profile and the intermediate metabolites should be the subject of future studies based on genomics and proteomics. Likewise, due to the legal and environmental limitations when monitoring industrial discharges and the distribution of azo dyes in the environment, it is necessary for the scientific community to provide innovative mechanisms in which monitoring discharges and bodies of water receptors are based on amine detection.
The results of this study suggest that the enzymes Azoreductase (2HPV) are potential degraders of azo dyes due to their stability, good quality of crystallographic structure, as they are intracellular, hydrophilic and thermostable. The high content of α helices indicates their thermal resistance, which, associated with their structural quality, makes them potential degraders of azo dyes.
The properties of Azoreductase (2HPV), whose origin is
This work would not have been possible without the help of the work team of the Clinical and Environmental Microbiology Group of the University of Cartagena.
Conflict of interest
The authors declare no conflict of interest.