|Year : 2023 | Volume
| Issue : 1 | Page : 83-92
Insight into biosorption of hexavalent chromium using isolated species Aspergillus Proliferans LA: A systemic and In silico studies
Abhimati Shukla, Lalit Kumar Singh
Department of Biochemical Engineering, School of Chemical Technology, Harcourt Butler Technical University, Kanpur, Uttar Pradesh, India
|Date of Submission||12-Nov-2022|
|Date of Decision||17-Jan-2023|
|Date of Acceptance||04-Feb-2023|
|Date of Web Publication||14-Mar-2023|
Lalit Kumar Singh
Department of Biochemical Engineering, School of Chemical Technology, Harcourt Butler Technical University, Kanpur - 208 002, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: The wastewater disposal into the water bodies without removing the toxic heavy metals and other industrial impurities is a major issue these days. These heavy metals cause serious health issues to the human and animal life and also harm the environment and reduce the productivity of crops. A potent microorganism resistant to hexavalent chromium was isolated. The activity of this isolated strain was analyzed using in silico studies. Methods: In this study, a chromium-resistant fungus was isolated from the soil of the dumping sites of the tanneries in Kanpur, UP, India, followed by isolation by serial dilution, plating method, and finally, genome sequencing. It was identified as Aspergillus proliferans LA that is submitted to the National Collection for Industrial Microorganisms (NCIM) database with accession no. NCIM-1473. In the current study, the comparative analysis of the protein sequence of A. proliferans (NCIM-1473) was done against the known 53 protein sequences of the fungus and bacterial strains already reported for their chromium-resistant nature. The physical and chemical parameters of the known and isolated chromium-resistant proteins were analyzed using the ProtParam tool. The comparative study on the sequence and structural alignment of known and isolated chromium-resistant protein was done using EMBOSS-NEEDLE and FATCAT, respectively. Results: In this analysis, the top 10 strains showing similarity with A. proliferans (NCIM-1473) were reported and among which ChrI, chromium regulatory protein Cupriavidus metallidurans CH34 was showing maximum similarity with isolated chromium resistant protein for all the analysis, namely ProtParam, sequence, and FATCAT analysis. This strain, Cupriavidus metallidurans CH34, has been reported resistant against eight heavy metals, one of which is chromate, and was first identified in the heavy-metal contaminated sludge in a settling tank of Belgium. Conclusion: These studies conclude that the strain isolated in our laboratory (accession no. NCIM-1473) is potentially chromium resistant and a unique strain.
Keywords: Aspergillus proliferans LA, chromium, FATCAT, ProtParam
|How to cite this article:|
Shukla A, Singh LK. Insight into biosorption of hexavalent chromium using isolated species Aspergillus Proliferans LA: A systemic and In silico studies. Biomed Biotechnol Res J 2023;7:83-92
|How to cite this URL:|
Shukla A, Singh LK. Insight into biosorption of hexavalent chromium using isolated species Aspergillus Proliferans LA: A systemic and In silico studies. Biomed Biotechnol Res J [serial online] 2023 [cited 2023 Jun 5];7:83-92. Available from: https://www.bmbtrj.org/text.asp?2023/7/1/83/371702
| Introduction|| |
The overexploitation of available resources and unregulated disposal of industrial waste are major issues arising due to rapid industrialization. A major global issue these days is heavy metal pollution varying in severity from one place to another. The major heavy metals known for their toxicity are Arsenic, Lead, Cadmium, Chromium, and Mercury. The hexavalent form of chromium is widely used in textile industries, tanneries, stainless steel industries, and electroplating processes. The effluents from these industries are discharged directly into the ecosystem without proper treatment, which in turn leads to contamination of the ecosystem. This hexavalent chromium pollution causes severe health issues to flora and fauna of the ecosystem. Hence, scientists are working on different techniques for the treatment of this contaminated water.
Various techniques are being used these days for the removal and recovery of hexavalent chromium such as membrane filtration, electroplating, chemical precipitation, biosorption, and bioremediation.
The mechanism of biosorption, bioaccumulation, and biotransformation of hexavalent chromium has been explored in several researches. Hexavalent chromium attaches through a chemical bond formed between the metal ion and functional groups present on the cell surface such as glycoproteins, glycolipids, and polysaccharides to the microbial cell. This hexavalent chromium on adsorption either converts into a trivalent form of chromium of precipitates on the microbial cell. This conversion to a trivalent form of chromium occurs in the presence of chromate reductase. A database, BacMet database has been curated, which contains information of bacterial genes supporting survival under environmental stress. This database mainly contains several chromate transporter genes (CHR), which have a major role in chromium efflux, transport, and binding.
The enzymatic mechanisms for microorganisms differ according to aerobic and anaerobic conditions. The aerobic Cr (VI) reductases are generally found in intercellular positions. Various bacteria have been found to contain intracellular soluble reductases such as ChrR, Yief, NemA, and AzoR. The reductases found can be usually classified into CHR, OYE, YieF, NfoR, and NfsA families. The electron donor used by these enzymes is NADP (H), and flavin is used as the cofactor. The efflux phenomenon also aides the microbes in resisting Cr (VI). The ChrA, an efflux protein, is found in various chromium resistance bacteria. In the case of fungus, ChrA homologous protein Chr-1 is also found to be associated with chromium resistance, but the efflux mechanism of the two differs. This protein is usually distributed in plasmids or chromosomes. In some microbes, the chrA gene is found adjacent to various other genes like chrB, a chromate sensing regulator, chrC, cellular oxidative stress reduction, chrE, involved in cleavage of the chromium-glutathione complexes, chrF, the function of these proteins is less known, chrL, chrI, and chrK. These efflux proteins present contribute to Cr (VI) resistance.
In this study, isolation of fungal strain was done, followed by 18sRNA sequencing performed by National Collection for Industrial Microorganisms (NCIM), Pune. The sequence information obtained was further used for the basic local alignment search tool (BLAST) and phylogenetic tree analysis using MEGA software. The information for the proteins involved in chromium biosorption was collected from the BacMEt database and National Centre for Biotechnology Information (NCBI). The BacMet database consists of information of experimentally confirmed genes responsible for heavy metal resistance and also contains information of genes similar to the experimentally confirmed genes, thus proving their role in heavy metal removal. This comparative analysis of model isolated organism (Aspergillus proliferans LA) was done against collected data of protein sequence involved in chromium biosorption. The various tools used in the analysis include EMBOSS-NEEDLE for pairwise alignment, ProtParam for sequence alignment, and FATCAT for structural alignment. These analyses were performed to study the properties of isolated strain A. proliferans (NCIM-1473).
| Methods|| |
Isolation of microorganism
The soil samples were collected from the highly chromium-contaminated areas around the dumping sites of tanneries. These areas were assumed to be rich in heavy metals, especially chromium, as it is used in the form of salts in the tanning of leather. The isolation of the fungal strain was performed using the serial dilution method-1 g of the soil sample was added to 9 ml sterile water, from this tube 1 ml was removed and added to a tube containing 9 ml sterile water. Similarly, the resultant dilutions were made by transferring to subsequent tubes. The media used for the isolation of the fungal strain was potato dextrose agar (PDA) with an initial chromium concentration of 20 ppm. These PDA plates were then incubated at 30°C for four days. After further purification, five colonies were obtained, of which three appeared to be fungal based on their morphological characteristics.
18s-RNA sequencing of the isolated strain
The purified fungal colonies obtained were transferred to agar slants for further analysis. The MALDI-TOF testing was then performed using VITEK-MS, an automated mass spectrometry microbial identification system that uses Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology. Based on the further analysis on different concentrations of chromium one fungal isolate was selected for further analysis. The colonies in the form of active slants were sent to NCIM-NCL, Pune, for 18s rRNA sequencing. The extraction of chromosomal DNA of the isolated species was done using spin column kit (HiMedia, India, or similar manufacturers). The amplication of the 18S rRNA gene (1500bp) was carried out using a polymerase chain reaction in a thermal cycler, followed by purification using Exonuclease I – Shrimp Alkaline Phosphatase (Exo-SAP). The amplicons were then sequenced using the Sanger method in ABI 3500xl genetic analyzer (Life Technologies, USA).
In silico analysis
These sequences were further analyzed using BLAST, with the closest culture sequence retrieved from the NCBI database that finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. The BLAST algorithms are used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. (i) Initial search to find potentially closely related type strain sequences using the BLASTN program, (ii) Pairwise alignment to calculate the sequence similarity values between the query sequence and the sequences identified in step (i). Therefore, each isolate is reported with the first five-ten hits observed in the said database. Further multiple sequence alignment and phylogenetic analysis were done for an accurate species prediction and prediction of evolutionary relationship.,,
The sequencing data and the isolated culture in lyophilized form have been deposited in the NCIM, Pune.
The various genes with a role in chromium resistance and absorption were searched and their information, i.e. gene name, accession no., protein name, protein sequence, superfamily, and the conserved sequence were collected from National Centre for Biological Information (NCBI) (https://www.ncbi.nlm.nih.gov/) and BacMet database. The information specified was collected for a total of 53 domains which was further used in the sequence analysis. This data were further used in the sequence alignment of the model organism (A. proliferans LA) with the various protein sequences obtained. The BacMet database is a manually curated database of metal resistance genes based on the review of scientific literature.
The similarities between the sequences of A. proliferans LA and the genes with a role in chromium resistance or absorption were evaluated using the EMBOSS-NEEDLE tool of EMBL's European Bioinformatics Institute. This tool writes the global sequence alignment of the two input sequences. In this tool, valid sequences in any format are uploaded in the first sequence and second sequence, respectively. The alignment options, i. e., protein or nucleotide, are then selected, followed by providing an additional information-gap open penalty, gap extends penalty, and list values. The job title and the email notification are then provided for the proper delivery of results directly by mail. On completion of the analysis, an email with the link of the results is sent to the email id specified.
The output in the form of a matrix is generated with the identity score, similarity score, and gap score values. A detailed alignment with the information of the sites of similarity, identity, and gaps is also generated with proper color coding for easy analysis. The pairwise alignment data generated on EMBOSS-NEEDLE were collected, and the sequences were ranked according to the scores generated.
The sequence information obtained was further used for the analysis of various physical and chemical parameters of the sequences using the ProtParam tool of EXPASY. This tool requires just the protein sequence information, which can be in the form of Accession no. or ID or in the form of raw sequences. The computations performed by ProtParam are either in the form of N-terminal amino acid or in the form of compositional data. The output generated by ProtParam includes the following parameters – molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficients which indicates the light a protein absorbs at a certain wavelength, estimated half-life, which predicts the time taken for half of the amount of protein in a cell to disappear after its synthesis in the cell, instability index estimates the stability of the protein in a test tube, aliphatic index defines the relative volume occupied by the aliphatic side chains and grand average of hydrapathicity (GRAVY) is computed as the sum of hydropathy values of all amino acids divided by the number of residues in the sequence.
The structural comparison between the protein structures of the genes found with a role in the adsorption of chromium in some form or the other with the protein structure of the isolated organism A. proliferans was performed using Flexible structure AlignmenT by Chaining Aligned fragment pairs allowing Twists (FATCAT) tool of protein structure alignment. This is a tool for flexible protein structure comparison. It mainly depends on two parameters, optimization of the alignment and minimization of the number of rigid-body movements (twists) around pivot points (hinges) introduced in the reference structure. In such alignment, translations and rotations between elements of one structure minimize the root mean square deviation (RMSD) between the compared structures.
The homology model of the isolated fungi A. proliferans was constructed using SWISS-MODEL structure modeling server and was uploaded in the structure 1 component of the pairwise alignment section of the FATCAT tool, in the second structure of the PDB structure of the known proteins with a role in chromium adsorption was uploaded. An online homology model was generated for the proteins whose PDB structure was not available using the SWISS-MODEL tool.
The output of the flexible structure comparison was generated, which consists of the following parameters, i.e. P value, which is used for evaluating the significance of the structural similarities detected. The flexible structure alignment is composed of an AFP chaining process allowing at most t twists. Dynamic programming is used in the chaining process, if S (k) denotes the best score of paths ending at the AFP k, then it can be calculated from the best score of paths ending at previous AFPs that can be connected to the k-th AFP, subject to the constraints. The FATCAT (chaining) score is the best of all S (k) in the alignment graph. Root Mean Square Deviation (RMSD) is the overall RMSD between the structures when one structure is rearranged at the positions where twists are detected by FATCAT.
| Results|| |
Isolation of microorganism
The isolation of fungal species was performed using the serial dilution method. The initial Cr (VI) concentration was kept as 20 ppm. This gave five purified colonies, of which three were found to be fungal based on their morphological characteristics, and two appeared to be bacterial. After further analysis, one fungal isolate showing good microbial growth on different concentrations of Cr (VI) was selected and used for further analysis. This fungal isolate was identified by NCL, Pune, as A. proliferans CBS121.45 with 99.9% similarity. The isolated strain and the sequence data are deposited in the general collection of microorganisms of the National Centre of Industrial Microorganisms (NCIM), and can be accessed by Aspergillus sp.(proliferans) (LA)/NCIM-1473.
The BLAST result for the isolated species with the top five hits is given in [Table 1].
|Table 1: National center for biological information-basic local alignment search tool hits|
Click here to view
The multiple sequence alignment was done using the sequence of the isolated species and the top 5 hits generated from BLAST by MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms software followed by phylogenetic tree building using the Maximum Likelihood method and Tamura-Nei model. The phylogenetic tree is given in [Figure 1].
|Figure 1: Phylogenetic tree showing the evolutionary relationship of the isolated species|
Click here to view
In silico analysis
A total of 53 domains with Cr (VI) reducing, transporter of Cr (VI) or regulatory proteins were identified in fungi and bacteria and reported in the BacMet database and NCBI database. These domains were searched in Bacmet and NCBI database and the information, such as gene name, accession no., PDB id, protein name, protein sequence, superfamily, and conserved sequences, were arranged in the tabular form given in [Table 2].
|Table 2: The sequential information of Cr (VI) reducing, transporter of Cr (VI) or regulatory proteins|
Click here to view
This information obtained was used for pairwise alignment study and ProtParam analysis. In the pairwise alignment study using EMBOSS-NEEDLE, the maximum score of 33 was seen in the chrI protein. The chrI protein is a chromium regulatory protein found in Cupriavidus metallidurans CH34. This is a Gram-negative bacterium and has been reported to be resistant to eight heavy metals, including Cr (VI). In the sequential analysis by ProtParam, the lowest instability index was 26.50 and 26.95 reported in the case of chrB and chrI proteins. The chrB protein is a chromate efflux transporter protein found in Bacillus amyloliquefaciens IT-45. [Table 3] and [Table 4] given below show the data obtained in pairwise alignment and ProtParam analysis.
|Table 3: Pairwise alignment analysis for the proteins involved in chromium biosorption|
Click here to view
The structural comparison between the proteins involved in chromium biosorption was made using the FATCAT tool of flexible protein comparison. This tool uses the protein structure for analysis and compares the protein structures and provides results in the form of FATCAT score, P value which is used for evaluating the significance of the structural similarities detected, Root Mean Square Deviation (RMSD) is the overall RMSD between the structures when one structure is rearranged at the positions where twists are detected by FATCAT. The FATCAT results are shown in [Table 5].
|Table 5: Flexible structure alignment by chaining aligned fragment pairs allowing twists analysis for the proteins involved in chromium biosorption|
Click here to view
[Figure 2] gives the modeled structure of the online homology model generation tool Swiss Model using five templates. The Ramachandran analysis of the modeled structure was done using the PROCHECK tool of the SAVES server. This gave 93.5% residues in the most favored region and 4.3% in the additional allowed region. The residues in the disallowed region were 2.2%.
|Figure 2: The model structure generated using SWISS-MODEL is shown above along with the Ramachandran analysis showing the no. of residues in the favourable region|
Click here to view
In the results obtained by pairwise alignment and ProtParam analysis, when the results were arranged in decreasing order of the pairwise alignment score and instability index, chrI was found common in both the cases with 33 pairwise alignment score and 26.95 instability index.
| Discussion|| |
Various researches have been carried out using fungal and bacterial isolates for the removal and recovery of heavy metals. In a study by Congeevaram et al., using heavy metal-resistant isolates of bacteria and fungi, Micrococcus sp. and Aspergillus sp., could tolerate high concentrations of chromium and nickel. Similarly, B. coagulans could tolerate up to 512 ppm concentration of hexavalent chromium.
Similarly, a comparison of the results of ProtParam analysis and FATCAT score was made, in which chrI was again found common in both FATCAT and ProtParam analysis, with a FATCAT score of 22.16, ceuE, enterochelin uptake substrate-binding protein, was also found common with a FATCAT score of 33.36 and instability index value of 27.20, chrA, an ABC-type enterochelin transport system, periplasmic component, was also found in both the cases with 27.97 instability index and 34.85 FATCAT score and AtWU_11493, putative chromate ion transporter (Eurofung), was also found with 21.53 FATCAT score and 29.40 instability index.
Similarly, the results of pairwise alignment and FATCAT analysis were also compared; chri was again found common in both FATCAT and pairwise alignment analysis with 33 pairwise alignment score and 22.16 FATCAT score, ANOM_003625, COesterase domain-containing protein, was also found common with 28.5 pairwise alignment score and 37.40 FATCAT score.
| Conclusion|| |
An economic and environmentally friendly characteristic for metal removal is microbial metal bioremediation. In this analysis, the microorganism was isolated from the soil near the tanneries using the serial dilution method on 20 ppm Cr (VI) concentration. The 18s-RNA sequencing and phylogeny analysis showed the similarity of the isolated strain to A. proliferans CBS121.45 and is deposited safely in NCIM-NCL, Pune and can be accessed by Aspergillus sp.(proliferans) (LA)/NCIM-1473. The in silico analysis of the sequence and structural information was done using the EMBOSS-NEEDLE, ProtParam, and FATCAT tools, and data were further analyzed in which it was concluded that ChrI, chromium regulatory protein C. metallidurans CH34 was found in all the analysis, i.e. sequence analysis, ProtParam analysis, and fatcat analysis. This strain, C. metallidurans CH34, has been found resistant to eight heavy metals, one of which is chromate, and was first identified in the heavy-metal contaminated sludge in a settling tank of Belgium.
Limitations of the study
The analysis done in this study mainly focused on the sequential analysis of the isolated microorganism; the docking and MD simulation studies for the same were not performed. The wet lab experiments for the data retrieved from the ProtParam analysis and FATCAT analysis should be performed for proper validation of the results.
Financial support and sponsorship
This work was supported by the third phase of the Technical Education Quality Improvement Program.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]