Unraveling the Stability of Halophilic Protease Enzymes: A Computational Study

Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.85 https://doi.org/10.5281/zenodo.17393614 Unraveling the Stability of Halophilic Protease Enzymes: A Computational Study Tabsum Chhetri1#, Saurav Kumar Mishra1#, Sarah Jacob 2, John J. Georrge1,2* 1 Department of Bioinformatics, University of North Bengal, District-Darjeeling, West Bengal734013, India 2 Department of Bioinformatics, Christ College, Rajkot, Gujarat, India #Consider as joint first authors *Corresponding author: [email protected] Abstract: Halophiles thrive in high salt concentrations, requiring more than 0.2 M NaCl for growth and resisting osmotic stress. They are classified based on their salinity concentration, i.e., slight (0.2-0.85 M or 2-5% of NaCl), moderate (0.85-3.4 M or 5-20% of NaCl), and extreme (3.4-5.1 M or 20-30% of NaCl) Halophiles. The number of acidic residues appears to be balanced by a reduced number of basic residues on a genome-wide scale. The main variance in the composition of halophiles and mesophiles was mainly attributed to the abundance of aspartic acid, lysine, etc. Therefore, discovering more amino acids can lead to new prospects for industrial applications. As Halophilic organisms producing the enzyme protease are being significantly used in detergent, pharmaceutical, and food manufacturing industries, this analysis can further lead to Genome sequencing, mutational studies, protein engineering, etc. Many bioinformatics approaches for proteases, such as PoPS, PROSPER, and PAL, have been used to identify the desired functional properties. Future studies on mutational stability and improving these halophilic proteases' stability can be rationalized through bioinformatics approaches. Keywords: Halophiles, Enzyme, Bioinformatics, Mesophilic Proteases 1. Introduction The living world is home to diverse microorganisms that thrive under varying environmental conditions, ranging from normal to extreme environments such as arid regions, coastal deepsea zones, and underground salt mines (Wani et al., 2022). Among these, halophiles are a fascinating group of microorganisms adapted to saline environments. Halophiles exist in all major domains of life: Archaea, Bacteria, and Eukarya. They are categorized into slight, moderate, and extreme halophiles based on their specific requirements for sodium chloride (NaCl). (Corral et al., 2019; Menon & Jobby, 2022). Halophilic microorganisms show significant diversity “(heterotrophic, phototrophic, and methanogenic archaea, as well as photosynthetic, lithotrophic, and heterotrophic bacteria)”, along with eukaryotes that were photosynthetic and heterotrophic. Notably, many halophiles possess unique adaptations that enable them to thrive in high-pH, high-salt environments, often under elevated temperatures, highlighting their exceptional resilience and ecology (Oren, 2002). This distinguishes them from mesophiles, which prefer moderate conditions and lose functionality in extreme environments due to denaturation; however, halophiles have shown resilience to such extreme conditions as they possess a high content of amino acid adaptation, enabling them to remain hydrated and operational even in extreme osmotic stress (Bowers & Wiegel, 2011). This specific characteristic of halophiles has helped them become one of the most common products used in industries for producing bioplastics, enzymes, etc., as they can remain functional in non-sterile, low-water, or high-temperature areas (Corral et al., 2019). Industrial bioinformatics aims to produce sustainable products, biofuels, and chemicals; however, it faces various challenges, such as high temperatures, freshwater, and the risk of contamination. Therefore, halophiles and their ability to survive and thrive in such environments have facilitated many processes and have been used to produce valuable products. In particular, Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.86 https://doi.org/10.5281/zenodo.17393614 halophilic enzymes have made the process easier (Litchfield, 2011). One such enzyme is a protease, which remains active and stable during extreme conditions. Such enzymes are vital for industrial applications in detergents, textiles, food processing, and pharmaceuticals, where high stability under harsh conditions is critical (Nwankwo et al., 2023). For instance, haloalkaliphilic proteases, such as those from Natronomonas pharaonis, can function at elevated pH and temperatures (Zhao et al., 2014). Meanwhile, Haloarchaea extremozymes are used in novel applications like restriction enzymes (Bonnaud et al., 2024). In an environment with high salt content, the halophilic protease enzyme remains stable and active, whereas other enzymes are known to denature. This happens because of their distinct structural characteristic, such as Hydrophobic surface reduction, negatively charged amino acids, and expanded ionpair networks. This characteristic of halophilic proteases has propelled their use in food processing, detergent composition, and biocatalysis, as these structural features help preserve integrity in high-salt settings (DasSarma & DasSarma, 2015). To maintain stability in the hypersaline environment, halophilic proteases use the "high-salt-in" technique to develop a structural feature that helps stop denaturation (Reang et al., 2024; Sinha & Khare, 2014). Another technique used is known as "low-salt, organic-solutes-in", which is the second tactic, in which organisms build up tiny organic molecules like proline, glycine, betaine, or others inside their cells. Protein structure and function can be effectively maintained in less salinised but complex settings by using these solutes, which work as stabilising agents without interfering with the enzyme's activity (Mahmood et al., 2019; Yaakop et al., 2016). Moreover, computational navigation in biological research has been used worldwide by researchers (Kachhadiya et al., 2024; Mishra & Georrge, 2023; Mishra et al., 2024; Mishra et al., 2025; Vakhariya Sakina et al., 2023). This research attempts to investigate the possibilities of halophilic proteases' ability to function in extreme saline environments. It also seeks to uncover unique enzymatic properties that enable halophiles to thrive under harsh conditions by contrasting them with mesophilic proteases. Additionally, computational tools are utilised to study sequence characteristics, aiding in identifying novel enzymes with improved stability and efficiency for biotechnological applications. 2. Materials and methods The methodology employed in this study is shown in Figure 1. 2.1 Retrieval of the organisms The data on halophilic species that produce proteases were retrieved in FASTA format from various online databases, such as NCBI Genome (https://www.ncbi.nlm.nih.gov/home/genomes/) and Protein (https://www.ncbi.nlm.nih.gov/protein/) databases (Schoch et al., 2020) and UniProt (https://www.uniprot.org/) database (Consortium, 2015). The databases contain genome information in the NCBI Genome database, and UniProt contains protein sequences from multiple sources. Similarly, Mesophilic species within a specific temperature range were also obtained in FASTA format from the NCBI database (Genome and Protein) and the UniProt protein primary sequence database (Naitam et al., 2024). Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.87 https://doi.org/10.5281/zenodo.17393614 Figure 1. Workflow of the steps followed for retrieving, classifying, and analysing halophilic and mesophilic proteases. 2.2 Classifications of the organisms Through the NCBI Taxonomy (https://www.ncbi.nlm.nih.gov/guide/taxonomy/), halophilic and mesophilic organisms previously obtained from NCBI and UniProt were categorised into two domains: Archaea and Bacteria. Further, the browser includes information on organism classification ranks so the organisms are classified according to their genus, family, and salinity. Based on salinity, halophilic organisms that produce proteases were divided into three classes: Extreme Halophiles, Moderate Halophiles, and Slight Halophiles (Hulshof et al., 2024). 2.3 Refinement and extraction of the sequences The previously screened Halophilic and Mesophilic sequences were subjected to CD-Hit to obtain non-redundant and unique sequences. Cluster Database at HIGH Identity with Tolerance, CD-HIT, is a program that takes a sequence as input, and the output consists of nonredundant sequences (Huang et al., 2010; Suha & Khan, 2024). Locus tags and CDs were obtained from NCBI by BLAST. UniProt was utilized to obtain details like gene name, protein name, E.C. No, etc. (Eser et al., 2014). 2.4 Comparative analysis of the organisms Finally, the characteristics of the sequence of the halophilic and mesophilic proteases were analysed, wherein non-redundant sequences were aligned and clustered, and the lengths of halophilic and mesophilic proteases were compared within similar domains and salinity classes. Frequencies of amino acid composition were analysed to identify the proteases' salinity adaptations (Fukuchi et al., 2003). Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.88 https://doi.org/10.5281/zenodo.17393614 3. Results and discussions 3.1 Retrieval of the organisms A total of 121 organisms from UniProt and 148 halophilic proteases were screened from the NCBI database. The whole genome sequence of 56 organisms, including 30 halophilic organisms producing proteases, was available. Similarly, 53 sequences of Proteases produced from Mesophilic organisms were retrieved. The collection of protein sequences and their information was also carried out. 3.2 Classification of the organisms Using the NCBI taxonomy browser, the study divided 148 halophilic protease-producing species from UniProt and NCBI into domain, archaea, and bacteria. There were 89 organisms in the archaea and 59 in the bacteria (Figure 2A). Most of them belonged to the family Natrialbaceae (Figure 2B). Protease-producing halophilic organisms were categorised by genus, with six belonging to the Halorubrum Genus (Figure 2C). The domains of bacteria and archaea were categorised as mild, moderate, and extreme halophilic, and salinity concentrations varied from 2-5% to 20-30% (Figure 2D). The number of retrieved sequences of mesophilic protease-producing species was 53, respectively. Out of that, 16 were Bacteria and 37 were Archaea. Figure 2. Classification Of Halophilic Organisms. (A) Halophilic Organisms Producing Protease Categorised Based on Archaea and Bacteria Domains. (B) Halophilic Organism Producing Protease Categorised Based on Family. (C) Halophilic Organisms Producing Protease Enzymes Were Categorised Based on Genus. (D) Classification Of the Organisms Based on Salinity. Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.89 https://doi.org/10.5281/zenodo.17393614 3.3 Refinement and extraction of the sequences The sequence of Halophilic organisms producing the enzyme protease was retrieved based on the salinity concentration of slight (Figure 3A), moderate (Figure 3B), and extreme Halophiles (Figure 3C), with the number of organisms being 23, 12, and 6, respectively. The sequence length of Halophilic organisms producing the enzyme protease ranged from 180 to 850 (Figure 3). The Protein sequences and their data were collected. The' length of the mesophilic proteases ' sequence ranged from 180 to 815 (Figure 5). Pseudolateromonas sp. and Photoferrotroph had lower values than Halomonas elongata and Marinobacter lipolyticus, which had the highest values in halophilic archaea (Figure 3). At the same time, Natrialba chahannaoensis had higher values for halophilic bacteria, with the highest numbers found in Natrinema versiforme and Halophilofulus naxiensis. Moderate variability was present in most species (Figure 4); for the mesophilic proteases Thermosphaera aggregans and Thermogladius caldera had the most significant values, whereas Thermobacterium thermoresistibile, Bacillus thermozeamaize, Geobacillus kaustophilus, Novibacillus humi, Bathymodiolus thermophilus, and Paenibacillus naphthalenovorans displayed the highest values (Figure 5). Figure 3. The Sequence Length of Halophilic Proteases in Archaea. (A) Sequence Length of Slight Halophilic Organisms Producing Enzyme Protease. (B) Sequence Length of Slight Halophilic Organisms Producing Enzyme Protease. (C) Sequence Length of Slight Halophilic Organisms Producing Enzyme Protease. Figure 4. The Sequence Length of Halophilic Proteases Bacteria. (A) Sequence Length of Slight Halophilic Organisms Producing Enzyme Protease. (B) Sequence Length of Slight Halophilic Organisms Producing Enzyme Protease. (C) Sequence Length of Slight Halophilic Organisms Producing Enzyme Protease. Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.90 https://doi.org/10.5281/zenodo.17393614 Figure 5. The Sequence Length of Mesophilic Organisms Producing Enzyme Protease. (A) Archaea. (B) Bacteria. 3.4 Comparative analysis of the organisms The amino acid frequencies of bacteria and archaea at various halophilic levels are compared. The expectation in the literature that halophilic proteins have lower amino acid frequencies than mesophilic proteins is followed mainly by archaea, with exceptions for amino acids G, T, and occasionally N (Figure 6). However, the levels of amino acids such as A, L, and G are higher in bacteria in halophilic environments. Regarding the frequency of various amino acids in bacteria, archaea generally follow patterns found in the literature, but bacteria often exhibit variances (Figure 7). Archaea have lower frequencies for the amino acids R (arginine) and H (histidine) in moderately halophilic environments. In contrast, bacteria have greater frequencies for the amino acids A (alanine) and L (leucine) (Figure 7). Archaea are more consistently aligned with R and H in extremely halophilic environments. Significant variations are also seen in bacteria for several amino acids, including Q, I, and P (Figure 8). In conclusion, archaea follow predicted patterns more closely, frequently exhibiting variation in halophilic circumstances. However, bacteria frequently exhibit variations in particular amino acids, such as R (arginine) and E (glutamic acid), which might not be consistent with the research. Figure 6. Comparisons of sequence length between Mesophilic Archaea & Bacteria with Slight Halophilic Archaea & Bacteria. Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.91 https://doi.org/10.5281/zenodo.17393614 Figure 7. Comparisons of sequence length between Mesophilic Archaea & Bacteria with Moderate Halophilic Archaea & Bacteria. Figure 8. Comparisons of sequence length between Mesophilic Archaea & Bacteria with Extreme Halophilic Archaea & Bacteria. 4. Conclusion Halophiles, often called salt-loving organisms, are widely utilised in the food and detergent manufacturing industries and flourish in saltwater settings. The halophilic archaea and nonhalophilic mesophiles were compared to analyse their amino acid compositions. This research can provide insights into the essential activities of amino acid residues. Fewer basic residues were present in halophilic proteins to offset a significant overabundance of acidic residues. Alanine, lysine, asparagine, threonine, and aspartic acid contributed substantially to the compositional distinctions between mesophiles and halophiles. However, only aspartic acid Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.92 https://doi.org/10.5281/zenodo.17393614 differed substantially from the quantity predicted by the halophile's genomic DNA sequence. 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Microbial adaptation to different environmental conditions: molecular perspective of evolved genetic and cellular systems. Archives of Microbiology, 204(2), 144. Yaakop, A. S., Chan, K.-G., Ee, R., Lim, Y. L., Lee, S.-K., Manan, F. A., & Goh, K. M. (2016). Characterization of the mechanism of prolonged adaptation to osmotic stress of Jeotgalibacillus malaysiensis via genome and transcriptome sequencing analyses. Scientific Reports, 6(1), 33660. How to cite this Book Chapter? APA Style Tabsum Chhetri, Saurav Kumar Mishra, Sarah Jacob, John J. Georrge (2024). Unraveling the Stability of Halophilic Protease Enzymes: A Computational Study. Recent Trends in Science and Technology-2024 (pp. 8593). ISBN: 9788197073274. Rajkot, Gujarat, India: Christ Publications. https://doi.org/10.5281/zenodo.17393614 MLA Style Tabsum Chhetri, Saurav Kumar Mishra, Sarah Jacob, John J. Georrge. “Unraveling the Stability of Halophilic Protease Enzymes: A Computational Study”. Recent Trends in Science and Technology-2024 (ISBN: 9788197073274). Rajkot, Gujarat, India: Christ Publications, 2024. pp. 85-93. https://doi.org/10.5281/zenodo.17393614 Chicago Style Tabsum Chhetri, Saurav Kumar Mishra, Sarah Jacob, John J. Georrge. “Unraveling the Stability of Halophilic Protease Enzymes: A Computational Study”. Recent Trends in Science and Technology-2024 (ISBN: 9788197073274), pp. 85-93. Rajkot, Gujarat, India: Christ Publications, 2024. https://doi.org/10.5281/zenodo.17393614