Structural and Functional Analysis of Tannase from Aspergillus niger: An Insilico Approach for Wine Industry Waste Treatment

Structural and Functional Analysis of Tannase from Aspergillus niger: An Insilico Approach for Wine Industry Waste Treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Structural and Functional Analysis of Tannase from Aspergillus niger : An Insilico Approach for Wine Industry Waste Treatment Muhammad Aamir Sharif, Khawar Sharif, Mumtaz Bibi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7384741/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The worldwide wine industry is one of the most significant and rapidly expanding sectors and contributes billions of dollars to the world economy each year. The waste produced as a result of wine production of great concern for the environment safety. Tannin is one of the materials present in the wine waste which can be hydrolyzed into gallic acid and glucose by an enzyme tannin acyl hydrolase which is extracted from a fungus aspergillus niger . Our effort involves computational study for analyzing the structural and functional characteristics of an enzyme tannin acyl hydrolase which is extracted from a fungus aspergillus niger. The protein sequence of tannin acyl hydrolase was taken from the RCSB-PDB database. Afterwards, physicochemical characteristics and primary structure analysis were determined using Protparam webserver. The online bioinformatics tool SOPMA was used to measure number of GRAVY, amino acids, aliphatic index, theoretical pI and instability index. CELLO tool helped to determine the subcellular localization. SAVES server v6.1 helped to obtain the overall quality factor, 3D-1D ratio, Z-score and other stereochemical properties. Ramachandran plot, local quality estimate, QMEAN and 3D structure assessment was determined with the help of SwissModel. The family classification of protein and domain study was performed using InterproScan software. Finally, STRING database helped to visualize protein-protein interactions for the functional study. Result: Tannin acyl hydrolase mostly constitutes acidic amino acids and possesses thermostability. Higher aliphatic index proved its property of being thermostable. The fact that over 90% of the amino acids are present in the Ramachandran plot's preferred region indicates that tannic acyl hydrolase is a stable protein. Conclusion: This analysis aims to provide useful insights for tannin acyl hydrolase to be used in bioremediation of wine industry waste and gives important knowledge about it for the experimental laboratory work. Tannase in silico Aspergillus niger wine industry waste treatment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Wine industry is continuously growing and there is increase in the demand of the wine globally. In the year 2025, wine industry is estimated to meet the demand, industries focus more on production and pay less attention to the waste treatment. The wastes produced as a result belong to different categories like alkaloids, antibiotics, phenolics, resins, saponins, sterols, tannins, terpenes, and volatile oils. Waste from the wine business includes grape pomace, seed peel, stalks, and vine leaves. Since these wastes contain organic matter with high concentrations of volatile and phenolic chemicals, they pollute the environment if left untreated or not recycled. For instance, some wastewaters can present biological oxygen demands (BOD) higher than 5 g/L, low pH (< 5), and active microbial populations( 3 ). Industry statistics show that, despite the inconvenience of alcohol, the waste produced during the winemaking process is typically employed as vineyard fertilizer and animal feed. The grape marc is also used for distillation in the alcohol winery. However, most of this waste is still disposed without any previous treatment resulting in damage to the environment as, for example, the contamination of surface and ground water( 14 , 16 ). Tannins are a group of water-soluble polyphenolic compounds (with molecular weight ranging from 0.5 − 20 kD) naturally found in plants and have been reported to be the fourth most abundant plant constituents after cellulose, hemicellulose and lignin( 22 , 11 ). They are amorphous, astringent substances occurring widely in the bark, wood, leaves, and resinous exudations of plants ( 17 ) ( 23 ) ( 21 , 9 ). Biochemically, tannins are sort of secondary metabolites predominantly available in plant-based foodstuffsand beverages grapes, blackberries, strawberries, walnuts, cashew nuts, hazelnuts, mangoes,and tea( 5 ). Tannins are considered defensive molecules to protect plant tissues from herbivorous attacks because of their astringent taste( 4 , 19 , 13 ). Tannins possess antioxidant activity. This property is related to their chemical structure as they possess phenolic rings able to bind to a wide range of molecules and act as electron scavengers to trap ions and radicals( 20 , 10 ). Galloyl ester, glucose, and gallic acid are produced through the hydrolysis of the ester link of hydrolyzable tannin by tannase (tannin acyl hydrolase, E.C 3.1.1.20). Numerous species, including bacteria, yeast, and fungi, biosynthesize the inducible enzyme tannase. Gallic acid is the major tannic acid hydrolytic product and is applied in cosmetics and in the synthesis of various antioxidants( 1 ) ( 2 ). Through the process of hydrolysis, it lowers the burden of tannins and yields glucose and gallic acid, which are used as fuel. The production of precipitates in beverages containing other molecules is facilitated by the high tannin content found in coffee-flavored beverages, fruit juices, wine, beer, and iced tea. Tannases are used to remove these undesired properties from the fruit juices( 12 ). It is considered an environmentally significant biocatalyst as well. The use of biomass from natural sources has not been fully examined in any of the reviews, despite the fact that they have all focused on the screening, manufacture, characterisation, and purification of tannase from diverse sources. Aspergillus niger's fungal tannase crystal structure is the first to be published. A bowl-shaped hemispherical shape with a surface concavity enclosed by N-linked glycans is formed by the enzyme's characteristic α/β-hydrolase-fold domain and a large inserted cap domain. Gallic acid forms two hydrogen-bonding networks with nearby residues at the intersection of the two domains within the concavity. One involves residues from the hydrolase-fold domain, including those from the catalytic triad, which is made up of Ser206, His485, and Asp439, and is formed around the carboxyl group of gallic acid. The latter is created around the compound's three hydroxyl groups and involves residues primarily from the cap domain, such as Gln238, Gln239, His242, and Ser441. Gallic acid forms a hydrophobic bond with Ile442 to form a sandwich-like bond. All of these residues are found to be highly conserved among fungal and yeast tannases( 6 ) . 2. Methodology 2.1. Sequence retrieval The RCSB-PDB database contains the experimentally determined sequences and structures of proteins. Tannin Acyl Hydrolase's sequence was obtained from the RCSB-PDB [ https://www.rcsb.org/ ] database and subsequently downloaded in PDB and FASTA formats. 2.2. Primary Structure Analysis and Subcellular Localization The computed parameters like molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (GRAVY) were all determined by the Protparam database ( 7 )[ https://web.expasy.org/protparam/ ]. CELLO subcellular localization predictor V2.5 [ https://cello.life.nctu.edu.tw/ ] was used to predict subcellular location. 2.3. Secondary Structure Analysis Nuclear Magnetic Resonance method is performed to predict the structure of protein normally in the laboratory work( 18 ). Using a web-based server Alignment-Based Self-Optimized Prediction Method, or SOPMA [ https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html ], the secondary structure of the recovered protein sequence was predicted based on the percentage of α-helices, β-turns, and β-sheets in the protein( 8 ). 2.4. Analysis of Tertiary Structures, Validation, and Homology Modeling X-ray crystallography is typically used for determining the proteins' tertiary structure, however, some online webservers like AlphaFold and Phyre 2.2 can be used to determine computationally. Phyre 2.2 ( https://www.sbg.bio.ic.ac.uk/phyre2/ ) was utilized in this particular situation. High resolution 3D protein structure was acquired by using SAVES server v6.1 [ https://saves.mbi.ucla.edu/ ]. In SAVES v.1, the overall quality factor for the crystallographic protein structure was confirmed; 3D-1D score was determined by VERIFY, different stereochemical parameters of residues were obtained using WHATCHECK and PROCHECK found out the quality of protein structure by evaluating residue-by-reside geometry and overall structure geometry( 15 ). Using SAVES, 3D structure was also viewed and downloaded. Ramachandran plot,QMEAN, solvation, local quality estimate and torsion were determined using a webserver of Structure Assessment by SwissModel [ https://swissmodel.expasy.org/assess ]. 2.5. Functional Analysis and Protein-protein interactions InterPro Go terms and PANTHER Go terms were acquired using the Interpro portal [ https://www.ebi.ac.uk/interpro/ ]. Protein-protein interactions with more closely related proteins were predicted with the use of the String database [ https://string-db.org/ ]. 3. Results 3.1. Sequence Retrieval The protein sequence of Tannin acyl hydrolase from asperillus niger 7K4O was obtained from RCSB-PDB database in FASTA and PDB formats. 3.2. Primary Structure Analysis Theoretical overview of the nature of the Tannin acyl hydrolase was determined by the computational analysis of its physicochemical qualities as Table 1 shows. Table 1 Comparative Physicochemical attributes of Enzymes Comparative Physicochemical attributes of Enzymes Serial No Enzyme Molecular Weight Amino Acids Instability Index Theoretical pI Aliphatic Index GRAVY 1 Tannin Acyl Hydrolase 60773.3 554 36.49 4.28 72.92 -0.29 As if the theoretical pI value is less than 7 then it is considered acidic and also there are more negatively charged amino acids therefore, Tannin acyl hydrolase consists mainly of acidic amino acids. Instability index of tannin acyl hydrolase is less than 40 which makes it a stable protein. If and only if the instability index value had been higher than 40, the protein would have been unstable. A higher aliphatic index score indicates greater thermotolerance and it shows the presence of thermostable globular proteins. If an enzyme has higher aliphatic index, it is considered for industrial use. Tannin acyl hydrolase has negative GRAVY value representing its hydrophilic nature and ultimately better water-protein interactions. Glycine(Gly), alanine (Ala) and threonine (Thr) appear to be the most abundant amino acids (9.0%, 8.3% and 8.3% respectively). Compared to the positively charged amino acids (Arg + Lys), the negatively charged amino acids (Asp + Glu) were significantly greater as shown in Fig. 1 and Fig. 2 . 3.3. Secondary Structure Analysis In general terms, protein's secondary structure is constructed based on its primary structure using wet lab techniques. In this analysis, computational method was applied for studying the tannic acyl hydrolase's secondary structure and a webserver SOPMA was used as is shown in Fig. 3 . Like laccases, tannin acyl hydrolase was also found to be rich in random coils. The presence of high concentration of random coils confirms the flexibility and conformational changes of the protein. The higher value of α-helices in tannin acyl hydrolase exhibits significant thermal resistance on the basis of its intrinsic stability (Fig. 4 ). 3.4. Analysis of tertiary structures, validation and homology modeling The Structure Analysis and Verification Server (SAVES v6.1) was used to compute the quality of the standard X-ray crystallographic protein structure. Tannin acyl hydrolase was verified as an excellent protein structure by the use of ERRAT, WhatCheck,, ProChcck and PROVES tools run by SAVES. Table 2 shows that this protein structure was evaluated as useful by both its quality and quantity. A 3D-1D ratio according to scores was also provided by this tool (Fig. 5 ). For the practical application of structure interpretation, local model quality was evaluated with SwissModel and Fig. 6 displays the target-template alignment prediction. The accuracy of the local model using the tannin acyl hydrolase residues along its sequence is accurately shown in the same figure. For homology modeling studies, SwissModel Structure Assessment tool estimated the good model quality wise. The findings of a comparison between tannin acyl hydrolase and the non-redundant PDB structure are displayed in Fig. 7 . Table 2 Quality Assessment Score Quality Assessment Score 3D–1D Compatibility (%) ERRAT Quality (%) QMEAN Z-Score Amino Acids in Favored Regions (Ramachandran Plot, %) 83.94 93.692 0.35 90.7 An ideal protein's QMEAN Z-score falls between 0 and 1, so here, tannin acyl hydrolase has QMEAN Z-score value is 0.35 as represented in Fig. 8 . ERRAT server also evaluated the error regions by examining the non-bonding atomic association statistics. 93.692 is considered a very satisfactory ERRAT value. The SAVES webserver's PROCHECK program yielded the Ramachandran plot between the amino acids' φ-Ψ torsion angles. Rare left-handed α-helices were among the conformations found in the allowed area in quadrant 1 of the Ramachandran plots. Quadrant 2 had the most advantageous sterically permitted β-strand conformations, Quadrant 3 had right-handed α-helices, and Quadrant 4 had conformations with the disfavored area. The percentage was residues was found to be 90.7% in in the most favored regions of Ramachandran plots. Hence, it proved the protein model was of satisfactory quality. A few residues present in the disallowed area confirmed the reliability of tannin acyl hydrolase as shown in Fig. 9 . 3.5. Functional Analysis and protein-protein Associations The tannin acyl hydrolase from Aspergillus niger (PDB ID: 7K4O) was functionally annotated using InterProScan. The analysis revealed that the enzyme belongs to the Feruloyl esterase B-related family and is functionally classified as a Tannase (Tannin acyl hydrolase). This classification supports its established role in the degradation of hydrolyzable tannins by cleaving gallic acid and glucose form ester linkages in tannic acid. In Fig. 10 , InterProScan analysis revealed two alpha/beta-hydrolase domains in the tannin acyl hydrolase (7K4O). The first domain spans approximately residues 40–250, and the second spans residues 290–520. These conserved domains are characteristic of esterases and contribute to the enzyme's catalytic function, likely forming a catalytic triad responsible for hydrolyzing ester bonds in tannin substrates. To explore the functional context of tannin acyl hydrolase (G3XLI5_ASPNA), a protein–protein interaction (PPI) network was generated using STRING-db. The resulting network shows that the enzyme has strong predicted interactions with other hydrolases, dehydrogenases (e.g., gpdA), and unknown hypothetical proteins (Fig. 11 ). These connections suggest that the enzyme may be involved in a larger enzymatic complex or pathway associated with the biodegradation of plant-derived polyphenols and energy metabolism. The network reveals both experimentally supported (pink edges) and computationally predicted (green, blue, black) interactions, indicating confidence in the protein’s role within a metabolically active hydrolase network. Notably, the association with glyceraldehyde-3-phosphate dehydrogenase (gpdA) hints at possible links between tannin degradation and primary carbon metabolism. 4. Discussion Tannin acyl hydrolase was chosen wisely for this study because of the reason that the native sequence does not need any ligand. Its presence in Aspergillus niger which is a thermostable fungus which can also withstand acidic environment. It makes it a good candidate for industrial processing like the cellulases and xylanases. As it is well known fact that the presence of cysteine residues reduce the flexibility of enzymes due to too many disulfide bonds, in the case of tannin acyl hydrolase there are very few cysteine residues which are ultimately necessary for stabilization of protein structure. Presence of huge amount of α-helices, tannin acyl hydrolase is considered thermostable enzyme. The Ramachandran plot showed that presence of over 90% of amino acids in the favored area, demonstrate the model's stability. The SAVES analysis also verified this result. The QMEAN analysis and ERRAT quality factor also lead to the fact that tannin acyl hydrolase shows the good quality features. InterproScan results gave information about the family classification and functions of the enzyme. The 2 domains of the enzyme were well suited for the hydrolatic activity of tannin. String analysis of tannin acyl hydrolase showed that because of the potential gene-neighbor connection or perhaps the expression of gene co-occurrence, the query protein showed good interacting connections. A known expressive interaction with proteins identified was determined as a result of the functional analysis. Aspergillus niger 's tannin acyl hydrolase is a recognized functionally stable enzyme for the breakdown of tannins. 5. Conclusion Our investigation indicates that tannin acyl hydrolase's model protein structure demonstrated good quality and possesses a stable crystallographic structures. This tannin degrading enzyme residing in the cytoplasm is intracellular, thermostable and hydrophilic in nature as revealed by its physicochemical characterization. High overall quality factor of 93.692 makes it the best enzyme for tannin degradation. Hence, it can be argued that the further studies can lead its utilization in the industrial sector. In addition to helping the researchers comprehend the structural and functional properties of a significant tannin-degrading enzyme, our study may also aid with the setup required in the lab to conduct experimental research and validate its activities. Declarations Conflict of interest The authors have no relevant financial or non-financial interests to disclose. Financial support No financial support for provided to the authors for this study. Acknowledgments Authors are grateful to Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University for supervising and guiding in this research. Author’s Contributions All authors contributed equally to the study conception and design. Literature review and selection of tools and data analysis were performed by Muhammad Aamir Sharif and Khawar Sharif. The first draft of the manuscript was written by Mumtaz Bibi and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Availability of Data and Materials All data generated or analyzed during this study are included in this published article. References Aguilar, C. N. and Gutierrez-Sanchez, G. 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Residues\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/eb622480b77f85f8e7bcfff3.png"},{"id":89350196,"identity":"d49f4d99-0a27-47a0-92f1-ce765ad1955e","added_by":"auto","created_at":"2025-08-19 06:00:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54607,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of different amino acids\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/c7b1a1c4e4e6d0652c0ab51f.png"},{"id":89350200,"identity":"ce928d75-1191-4936-a9d7-054fb6937f1e","added_by":"auto","created_at":"2025-08-19 06:00:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106121,"visible":true,"origin":"","legend":"\u003cp\u003eSOPMA Analysis of 7K4O\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/ccdc2e41d9aeb9e045ffdd2c.png"},{"id":89349511,"identity":"dc7cd102-b535-4d0b-86c0-592710f6c498","added_by":"auto","created_at":"2025-08-19 05:52:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47461,"visible":true,"origin":"","legend":"\u003cp\u003eSecondary Structure Composition\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/d2722682d50b4c91b04d5de0.png"},{"id":89350198,"identity":"7809d335-ea29-4694-95bb-41e93acb5532","added_by":"auto","created_at":"2025-08-19 06:00:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":196714,"visible":true,"origin":"","legend":"\u003cp\u003eVerify 3D Plot for 7K4O\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/5ff04f75fbbd68cd3537f4c3.png"},{"id":89351192,"identity":"61ce64df-da52-4e11-8a43-1b904528b41e","added_by":"auto","created_at":"2025-08-19 06:16:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":106763,"visible":true,"origin":"","legend":"\u003cp\u003eLocal Quality Estimate\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/f9767e832175b5ea7b71e1c2.png"},{"id":89350449,"identity":"e87aee5b-776e-4701-82f5-873f2c2e9a30","added_by":"auto","created_at":"2025-08-19 06:08:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":117582,"visible":true,"origin":"","legend":"\u003cp\u003eQuality Comparison with non-redundant set of PDB Structures\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/e0e571bf13ec84d7c193b5d4.png"},{"id":89349517,"identity":"2d716d93-3f17-44df-8d09-5d136c47c71b","added_by":"auto","created_at":"2025-08-19 05:52:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":142148,"visible":true,"origin":"","legend":"\u003cp\u003eQMEAN Z-Score for 7K4O\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/fc067922d35d737d4429cc8a.png"},{"id":89350451,"identity":"084f719d-e638-4172-b94f-52611b17d473","added_by":"auto","created_at":"2025-08-19 06:08:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":451809,"visible":true,"origin":"","legend":"\u003cp\u003eRamachandran Plot\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/68fcd0728b4511e6ed2c66ff.png"},{"id":89349522,"identity":"91704ae5-5a22-4fd1-84b2-0acf6bb2c98a","added_by":"auto","created_at":"2025-08-19 05:52:07","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":96524,"visible":true,"origin":"","legend":"\u003cp\u003eInterpro Scan showing domains of Tannase\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/999b69ff9ad727578c6056c1.png"},{"id":89349524,"identity":"71e4af8e-5d14-4df8-9ff6-9809b879bfb5","added_by":"auto","created_at":"2025-08-19 05:52:07","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":150714,"visible":true,"origin":"","legend":"\u003cp\u003eProtein-Protein associations for Tannin acyl hydrolase\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/13e35554595a12fd4b9f68f4.png"},{"id":89351773,"identity":"613c89bc-80b1-4afb-9a3c-da3102e6c5c0","added_by":"auto","created_at":"2025-08-19 06:24:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2150646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7384741/v1/3aa2bf56-968b-43d1-a7d1-44589058e51f.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eStructural and Functional Analysis of Tannase from \u003cem\u003eAspergillus niger\u003c/em\u003e: An Insilico Approach for Wine Industry Waste Treatment\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWine industry is continuously growing and there is increase in the demand of the wine globally. In the year 2025, wine industry is estimated to meet the demand, industries focus more on production and pay less attention to the waste treatment. The wastes produced as a result belong to different categories like alkaloids, antibiotics, phenolics, resins, saponins, sterols, tannins, terpenes, and volatile oils. Waste from the wine business includes grape pomace, seed peel, stalks, and vine leaves. Since these wastes contain organic matter with high concentrations of volatile and phenolic chemicals, they pollute the environment if left untreated or not recycled. For instance, some wastewaters can present biological oxygen demands (BOD) higher than 5 g/L, low pH (\u0026lt;\u0026thinsp;5), and active microbial populations(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIndustry statistics show that, despite the inconvenience of alcohol, the waste produced during the winemaking process is typically employed as vineyard fertilizer and animal feed. The grape marc is also used for distillation in the alcohol winery. However, most of this waste is still disposed without any previous treatment resulting in damage to the environment as, for example, the contamination of surface and ground water(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTannins are a group of water-soluble polyphenolic compounds (with molecular weight ranging from 0.5 \u0026minus;\u0026thinsp;20 kD) naturally found in plants and have been reported to be the fourth most abundant plant constituents after cellulose, hemicellulose and lignin(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). They are amorphous, astringent substances occurring widely in the bark, wood, leaves, and resinous exudations of plants (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Biochemically, tannins are sort of secondary metabolites predominantly available in plant-based foodstuffsand beverages grapes, blackberries, strawberries, walnuts, cashew nuts, hazelnuts, mangoes,and tea(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Tannins are considered defensive molecules to protect plant tissues from herbivorous attacks because of their astringent taste(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Tannins possess antioxidant activity. This property is related to their chemical structure as they possess phenolic rings able to bind to a wide range of molecules and act as electron scavengers to trap ions and radicals(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGalloyl ester, glucose, and gallic acid are produced through the hydrolysis of the ester link of hydrolyzable tannin by tannase (tannin acyl hydrolase, E.C 3.1.1.20). Numerous species, including bacteria, yeast, and fungi, biosynthesize the inducible enzyme tannase. Gallic acid is the major tannic acid hydrolytic product and is applied in cosmetics and in the synthesis of various antioxidants(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Through the process of hydrolysis, it lowers the burden of tannins and yields glucose and gallic acid, which are used as fuel. The production of precipitates in beverages containing other molecules is facilitated by the high tannin content found in coffee-flavored beverages, fruit juices, wine, beer, and iced tea. Tannases are used to remove these undesired properties from the fruit juices(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). It is considered an environmentally significant biocatalyst as well. The use of biomass from natural sources has not been fully examined in any of the reviews, despite the fact that they have all focused on the screening, manufacture, characterisation, and purification of tannase from diverse sources.\u003c/p\u003e\u003cp\u003eAspergillus niger's fungal tannase crystal structure is the first to be published. A bowl-shaped hemispherical shape with a surface concavity enclosed by N-linked glycans is formed by the enzyme's characteristic α/β-hydrolase-fold domain and a large inserted cap domain. Gallic acid forms two hydrogen-bonding networks with nearby residues at the intersection of the two domains within the concavity. One involves residues from the hydrolase-fold domain, including those from the catalytic triad, which is made up of Ser206, His485, and Asp439, and is formed around the carboxyl group of gallic acid. The latter is created around the compound's three hydroxyl groups and involves residues primarily from the cap domain, such as Gln238, Gln239, His242, and Ser441. Gallic acid forms a hydrophobic bond with Ile442 to form a sandwich-like bond. All of these residues are found to be highly conserved among fungal and yeast tannases(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) .\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Sequence retrieval\u003c/h2\u003e\u003cp\u003eThe RCSB-PDB database contains the experimentally determined sequences and structures of proteins. Tannin Acyl Hydrolase's sequence was obtained from the RCSB-PDB [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e] database and subsequently downloaded in PDB and FASTA formats.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Primary Structure Analysis and Subcellular Localization\u003c/h2\u003e\u003cp\u003eThe computed parameters like molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (GRAVY) were all determined by the Protparam database (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)[\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCELLO subcellular localization predictor V2.5 [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cello.life.nctu.edu.tw/\u003c/span\u003e\u003cspan address=\"https://cello.life.nctu.edu.tw/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e] was used to predict subcellular location.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Secondary Structure Analysis\u003c/h2\u003e\u003cp\u003eNuclear Magnetic Resonance method is performed to predict the structure of protein normally in the laboratory work(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Using a web-based server Alignment-Based Self-Optimized Prediction Method, or SOPMA [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html\u003c/span\u003e\u003cspan address=\"https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e], the secondary structure of the recovered protein sequence was predicted based on the percentage of α-helices, β-turns, and β-sheets in the protein(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Analysis of Tertiary Structures, Validation, and Homology Modeling\u003c/h2\u003e\u003cp\u003eX-ray crystallography is typically used for determining the proteins' tertiary structure, however, some online webservers like AlphaFold and Phyre 2.2 can be used to determine computationally. Phyre 2.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sbg.bio.ic.ac.uk/phyre2/\u003c/span\u003e\u003cspan address=\"https://www.sbg.bio.ic.ac.uk/phyre2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized in this particular situation. High resolution 3D protein structure was acquired by using SAVES server v6.1 [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://saves.mbi.ucla.edu/\u003c/span\u003e\u003cspan address=\"https://saves.mbi.ucla.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e]. In SAVES v.1, the overall quality factor for the crystallographic protein structure was confirmed; 3D-1D score was determined by VERIFY, different stereochemical parameters of residues were obtained using WHATCHECK and PROCHECK found out the quality of protein structure by evaluating residue-by-reside geometry and overall structure geometry(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Using SAVES, 3D structure was also viewed and downloaded.\u003c/p\u003e\u003cp\u003eRamachandran plot,QMEAN, solvation, local quality estimate and torsion were determined using a webserver of Structure Assessment by SwissModel [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/assess\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/assess\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Functional Analysis and Protein-protein interactions\u003c/h2\u003e\u003cp\u003eInterPro Go terms and PANTHER Go terms were acquired using the Interpro portal [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e]. Protein-protein interactions with more closely related proteins were predicted with the use of the String database [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Sequence Retrieval\u003c/h2\u003e\u003cp\u003eThe protein sequence of Tannin acyl hydrolase from \u003cem\u003easperillus niger\u003c/em\u003e 7K4O was obtained from RCSB-PDB database in FASTA and PDB formats.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Primary Structure Analysis\u003c/h2\u003e\u003cp\u003eTheoretical overview of the nature of the Tannin acyl hydrolase was determined by the computational analysis of its physicochemical qualities as Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparative Physicochemical attributes of Enzymes\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003eComparative Physicochemical attributes of Enzymes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSerial No\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEnzyme\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMolecular Weight\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAmino Acids\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInstability Index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTheoretical pI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAliphatic Index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eGRAVY\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTannin Acyl Hydrolase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60773.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e554\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e72.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-0.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAs if the theoretical pI value is less than 7 then it is considered acidic and also there are more negatively charged amino acids therefore, Tannin acyl hydrolase consists mainly of acidic amino acids. Instability index of tannin acyl hydrolase is less than 40 which makes it a stable protein. If and only if the instability index value had been higher than 40, the protein would have been unstable. A higher aliphatic index score indicates greater thermotolerance and it shows the presence of thermostable globular proteins. If an enzyme has higher aliphatic index, it is considered for industrial use. Tannin acyl hydrolase has negative GRAVY value representing its hydrophilic nature and ultimately better water-protein interactions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGlycine(Gly), alanine (Ala) and threonine (Thr) appear to be the most abundant amino acids (9.0%, 8.3% and 8.3% respectively). Compared to the positively charged amino acids (Arg\u0026thinsp;+\u0026thinsp;Lys), the negatively charged amino acids (Asp\u0026thinsp;+\u0026thinsp;Glu) were significantly greater as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Secondary Structure Analysis\u003c/h2\u003e\u003cp\u003eIn general terms, protein's secondary structure is constructed based on its primary structure using wet lab techniques. In this analysis, computational method was applied for studying the tannic acyl hydrolase's secondary structure and a webserver SOPMA was used as is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Like laccases, tannin acyl hydrolase was also found to be rich in random coils. The presence of high concentration of random coils confirms the flexibility and conformational changes of the protein. The higher value of α-helices in tannin acyl hydrolase exhibits significant thermal resistance on the basis of its intrinsic stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Analysis of tertiary structures, validation and homology modeling\u003c/h2\u003e\u003cp\u003eThe Structure Analysis and Verification Server (SAVES v6.1) was used to compute the quality of the standard X-ray crystallographic protein structure. Tannin acyl hydrolase was verified as an excellent protein structure by the use of ERRAT, WhatCheck,, ProChcck and PROVES tools run by SAVES. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that this protein structure was evaluated as useful by both its quality and quantity. A 3D-1D ratio according to scores was also provided by this tool (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For the practical application of structure interpretation, local model quality was evaluated with SwissModel and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e displays the target-template alignment prediction. The accuracy of the local model using the tannin acyl hydrolase residues along its sequence is accurately shown in the same figure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor homology modeling studies, SwissModel Structure Assessment tool estimated the good model quality wise. The findings of a comparison between tannin acyl hydrolase and the non-redundant PDB structure are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQuality Assessment Score\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003eQuality Assessment Score\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3D\u0026ndash;1D Compatibility (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eERRAT Quality (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eQMEAN Z-Score\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAmino Acids in Favored Regions (Ramachandran Plot, %)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e83.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e93.692\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e90.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAn ideal protein's QMEAN Z-score falls between 0 and 1, so here, tannin acyl hydrolase has QMEAN Z-score value is 0.35 as represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. ERRAT server also evaluated the error regions by examining the non-bonding atomic association statistics. 93.692 is considered a very satisfactory ERRAT value. The SAVES webserver's PROCHECK program yielded the Ramachandran plot between the amino acids' φ-Ψ torsion angles. Rare left-handed α-helices were among the conformations found in the allowed area in quadrant 1 of the Ramachandran plots. Quadrant 2 had the most advantageous sterically permitted β-strand conformations, Quadrant 3 had right-handed α-helices, and Quadrant 4 had conformations with the disfavored area. The percentage was residues was found to be 90.7% in in the most favored regions of Ramachandran plots. Hence, it proved the protein model was of satisfactory quality. A few residues present in the disallowed area confirmed the reliability of tannin acyl hydrolase as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Functional Analysis and protein-protein Associations\u003c/h2\u003e\u003cp\u003eThe tannin acyl hydrolase from \u003cem\u003eAspergillus niger\u003c/em\u003e (PDB ID: 7K4O) was functionally annotated using InterProScan. The analysis revealed that the enzyme belongs to the Feruloyl esterase B-related family and is functionally classified as a Tannase (Tannin acyl hydrolase). This classification supports its established role in the degradation of hydrolyzable tannins by cleaving gallic acid and glucose form ester linkages in tannic acid.\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, InterProScan analysis revealed two alpha/beta-hydrolase domains in the tannin acyl hydrolase (7K4O). The first domain spans approximately residues 40\u0026ndash;250, and the second spans residues 290\u0026ndash;520. These conserved domains are characteristic of esterases and contribute to the enzyme's catalytic function, likely forming a catalytic triad responsible for hydrolyzing ester bonds in tannin substrates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo explore the functional context of tannin acyl hydrolase (G3XLI5_ASPNA), a protein\u0026ndash;protein interaction (PPI) network was generated using STRING-db. The resulting network shows that the enzyme has strong predicted interactions with other hydrolases, dehydrogenases (e.g., gpdA), and unknown hypothetical proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). These connections suggest that the enzyme may be involved in a larger enzymatic complex or pathway associated with the biodegradation of plant-derived polyphenols and energy metabolism.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe network reveals both experimentally supported (pink edges) and computationally predicted (green, blue, black) interactions, indicating confidence in the protein\u0026rsquo;s role within a metabolically active hydrolase network. Notably, the association with glyceraldehyde-3-phosphate dehydrogenase (gpdA) hints at possible links between tannin degradation and primary carbon metabolism.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTannin acyl hydrolase was chosen wisely for this study because of the reason that the native sequence does not need any ligand. Its presence in \u003cem\u003eAspergillus niger\u003c/em\u003e which is a thermostable fungus which can also withstand acidic environment. It makes it a good candidate for industrial processing like the cellulases and xylanases. As it is well known fact that the presence of cysteine residues reduce the flexibility of enzymes due to too many disulfide bonds, in the case of tannin acyl hydrolase there are very few cysteine residues which are ultimately necessary for stabilization of protein structure. Presence of huge amount of α-helices, tannin acyl hydrolase is considered thermostable enzyme. The Ramachandran plot showed that presence of over 90% of amino acids in the favored area, demonstrate the model's stability. The SAVES analysis also verified this result. The QMEAN analysis and ERRAT quality factor also lead to the fact that tannin acyl hydrolase shows the good quality features. InterproScan results gave information about the family classification and functions of the enzyme. The 2 domains of the enzyme were well suited for the hydrolatic activity of tannin. String analysis of tannin acyl hydrolase showed that because of the potential gene-neighbor connection or perhaps the expression of gene co-occurrence, the query protein showed good interacting connections. A known expressive interaction with proteins identified was determined as a result of the functional analysis. \u003cem\u003eAspergillus niger\u003c/em\u003e's tannin acyl hydrolase is a recognized functionally stable enzyme for the breakdown of tannins.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur investigation indicates that tannin acyl hydrolase's model protein structure demonstrated good quality and possesses a stable crystallographic structures. This tannin degrading enzyme residing in the cytoplasm is intracellular, thermostable and hydrophilic in nature as revealed by its physicochemical characterization. High overall quality factor of 93.692 makes it the best enzyme for tannin degradation. Hence, it can be argued that the further studies can lead its utilization in the industrial sector. In addition to helping the researchers comprehend the structural and functional properties of a significant tannin-degrading enzyme, our study may also aid with the setup required in the lab to conduct experimental research and validate its activities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eFinancial support\u003c/h2\u003e\n\u003cp\u003eNo financial support for provided to the authors for this study.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eAuthors are grateful to Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University for supervising and guiding in this research.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor\u0026rsquo;s Contributions\u003c/h2\u003e\n\u003cp\u003eAll authors contributed equally to the study conception and design. Literature review and selection of tools and data analysis were performed by Muhammad Aamir Sharif and Khawar Sharif. The first draft of the manuscript was written by Mumtaz Bibi and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAguilar, C. N. and Gutierrez-Sanchez, G. (2001) Review: Sources, Properties, Applications and Potential uses of Tannin Acyl Hydrolase. Food Science and Technology International\u003cem\u003e,\u003c/em\u003e \u003cstrong\u003e7,\u003c/strong\u003e 373-382.\u003c/li\u003e\n\u003cli\u003eAithal, M. and Belur, P. D. 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Technol\u003cem\u003e,\u003c/em\u003e \u003cstrong\u003e38,\u003c/strong\u003e 183\u0026ndash;197.\u003c/li\u003e\n\u003cli\u003eVuolo, M. M., Lima, V. S. and Mar\u0026oacute;stica, J. M. R. (2018) Phenolic compounds: structure, classification, and antioxidant power. In Bioactive Compounds: Health Benefits and Potential Applications. Elsevier Inc, Amsterdam Netherlands\u003cstrong\u003e,\u003c/strong\u003e 33-50.\u003c/li\u003e\n\u003cli\u003eZivkovic, J., Mujic, I., Zekovic, Z., Nikolic, G., Vidovic, S. and Mujic, A. (2009) Extraction and analysis of condensed tannins in Castanea sativa Mill. Journal of Central European. Agriculture\u003cem\u003e,\u003c/em\u003e \u003cstrong\u003e10,\u003c/strong\u003e 283\u0026ndash;288.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Bahauddin Zakariya University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tannase, in silico, Aspergillus niger, wine industry, waste treatment","lastPublishedDoi":"10.21203/rs.3.rs-7384741/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7384741/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003eThe worldwide wine industry is one of the most significant and rapidly expanding sectors and contributes billions of dollars to the world economy each year. The waste produced as a result of wine production of great concern for the environment safety. Tannin is one of the materials present in the wine waste which can be hydrolyzed into gallic acid and glucose by an enzyme tannin acyl hydrolase which is extracted from a fungus \u003cem\u003easpergillus niger\u003c/em\u003e. Our effort involves computational study for analyzing the structural and functional characteristics of an enzyme tannin acyl hydrolase which is extracted from a fungus \u003cem\u003easpergillus niger.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe protein sequence of tannin acyl hydrolase was taken from the RCSB-PDB database. Afterwards, physicochemical characteristics and primary structure analysis were determined using Protparam webserver. The online bioinformatics tool SOPMA was used to measure number of GRAVY, amino acids, aliphatic index, theoretical pI and instability index. CELLO tool helped to determine the subcellular localization. SAVES server v6.1 helped to obtain the overall quality factor, 3D-1D ratio, Z-score and other stereochemical properties. Ramachandran plot, local quality estimate, QMEAN and 3D structure assessment was determined with the help of SwissModel. The family classification of protein and domain study was performed using InterproScan software. Finally, STRING database helped to visualize protein-protein interactions for the functional study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult:\u003c/strong\u003eTannin acyl hydrolase mostly constitutes acidic amino acids and possesses thermostability. Higher aliphatic index proved its property of being thermostable. The fact that over 90% of the amino acids are present in the Ramachandran plot's preferred region indicates that tannic acyl hydrolase is a stable protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003eThis analysis aims to provide useful insights for tannin acyl hydrolase to be used in bioremediation of wine industry waste and gives important knowledge about it for the experimental laboratory work.\u003c/p\u003e","manuscriptTitle":"Structural and Functional Analysis of Tannase from Aspergillus niger: An Insilico Approach for Wine Industry Waste Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 05:52:02","doi":"10.21203/rs.3.rs-7384741/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1aba66fd-dbb8-4a49-afd2-6614c3e43e2e","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-19T05:52:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 05:52:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7384741","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7384741","identity":"rs-7384741","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}