Thermostable proteases are one of the pivotal enzymatic groups which play fundamental roles in biotechnologyrelated industries. The identification of bacterial thermostable enzymes through screening programs is a time and cost consuming process. So, extensive bioinformatics and experimental studies have been conducted to reveal thermo stabilizing factors. The current study was aimed to evaluate distinctive indicators among 33 thermostable and 10 mesostable proteolytic enzymes. The frequency of individual amino acids, aliphatic indexes, melting temperatures, isoelectric points, as well as, the frequency of AXXXA and GXXXG motifs were determined and compared among these enzymes. In addition, types of proteolytic enzymes and their active sites were assigned. Moreover, the frequency of alpha helixes, polar surface regions, and packing volumes of these enzymes with the known structures were characterized. Results showed that the frequency of Ala and AXXXA motifs were significantly higher in thermostable proteolytic enzymes, while they possess lower contents of Met, His, Lys and Leu in comparison to mesostable enzymes (P<0.05). According to statistical analysis, thermostable proteolytic enzymes indicated meaningful lower packing volumes than mesostable enzymes (P<0.05). Findings of the current study in addition to more detailed investigations on the thermostability mechanisms of various protein families are essential for designing more efficient industrial enzymes with functional properties at high temperatures.
Bioinformatics analysis, Protein engineering, Proteolytic enzyme, Thermostability
Argos P., Rossmann M. G., Grau U. M., Zuber H., Frank G. and Tratschin J. D. (1979) Thermal stability and protein structure. Biochemistry 18:25, 5698-5703.
Atlan D., Gilbert C., Blanc B. and Portalier R. (1994) Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397. Microbiology 140:3, 527-535.
Banbula A., Mak P., Bugno M., Silberring J., Dubin A., Nelson D., et al. (1999) Prolyl tripeptidyl peptidase from Porphyromonas gingivalis. A novel enzyme with possible pathological implications for the development of periodontitis. The Journal of Biological Chemistry 274:14, 9246-9252.
Berman H. M., Westbrook j., Feng Z., Gilliland G., Bhat T. N., Weissig H., et al. (2000) The protein data bank. Nucleic Acids Research 28:235-242.
Barzkar N., Homaei A., Hemmati R. and Patel S. (2018) Thermostable marine microbial proteases for industrial applications: scopes and risks. Extremophiles 22:3, 335-346.
Benjelloun-Touimi Z., Tahar M. S., Montecucco C., Sansonetti P. J. and Parsot C. (1998) SepA, the 110 kDa protein secreted by Shigella flexneri: twodomain structure and proteolytic activity. Microbiology 144:7, 1815-1822.
Bjelke J. R., Christensen J., Nielsen P. F., Branner S., Kanstrup A. B., Wagtmann N., et al. (2006) Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV. Biochemical Journal 396:2, 391-399.
Cambillau C. and Claverie J. M. (2000) Structural and genomic correlates of hyperthermostability. Journal of Biological Chemistry 275:42, 32383- 32386.
Catanzano F., Barone G., Graziano G. and Capasso S. (1997) Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A. Protein Science 6:8, 1682-1693.
Chakravarty S. and Varadarajan R. (2000) Elucidation of determinants of protein stability through genome sequence analysis. FEBS Letters 470:1, 65-69.
Chakravorty D., Parameswaran S., Dubey V. K. and Patra S. (2011) In silico characterization of thermostable lipases. Extremophiles 15:1, 89-103.
Consortium U. (2018) UniProt: a worldwide hub of protein knowledge. Nucleic Acids Research 47:D1, D506-D515.
Creighton T. E. (1993) Proteins: structures and molecular properties. Second ed. W. H. Freeman and Co. New York, 80 100 pp.
Dadshahi Z., Homaei A., Zeinali F., Sajedi R. H. and Khajeh K. (2016) Extraction and purification of a highly thermostable alkaline caseinolytic protease from wastes Penaeus vannamei suitable for food and detergent industries. Food Chemistry 202: 110-115.
Das R. and Gerstein M. (2000) The stability of thermophilic proteins: a study based on comprehensive genome comparison. Functional and Integrative Genomics 1:1, 76-88.
Deng A., Wu J., Zhang Y., Zhang G. and Wen T. (2010) Purification and characterization of a surfactant-stable high alkaline protease from Bacillus sp. B001. Bioresource Technology 101:18, 7100-7106.
Dill K. A. (1990) Dominant forces in protein folding. Biochemistry 29:31, 7133-7155.
Esakkiraj P., Meleppat B., Lakra A. K., Ayyanna R. and Arul V. (2016) Cloning, expression, characterization and application of protease produced by Bacillus cereus PMW8. RSC Advances 6:45, 38611-38616.
Fenster K. M., Parkin K. L. and Steele J. L. (1997) Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32. Journal of Bacteriology 179:8, 2529-2533.
Fujii M., Takagi M., Imanaka T. and Aiba S. (1983) Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis. Journal of Bacteriology 154:2, 831-837.
Gödde C., Sahm K., Brouns S. J., Kluskens L. D., van der Oost J., de Vos W. M., et al. (2005) Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli. Applied and Environmental Microbiology 71:7, 3951-3958.
Goldstein J., Kordula T., Moon J., Mayo J. and Travis J. (2005) Characterization of an extracellular dipeptidase from Streptococcus gordonii FSS2. Infection and Immunity 73:2, 1256-1259.
Gromiha M. M. and Suresh M. X. (2008) Discrimination of mesophilic and thermophilic proteins using machine learning algorithms. Proteins 70:4, 1274-1279.
Grys T. E., Walters L. L. and Welch R. A. (2006) Characterization of the StcE protease activity of Escherichia coli O157: H7. Journal of Bacteriology 188:13, 4646-4653.
Haddar A., Fakhfakh-Zouari N., Hmidet N., Frikha F., Nasri M. and Kamoun A. S. (2010) Low-cost fermentation medium for alkaline protease production by Bacillus mojavensis A21 using hulled grain of wheat and sardinella peptone. Journal of Bioscience and Bioengineering 110:3, 288-294.
Haney P. J., Badger J. H., Buldak G. L., Reich C. I., Woese C. R. and Olsen G. J. (1999) Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species. Proceedings of the National Academy of Sciences 96:7, 3578- 3583.
Hellendoorn M. A., Franke-Fayard B. M., Mierau I., Venema G. and Kok J. (1997) Cloning and analysis of the pepV dipeptidase gene of Lactococcus lactis MG1363. Journal of Bacteriology 179:11, 3410- 3415.
Homaei A. and Etemadipour R. (2015) Improving the activity and stability of actinidin by immobilization on gold nanorods. International Journal of Biological Macromolecules 72:1176- 1181.
Homaei A. A., Sajedi R. H., Sariri R., Seyfzadeh S. and Stevanato R. (2010) Cysteine enhances activity and stability of immobilized papain. Amino Acids 38:3, 937-942.
Iqbalsyah T. M., Malahayati, Atikah, and Febriani. (2019) Purification and partial characterization of a thermo-halostable protease produced by Geobacillus sp. strain PLS A isolated from undersea fumaroles. Journal of Taibah University for Science 13:1, 850- 857.
Jasilionis A., Kaupinis A., Ger M., Valius M., Chitavichius D. and Kuisiene N. (2012) Gene expression and activity analysis of the first thermophilic U32 peptidase. Central European Journal of Biology 7:4, 587-595.
Jasilionis A. and Kuisiene N. (2015) Characterization of a novel thermostable oligopeptidase from Geobacillus thermoleovorans DSM 15325. Journal of Microbiology and Biotechnology 25:7, 1070-1083.
Kamal M., Höög J. O., Kaiser R., Shafqat J., Razzaki T., Zaidi Z. H., et al. (1995) Isolation, characterization and structure of subtilisin from a thermostable Bacillus subtilis isolate. FEBS Letters 374:3, 363-366.
Kaur I., Kocher G. S. and Gupta V. (2012) Molecular cloning and nucleotide sequence of the gene for an alkaline protease from Bacillus circulans MTCC 7906. Indian Journal of Microbiology 52:4, 630-637.
Kleiger G., Grothe R., Mallick P. and Eisenberg D. (2002) GXXXG and AXXXA: common α-helical interaction motifs in proteins, particularly in extremophiles. Biochemistry 41:19, 5990-5997.
Kobayashi T., Hakamada Y., Adachi S., Hitomi J., Yoshimatsu T., Koike K., et al. (1995) Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Applied Microbiology and Biotechnology 43:3, 473-481.
Kubo M. and Imanaka T. (1988) Cloning and nucleotide sequence of the highly thermostable neutral protease gene from Bacillus stearothermophilus. Microbiology 134:7, 1883- 1892.
Kȕhn S. and Fortnagel P. (1993) Molecular cloning and nucleotide sequence of the gene encoding a calcium-dependent exoproteinase from Bacillus megaterium ATCC 14581. Microbiology 139:1, 39- 47.
Kumar C. (2002) Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus. Letters in Applied Microbiology. 34:1, 13-17.
Kumar S., Tsai C. J. and Nussinov R. (2000) Factors enhancing protein thermostability. Protein Engineering 13:3, 179-191.
Ladbury J. E., Wynn R., Thomson J. A. and Sturtevant J. M. (1995) Substitution of charged residues into the hydrophobic core of Escherichia coli thioredoxin results in a change in heat capacity of the native protein. Biochemistry 34:7, 2148-2152.
Lee H. S., Kim Y. J., Bae S. S., Jeon J. H., Lim J. K., Kang S. G., et al. (2006) Overexpression and characterization of a carboxypeptidase from the hyperthermophilic archaeon Thermococcus sp. NA1. Bioscience, Biotechnology, and Biochemistry 70:5, 1140-1147.
Lee J. K., Kim Y. O., Kim H. K., Park Y. S. and Oh T. K. (1996) Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene. Bioscience, Biotechnology, and Biochemistry 60:5, 840-846.
Lee S. H., Minagawa E., Taguchi H., Matsuzawa H., Ohta T., Kaminogawa S., et al. (1992) Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1. Bioscience, Biotechnology, and Biochemistry 56:11, 1839-1844.
Li A. N. and Li D. C. (2009) Cloning, expression and characterization of the serine protease gene from Chaetomium thermophilum. Journal of Applied Microbiology 106:2, 369-380.
Liang H. K., Huang C. M., Ko M. T. and Hwang J. K. (2005) Amino acid coupling patterns in thermophilic proteins. Proteins 59:1, 58-63.
Maciver B., McHale R. H., Saul D. J. and Bergquist P. L. (1994) Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli. Applied and Environmental Microbiology 60:11, 3981-3988.
Marchler-Bauer A., Bo Y., Han L., He J., Lanczycki C. J., Lu S., et al. (2016) CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Research 45:D1, D200-D203.
Medrano F., Alonso J., Garcia J., Romero A., Bode W. and Gomis????Ruth F. (1998) Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family. The EMBO Journal 17:1, 1-9.
Morya V. K., Yadav S., Kim E. K. and Yadav D. (2012) In silico characterization of alkaline proteases from different species of Aspergillus. Applied Biochemistry and Biotechnology 166:1, 243-257.
Motoshima H., Azuma N., Kaminogawa S., Ono M., Minagawa E., Matsuzawa H., et al. (1990) Molecular cloning and nucleotide sequence of the aminopeptidase T gene of Thermus aquaticus YT-1 and its high-level expression in Escherichia coli. Agricultural and biological chemistry 54:9, 2385- 2392.
Munro G. K., McHale R. H., Saul D. J., Reeves R. A. and Bergquist P. L. (1995) A gene encoding a thermophilic alkaline serine proteinase from Thermus sp. strain Rt41A and its expression in Escherichia coli. Microbiology 141:7, 1731-1738.
Ohara-Nemoto Y., Rouf S. M., Naito M., Yanase A., Tetsuo F., Ono T., et al. (2014) Identification and characterization of prokaryotic dipeptidyl-peptidase 5 from Porphyromonas gingivalis. The Journal of Biological Chemistry 289:9, 5436-5448.
Pack S. P., Kang T. J. and Yoo Y. J. (2013) Protein thermostabilizing factors: high relative occurrence of amino acids, residual properties, and secondary structure type in different residual state. Applied Biochemistry and Biotechnology 171:5, 1212-1226.
Pack S. P. and Yoo Y. J. (2004) Protein thermostability: structure-based difference of amino acid between thermophilic and mesophilic proteins. Journal of Biotechnology 111:3, 269-277.
Panja A. S., Bandopadhyay, B. and Maiti, S. (2015) Protein thermostability is owing to their preferences to non-polar smaller volume amino acids, variations in residual physico-chemical properties and more salt-bridges. PLoS ONE 10:7, e0131495.
Patel S. (2017) A critical review on serine protease: key immune manipulator and pathology mediator. Allergologia et Immunopathologia 45:6, 579-591.
Pellegrini M., Marcotte E. M., Thompson M. J., Eisenberg D. and Yeates T. O. (1999) Assigning protein functions by comparative genome analysis: protein phylogenetic profiles. Proceedings of the National Academy of Sciences 96:8, 4285-4288.
Pombejra S. N., Jamklang M., Uhrig J. P., Vu K. and Gelli A. (2018) The structure-function analysis of the Mpr1 metalloprotease determinants of activity during migration of fungal cells across the bloodbrain barrier. PLoS ONE 13:8, e0203020.
Ramadurai L., Lockwood K. J., Lockwood J., Nadakavukaren M. J. and Jayaswal R. K. (1999) Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus. Microbiology 145:4, 801-808.
Ramakrishnan V., Thambidurai Y., Rajasekharan S. K. and Mohanvel S. K. (2017) Partial characterization and cloning of protease from Bacillus. Asian Journal of Pharmaceutical and Clinical Research 10:10, 187-191.
Ran L. Y., Su H. N., Zhou M. Y., Wang L., Chen X. L., Xie B. B., et al. (2014) Characterization of a novel subtilisin-like protease myroicolsin from deep sea bacterium Myroides profundi D25 and molecular insight into its collagenolytic mechanism. The Journal of Biological Chemistry 289:9, 6041-6053.
Raveendran S., Parameswaran B., Beevi Ummalyma S., Abraham A., Kuruvilla Mathew A., Madhavan A., et al. (2018) Applications of microbial enzymes in food industry. Food Technology and Biotechnology 56:1, 16-30.
Rawlings N. D., Barrett A. J., Thomas P. D., Huang X., Bateman A. and Finn R.D. (2017) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Research 46:D1, D624-D632.
Rawlings N. D., Morton F. R., Kok C. Y., Kong J. and Barrett A. J. (2007) MEROPS: the peptidase database. Nucleic Acids Research 36:suppl_1, D320-D325.
Razvi A. and Scholtz J. M. (2006) Lessons in stability from thermophilic proteins. Protein Science 15:7, 1569-1578.
Razzaq A., Shamsi S., Ali A., Ali Q., Sajjad M., Malik A. and Ashraf M. (2019) Microbial proteases applications. Frontiers in Bioengineering and Biotechnology 7:110.
Reddi R., Arya T., Kishor C., Gumpena R., Ganji R. J., Bhukya S., et al. (2014) Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents. The FEBS Journal 281:18, 4240-4248.
Russell R. J., Hough D. W., Danson M. J. and Taylor G. L. (1994) The crystal structure of citrate synthase from the thermophilic archaeon, Thermoplasma acidophilum. Structure 2:12, 1157-1167.
Sadeghi M., Naderi-Manesh H., Zarrabi M. and Ranjbar B. (2006) Effective factors in thermostability of thermophilic proteins. Biophysical Chemistry 119:3, 256-270.
Salarizadeh N., Hasannia S., Akbari Noghabi K. and Hassan Sajedi R. (2014) Purification and characterization of 50 kDa extracellular metalloprotease from Serratia sp. ZF03. Iranian Journal of Biotechnology12:3, 18-27.
Saul D. J., Williams L. C., Toogood H. S., Daniel R. M. and Bergquist P. L. (1996) Sequence of the gene encoding a highly thermostable neutral proteinase from Bacillus sp. strain EA1: expression in Escherichia coli and characterisation. Biochimica et Biophysica Acta 1308:1, 74-80.
Savijoki K. and Palva A. (2000) Purification and molecular characterization of a tripeptidase (PepT) from Lactobacillus helveticus. Applied and Environmental Microbiology 66:2, 794-800.
Seo J. M., Ji G. E., Cho S. H., Park M. S. and Lee H. J. (2007) Characterization of a Bifidobacterium longum BORI dipeptidase belonging to the U34 family. Applied and Environmental Microbiology 73:17, 5598-5606.
Sharipova M., Balaban N., Kayumov A., Kirillova Y., Mardanova A., Gabdrakhmanova L., et al. (2008) The expression of the serine proteinase gene of Bacillus intermedius in Bacillus subtilis. Microbiological Research 163:1, 39-50.
Simossis V. A. and Heringa J. (2005) PRALINE: a multiple sequence alignment toolbox that integrates homology-extended and secondary structure information. Nucleic Acids Research 33:suppl_2, W289-W294.
Souza P. M. D., Bittencourt, M. L. D. A., Caprara, C. C., Freitas, M. D., Almeida, R. P. C. D., Silveira, D., et al. (2015) A biotechnology perspective of fungal proteases. Brazilian Journal of Microbiology 46:2, 337-346.
Szilágyi A. and Závodszky P. (2000) Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure 8:5, 493-504.
Takami H., Kobayashi T., Aono R. and Horikoshi K. (1992) Molecular cloning, nucleotide sequence and expression of the structural gene for a thermostable alkaline protease from Bacillus sp. no. AH-101. Applied Microbiology and Biotechnology 38:1, 101- 108.
Tavano O. L., Berenguer????Murcia A., Secundo F. and Fernandez????Lafuente R. (2018) Biotechnological applications of proteases in food technology. Comprehensive Reviews in Food Science and Food Safety 17:2, 412-436.
Taylor T. J. and Vaisman I. I. (2010) Discrimination of thermophilic and mesophilic proteins. BMC Structural Biology10:1, S5.
Tekaia F., Yeramian E. and Dujon B. (2002) Amino acid composition of genomes, lifestyles of organisms, and evolutionary trends: a global picture with correspondence analysis. Gene 297:1-2, 51-60.
Tomazic S. J. and Klibanov A. M. (1988) Mechanisms of irreversible thermal inactivation of Bacillus alpha-amylases. Journal of Biological Chemistry 263:7, 3086-3091.
Tran L., Wu X. and Wong S. (1991) Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis. Journal of Bacteriology 173:20, 6364 6372.
Tsuchiya K., Ikeda I., Tsuchiya T. and Kimura T. (1997) Cloning and expression of an intracellular alkaline protease gene from alkalophilic Thermoactinomyces sp. HS682. Bioscience, Biotechnology, and Biochemistry 61:2, 298-303.
Van den Burg B., Enequist H., Van der Haar M., Eijsink V., Stulp B. and Venema G. (1991) A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product. Journal of Bacteriology 173:13, 4107-4115.
Vecerek B. and Kyslik P. (1995) Cloning and sequencing of the neutral protease-encoding gene from a thermophilic strain of Bacillus sp. Gene 158:1, 147-148.
Vogt G., Woell S. and Argos P. (1997) Protein thermal stability, hydrogen bonds, and ion pairs. Journal of Molecular Biology 269:4, 631-643.
Wakarchuk W. W., Sung W. L., Campbell R. L., Cunningham A., Watson D. C. and Yaguchi M. (1994) Thermostabilization of the Bacillus circulans xylanase by the introduction of disulfide bonds. Protein Engineering, Design and Selection 7:11, 1379-1386.
Walker N. D., McEwan N. R. and Wallace R. J. (2003) Cloning and functional expression of dipeptidyl peptidase IV from the ruminal bacterium Prevotella albensis M384T. Microbiology 149:8, 2227-2234.
Wang S. L., Wang C. Y. and Huang T. Y. (2008) Microbial reclamation of squid pen for the production of a novel extracellular serine protease by Lactobacillus paracasei subsp paracasei TKU012. Bioresource Technology 99:9, 3411-3417.
Wani A. H., Sharma M., Salwan R., Singh G., Chahota R. and Verma S. (2016) Cloning, expression, and functional characterization of serine protease Aprv2 from virulent isolate Dichelobacter nodosus of Indian origin. Applied Biochemistry and Biotechnology 180:3, 576-587.
Willard L., Ranjan A., Zhang H., Monzavi H., Boyko R. F., Sykes B. D., et al. (2003) VADAR: a web server for quantitative evaluation of protein structure quality. Nucleic Acids Research 31:13, 3316-3319.
Wu J., Bian Y., Tang B., Chen X., Shen P. and Peng Z. (2004) Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops. FEMS Microbiology Letters 230:2, 251-258.
Xin Y., Sun Z., Chen Q., Wang J., Wang Y., Luogong L., et al. (2015) Purification and characterization of a novel extracellular thermostable alkaline protease from Streptomyces sp. M30. Journal of Microbiology and Biotechnology 25:11, 1944-1953.
Xu Z., Liu Y., Yang Y., Jiang W., Arnold E. and Ding J. (2003) Crystal structure of D-hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability. Journal of Bacteriology 185:14, 4038-4049.
Yamagata Y. and Ichishima E. (1995) A new alkaline serine protease from alkalophilic Bacillus sp.: cloning, sequencing, and characterization of an intracellular protease. Current Microbiology 30:6, 357-366.
Zhang X., Chen S., Hu Z., Zhang L. and Wang H. (2009) Expression and characterization of two functional methionine aminopeptidases from Mycobacterium tuberculosis H37Rv. Current Microbiology 59:5, 520-525.
Zhou X. X., Wang Y. B., Pan Y. J. and Li W. F. (2008) Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins. Amino Acids 34:1, 25-33.
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