Abstract
A halophilic bacterium, Virgibacillus sp. strain CD6, was isolated from salted fish and its extracellular protease was characterized. Protease production was found to be highest when yeast extract was used as nitrogen source for growth. The protease exhibited stability at wide range of salt concentration (0–12.5%, w/v), temperatures (20–60 °C), and pH (4–10) with maximum activity at 10.0% (w/v) NaCl, 60 °C, pH 7 and 10, indicating its polyextremophilicity. The protease activity was enhanced in the presence of Mg2+, Mn2+, Cd2+, and Al3+ (107–122% relative activity), and with retention of activity > 80% for all of other metal ions examined (K+, Ca2+, Cu2+, Co2+, Ni2+, Zn2+, and Fe3+). Both PMSF and EDTA inhibited protease activity, denoting serine protease and metalloprotease properties, respectively. High stability (> 70%) was demonstrated in the presence of organic solvents and detergent constituents, and the extracellular protease from strain CD6 was also found to be compatible in commercial detergents. Proteinaceous stain removal efficacy revealed that crude protease of strain CD6 could significantly enhance the performance of commercial detergent. The protease from Virgibacillus sp. strain CD6 could serve as a promising alternative for various applications, especially in detergent industry.
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Abraham J, Gea T, Sánchez A (2014) Substitution of chemical dehairing by proteases from solid-state fermentation of hair wastes. J Clean Prod 74:191–198
Cao Z-J, Zhang Q, Wei D-K, Chen L, Wang J, Zhang X-Q, Zhou M-H (2009) Characterization of a novel Stenotrophomonas isolate with high keratinase activity and purification of the enzyme. J Ind Microbiol Biotechnol 36:181–188
Chamroensaksri N, Akaracharanya A, Visessanguan W, Tanasupawat S (2008) Characterization of halophilic bacterium NB2-1 from pla-ra and its protease production. J Food Biochem 32:536–555
Dalmaso GZL, Ferreira D, Vermelho AB (2015) Marine extremophiles: a source of hydrolases for biotechnological applications. Mar Drugs 13:1925–1965
Dammak DF, Smaoui SM, Ghanmi F, Boujelben I, Maalej S (2016) Characterization of halo-alkaline and thermostable protease from Halorubrum ezzemoulense strain ETR14 isolated from Sfax solar saltern in Tunisia. J Basic Microbiol 56:337–346
DasSarma S, DasSarma P (2015) Halophiles and their enzymes: negativity put to good use. Curr Opin Microbiol 25:120–126
Debette J (1991) Isolation and characterization of an extracellular proteinase produced by a soil strain of Xanthomonas maltophilia. Curr Microbiol 22:85–90
Edbeib MF, Wahab RA, Huyop F (2016) Halophiles: biology, adaptation, and their role in decontamination of hypersaline environments. World J Microbiol Biotechnol 32:1–23
Garabito MJ, Arahal DR, Mellado E, Márquez MC, Ventosa A (1997) Bacillus salexigens sp. nov., a new moderately halophilic Bacillus species. Int J Syst Evol Microbiol 47:735–741
Gupta R, Beg Q, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59:15–32
Gupta A, Joseph B, Mani A, Thomas G (2008) Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus. World J Microbiol Biotechnol 24:237–243
Gupta R, Sharma R, Beg QK (2013) Revisiting microbial keratinases: next generation proteases for sustainable biotechnology. Crit Rev Biotechnol 33:216–228
Haddar A, Agrebi R, Bougatef A, Hmidet N, Sellami-Kamoun A, Nasri M (2009) Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and potential application as a laundry detergent additive. Bioresour Technol 100:3366–3373
Heyrman J et al (2003) Virgibacillus carmonensis sp. nov., Virgibacillus necropolis sp. nov. and Virgibacillus picturae sp. nov., three novel species isolated from deteriorated mural paintings, transfer of the species of the genus Salibacillus to Virgibacillus, as Virgibacillus marismortui comb. nov. and Virgibacillus salexigens comb. nov., and emended description of the genus Virgibacillus. Int J Sys Evol Microbiol 53:501–511. https://doi.org/10.1099/ijs.0.02371-0
Homaei A, Lavajoo F, Sariri R (2016) Development of marine biotechnology as a resource for novel proteases and their role in modern biotechnology. Int J Biol Macromol 88:542–552
Karn SK, Kumar A (2015) Hydrolytic enzyme protease in sludge: recovery and its application. Biotechnol Bioprocess Eng 20:652–661
Khelaifia S et al (2015) Noncontiguous finished genome sequence and description of Virgibacillus massiliensis sp. nov., a moderately halophilic bacterium isolated from human gut. New Microbes New Infect 8:78–88
Kumar CG, Takagi H (1999) Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv 17:561–594
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874
Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 125–175
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Madigan MT, Martinko JM, Dunlap PV, Clark DP (2009) Brock biology of microorganisms, 12th edn. Benjamin Cummings, San Francisco
Montriwong A, Rodtong S, Yongsawatdigul J (2015) Detergent-stable salt-activated proteinases from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation. Appl Biochem Biotechnol 176:505–517
Niyonzima FN, More S (2015a) Detergent-compatible proteases: microbial production, properties, and stain removal analysis. Prep Biochem Biotechnol 45:233–258
Niyonzima FN, More SS (2015b) Coproduction of detergent compatible bacterial enzymes and stain removal evaluation. J Basic Microbiol 55:1149–1158
Nokhal T-H, Schlegel HG (1983) Taxonomic Study of Paracoccus denitrificans. Int J Sys Evol Microbiol 33:26–37. https://doi.org/10.1099/00207713-33-1-26
North MJ (1982) Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiol Rev 46:308
Rajeswari VD, Jayaraman G, Sridharan T (2012) Purification and characterization of extracellular protease from halotolerant bacterium Virgibacillus dokdonensis VITP14. Asian J Biochem 7:123–132
Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Sathishkumar R, Ananthan G, Raghunathan C (2015) Production and characterization of haloalkaline protease from ascidian-associated Virgibacillus halodenitrificans RSK CAS1 using marine wastes. Ann Microbiol 65:1481–1493
Secades P, Guijarro J (1999) Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production. Appl Environ Microbiol 65:3969–3975
Setyorini E, Takenaka S, Murakami S, Aoki K (2006) Purification and characterization of two novel halotolerant extracellular proteases from Bacillus subtilis strain FP-133. Biosci Biotechnol Biochem 70:433–440
Singh R, Kumar M, Mittal A, Mehta PK (2016) Microbial enzymes: industrial progress in 21st century. 3 Biotech 6:174
Sinsuwan S, Rodtong S, Yongsawatdigul J (2007) NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation. J Food Sci 72:C264–C269
Sinsuwan S, Rodtong S, Yongsawatdigul J (2008) Production and characterization of NaCl-activated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation. Process Biochem 43:185–192
Sinsuwan S, Rodtong S, Yongsawatdigul J (2009) Purification and characterization of a salt-activated and organic solvent-stable heterotrimer proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce. J Agric Food Chem 58:248–256
Tavano OL (2013) Protein hydrolysis using proteases: an important tool for food biotechnology. J Mol Catal B Enzym 90:1–11
Yin J, Chen J-C, Wu Q, Chen G-Q (2015) Halophiles, coming stars for industrial biotechnology. Biotechnol Adv 33:1433–1442
Acknowledgements
The authors would like to thank Universiti Teknologi Malaysia for providing facilities to carry out this research work. This work was supported financially by Ministry of Education Malaysia and Universiti Teknologi Malaysia RU grant under project number of 4F265 and 07H43, respectively. Ming Quan Lam acknowledges MyBrain15 scholarship from Ministry of Higher Education Malaysia. Suganthi Thevarajoo and Chitra Selvaratnam acknowledge PhD Zamalah scholarship from Universiti Teknologi Malaysia.
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Lam, M.Q., Nik Mut, N.N., Thevarajoo, S. et al. Characterization of detergent compatible protease from halophilic Virgibacillus sp. CD6. 3 Biotech 8, 104 (2018). https://doi.org/10.1007/s13205-018-1133-2
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DOI: https://doi.org/10.1007/s13205-018-1133-2