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Production and characterization of haloalkaline protease from ascidian-associated Virgibacillus halodenitrificans RSK CAS1 using marine wastes

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Abstract

A marine ascidian-associated bacterium, Virgibacillus halodenitrificans RSK CAS1, was optimized for protease production by response surface methodology using marine waste as substrate. The central composite design was employed, and the optimal medium constituents for maximum protease production (1461.11 U/ml) were determined to be shrimp shell powder (15.32 g/l), casein (5.37 g/l), MgSO4 (3.0 g/l) and NaCl (55.31 g/l). The protease was purified from the culture supernatant to homogeneity in a three-step procedure consisting of ammonium sulfate precipitation, ion exchange chromatography (DEAE-cellulose column) and gel-filtration chromatography (Sephadex G-75 column), resulting in a 8.7-fold-change in purified protein. This protein had a specific activity of 1,086.78 U/mg and a molecular weight of 21 kDa. It exhibited optimal activity at 50 °C, pH 9 and 25 % NaCl. The significant stability of this protein at higher levels of salt, metal ions, organic solvents and commercial detergents and at higher, temperature, as well as its application as a cleaning additive in blood stain removal, suggests its possible use the laundry detergent industry.

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References

  • Annamalai N, Rajeswari MV, Vijayalakshmi S, Balasubramanian T (2011) Purification and characterization of chitinase from Alcaligenes faecalis AU02 by utilizing marine wastes and its antioxidant activity. Ann Microbiol 61:801–807

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Annamalai N, Rajeswari MV, Thavasi R, Vijayalakshmi S, Balasubramanian T (2013a) Optimization, purification and characterization of novel thermostable, haloalkaline, solvent stable protease from Bacillus halodurans CAS6 using marine shellfish wastes: a potential additive for detergent and antioxidant synthesis. Bioprocess Biosyst Eng 7:873–783

    Article  Google Scholar 

  • Annamalai N, Rajeswari MV, Balasubramanian T (2013b) Extraction, purification and application of thermostable and halostable alkaline protease from Bacillus alveayuensis CAS5 using marine wastes. Food Bioprod Process. doi:10.1016/j.fbp.2013.08.009

    Google Scholar 

  • Arulmani M, Aparanjini K, Vasanthi K, Arumugam P, Arivuchelvi M, Kalaichelvan T (2006) Purification and partial characterization of serine protease from thermostable alkalophilic Bacillus laterosporus-AK1. World J Microbiol Biotechnol 23:475–481

    Article  Google Scholar 

  • Bhaskar N, Sudeepa ES, Rashmi HN, Selvi AT (2007) Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities. Bioresour Technol 98:2758–2764

    Article  CAS  PubMed  Google Scholar 

  • Chu IM, Lee C, Li TS (1992) Production and degradation of alkaline protease in batch cultures of Bacillus subtilis ATCC 14416. Enzym Microb Technol 4:55–61

    Google Scholar 

  • Deng A, Wu J, Zhang Y, Zhang G, Wen T (2010) Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001. Bioresour Technol 101:7100–7106

    Article  CAS  Google Scholar 

  • Esakkiraj P, Sankaralingam S, Usha R, Palavesam A, Immanuel G (2011) Solid-state protease production using anchovy waste meal by moderate halophile Serratia proteamaculans AP-CMST isolated from fish intestine. Ann Microbiol 61:749–755

    Article  CAS  Google Scholar 

  • Garrity GM, Boone DR, Castenholz RW (2001) Bergey’s manual of systematic bacteriology, 2nd edn. Springer, New York

    Google Scholar 

  • Gupta A, Khare SK (2007) A protease stable in organic solvents from solvent tolerant strain of Pseudomonas aeruginosa. Bioresour Technol 97:1788–1793

    Article  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Islam MS, Khan S, Tanaka M (2004) Waste loading in shrimp and fish processing effluents: potential source of hazards to the coastal and near shore environments. Mar Pollut Bull 49:103–110

    Article  CAS  PubMed  Google Scholar 

  • Johnvesly B, Naik GR (2001) Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochem 37:139–144

    Article  CAS  Google Scholar 

  • Karbalaei-Heidari HR, Ziaee A, Schaller J, Amoozegar MA (2007) Purification and characterization of an extracellular haloalkaline protease produced by the moderately halophilic bacterium, Salinivibrio sp. strain AF-2004. Enzym Microb Technol 40:266–272

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lagzian M, Asoodeh A (2012) An extremely thermotolerant, alkaliphilic subtilisin-like protease from hyperthermophilic Bacillus sp. MLA64. Int J Biol Macromolec 51:960–967

    Article  CAS  Google Scholar 

  • Liu BL, Kao PM, Tzeng YM, Feng KC (2003) Production of chitinase from Verticillium lecanii F091 using submerged fermentation. Enzym Microb Technol 33:410–415

    Article  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 93:265–275

    Google Scholar 

  • Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. Mol Biol J 3:208–218

    Article  CAS  Google Scholar 

  • Maruthiah T, Esakkiraj P, Prabakaran G, Palavesam A, Immanuel G (2013) Purification and characterization of moderately halophilic alkaline serine protease from marine Bacillus subtilis AP-MSU6. Biocatal Agric Biotechnol 2:116–119

    Google Scholar 

  • Maurer KH (2004) Detergent proteases. Curr Opin Biotechnol 15:330–334

    Article  CAS  PubMed  Google Scholar 

  • Nascimento WCA, Martins MLL (2004) Production and properties of an extracellular protease from thermophilic Bacillus sp. Braz J Microbiol 35:91–96

  • Nawani NN, Prakash D, Kapadnis BP (2010) Extraction, purification and characterization of an antioxidant from marine waste using protease and chitinase cocktail. World J Microbiol Biotechnol 26:1509–1517

    Article  CAS  Google Scholar 

  • Rai SK, Mukherjee AK (2009) Ecological significance and some biotechnological application of an organic solvent stable alkaline serine protease from Bacillus subtilis strain DM-04. Bioresour Technol 100:2642–2645

    Article  CAS  PubMed  Google Scholar 

  • Reddy LVA, Wee YJ, Yun JS, Ryu HW (2008a) Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through Plackett–Burman and response surface methodological approaches. Bioresour Technol 99:2242–2249

    Article  CAS  PubMed  Google Scholar 

  • Reddy LVA, Wee YJ, Ryu HW (2008b) Purification and characterization of an organic solvent and detergent-tolerant novel protease produced by Bacillus sp. RKY3. J Chem Technol Biotechnol 83:1526–1533

    Article  CAS  Google Scholar 

  • Saeki K, Ozaki K, Kobayashi T, Ito S (2007) Detergent alkaline proteases: enzymatic properties, genes and crystal structures. J Biosci Bioeng 103:501–508

    Article  CAS  PubMed  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  • Sareen R, Mishra P (2008) Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37. Appl Microbiol Biotechnol 79:399–405

    Article  CAS  PubMed  Google Scholar 

  • Sathishkumar R, Ananthan G, Selva Prabhu A (2014) Optimization of medium composition for alkaline protease production by Marinobacter sp. GACAS9 using response surface methodology—A statistical approach. Biocatal Agric Biotechnol 3:191–107

    Google Scholar 

  • Shivanand P, Jayaraman G (2009) Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast. Process Biochem 44:1088–1094

    Article  CAS  Google Scholar 

  • Sousa F, Jus S, Erbel A, Kokol V, Cavaco-Paulo A, Gubitz GM (2007) A novel metalloprotease from Bacillus cereus for protein fibre processing. Enzym Microb Technol 40:1772–1781

    Article  CAS  Google Scholar 

  • Tari C, Genckal H, Tokatli F (2006) Optimization of a growth medium using a statistical approach for the production of an alkaline protease from a newly isolated Bacillus sp. L21. Process Biochem 41:659–665

    Article  CAS  Google Scholar 

  • Todd EW (1949) Quantitative studies on the total plasmin and trypsin inhibitor of human blood serum. J Exp Med 39:295–308

    Article  Google Scholar 

  • Wang SL, Yang CH, Liang TW, Yen YH (2008) Optimization of conditions for protease production by Chryseobacterium taeanense TKU001. Bioresour Technol 99:3700–3707

    Article  CAS  PubMed  Google Scholar 

  • Wang SL, Chao CH, Liang TW, Chen CC (2009) Purification and characterization of protease and chitinase from Bacillus cereus TKU006 and conversion of marine wastes by these enzymes. Mar Biotechnol 11:334–344

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank the Dean and Director of CAS in Marine Biology and the authorities of Annamalai University for providing the facilities. The authors are also very grateful to the Ministry of Environment and Forest (MoEn&F) for funding and providing fellowship.

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Authors and Affiliations

  1. Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, 608 502, TN, India

    Ramamoorthy Sathishkumar & Gnanakkan Ananthan

  2. Zoological Survey of India, Andaman and Nicobar Islands Regional Station, Port Blair, Andaman and Nicobar Islands, Union Territory, India

    Chelladurai Raghunathan

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  1. Ramamoorthy Sathishkumar
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  2. Gnanakkan Ananthan
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  3. Chelladurai Raghunathan
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Correspondence to Gnanakkan Ananthan.

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Sathishkumar, R., Ananthan, G. & Raghunathan, C. Production and characterization of haloalkaline protease from ascidian-associated Virgibacillus halodenitrificans RSK CAS1 using marine wastes. Ann Microbiol 65, 1481–1493 (2015). https://doi.org/10.1007/s13213-014-0987-8

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  • DOI: https://doi.org/10.1007/s13213-014-0987-8

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