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Current Bioactive Compounds

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ISSN (Print): 1573-4072
ISSN (Online): 1875-6646

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Research Article

Production of Fibrinolytic Protease from a Halobacterium Bacillus licheniformis VITLMS Isolated from Marine Sponges of Rameshwaram Coast, India

Author(s): Lalith K.C. Kumar, Merlyn K. Samuel, Hemalatha Mooventhan, Mohanapriya Arumugam, Mohanasrinivasan Vaithialingam and Subathra D. Chandrasekaran*

Volume 17, Issue 2, 2021

Published on: 30 March, 2020

Page: [165 - 173] Pages: 9

DOI: 10.2174/1573407216666200330112127

Price: $65

Abstract

Background: Marine bacteria serve as excellent sources of therapeutic enzymes, metabolites and natural products, which possess novel therapeutic properties. Increasing death rates due to cardiovascular diseases urge for cost-effective production of the fibrinolytic enzyme.

Methods: In this study, marine sponge samples were screened for potent fibrinolytic producing bacteria. The primary screening was done for protease production, and clot lysis activity. The secondary screening was done for casein plasminogen activity and fibrinolytic activity. The strain which had potent fibrinolytic activity among them was further subjected to morphological, biochemical and molecular characterization. Media optimization was carried out to enhance enzyme production. The enzyme produced was subjected to purification using ammonium sulfate precipitation, gel filtration and characterized using HPLC and FTIR analysis.

Results: Sponge was identified to be Desmapsamma anchorata. Thirteen bacterial isolates were isolated from the sponge sample. The 16S rRNA sequencing revealed that the potential strain had 99% similarity with Bacillus licheniformis. Amongst the isolates, most were found to be morphologically identical to the Bacillus genus. Gram’s staining and SEM analysis of the potent isolate were performed to identify the spore formation and rod-shaped morphology of the bacteria. The optimal temperature and pH for the production of the enzyme were 37°C and 8, respectively. The carbon source maltose and nitrogen sources were malt extract and yeast extract that were found to be optimal. The optimum incubation time was found to be 4 to 5 days. The crude supernatant was purified with ammonium sulfate precipitation and gel filtration chromatography. The retention time of 11.3 min and the presence of functional groups show the purity of the enzyme. The partially purified enzyme showed 96.4% clot lysis in artificial clot lysis activity.

Conclusion: Although the secretion of fibrinolytic enzymes from Bacillus species is not new, based on our investigation, there are no reports regarding Bacillus licheniformis being isolated from marine sponges. However, there are reports of Bacillus licheniformis secreting fibrinolytic enzymes isolated from fermented food samples. This study identifies the marine environment as a potential source of new exploration for drug discovery.

Keywords: Desmapsamma anchorata, Bacillus licheniformis, clot lysis, fibrinolytic, halobacterium, Plasmin.

Graphical Abstract

[1]
Deepak, V.; Ilangovan, S.; Sampathkumar, M.V.; Victoria, M.J.; Pasha, S.P.B.S.; Pandian, S.B.R.K.; Gurunathan, S. Medium optimization and immobilization of purified fibrinolytic URAK from Bacillus cereus NK1 on PHB nanoparticles. Enzyme Microbial Technol., 2010, 47(6), 297-304.
[http://dx.doi.org/10.1016/j.enzmictec.2010.07.004]
[2]
Mahajan, P.M.; Nayak, S.; Lele, S.S. Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: Media optimization, purification and characterization. J. Biosci. Bioeng., 2012, 113(3), 307-314.
[http://dx.doi.org/10.1016/j.jbiosc.2011.10.023] [PMID: 22137041]
[3]
Ivanova, E.P.; Vysotskii, M.V.; Svetashev, V.I.; Nedashkovskaya, O.I.; Gorshkova, N.M.; Mikhailov, V.V.; Yumoto, N.; Shigeri, Y.; Taguchi, T.; Yoshikawa, S. Characterization of Bacillus strains of marine origin. Int. Microbiol., 1999, 2(4), 267-271.
[PMID: 10943423]
[4]
Kim, S.B.; Lee, D.W.; Cheigh, C.I.; Choe, E.A.; Lee, S.J.; Hong, Y.H.; Choi, H.J.; Pyun, Y.R. Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh. J. Ind. Microbiol. Biotechnol., 2006, 33(6), 436-444.
[http://dx.doi.org/10.1007/s10295-006-0085-4] [PMID: 16470353]
[5]
Santavy, D.L.; Willenz, P.; Colwell, R.R. Phenotypic study of bacteria associated with the caribbean sclerosponge, Ceratoporella nicholsoni. Appl. Environ. Microbiol., 1990, 56(6), 1750-1762.
[http://dx.doi.org/10.1128/AEM.56.6.1750-1762.1990] [PMID: 2383012]
[6]
Ravel, J.; Schrempf, H.; Hill, R.T. Mercury resistance is encoded by transferable giant linear plasmids in two chesapeake bay Streptomyces strains. Appl. Environ. Microbiol., 1998, 64(9), 3383-3388.
[http://dx.doi.org/10.1128/AEM.64.9.3383-3388.1998] [PMID: 9726886]
[7]
Chantawannakul, P.; Oncharoen, A.; Klanbut, K.; Chukeatirote, E.; Lumyong, S. Characterization of proteases of Bacillus subtilis strain 38 isolated from traditionally fermented soybean in Northern Thailand. Sci. Asia, 2002, 28(4), 241-245.
[http://dx.doi.org/10.2306/scienceasia1513-1874.2002.28.241]
[8]
Mander, P.; Cho, S.S.; Simkhada, J.R.; Choi, Y.H.; Yoo, J.C. A low molecular weight chymotrypsin-like novel fibrinolytic enzyme from Streptomyces sp. CS624. Process Biochem., 2011, 46(7), 1449-1455.
[http://dx.doi.org/10.1016/j.procbio.2011.03.016]
[9]
Ghani, M.; Ansari, A.; Aman, A.; Zohra, R.R.; Siddiqui, N.N.; Qader, S.A.U. Isolation and characterization of different strains of Bacillus licheniformis for the production of commercially significant enzymes. Pak. J. Pharm. Sci., 2013, 26(4), 691-697.
[PMID: 23811444]
[10]
Louden, B. C.; Haarmann, D.; Lynne, A. M. Use of blue agar CAS assay for siderophore detection. J. Microbiol. Biol. Educ., 2011, 12(1), 51.
[11]
Javadi, A.; Shamaei, M.; Mohammadi Ziazi, L.; Pourabdollah, M.; Dorudinia, A.; Seyedmehdi, S.M.; Karimi, S. Qualification study of two genomic DNA extraction methods in different clinical samples. Tanaffos, 2014, 13(4), 41-47.
[PMID: 25852760]
[12]
Wilson, D.A.; Reischl, U.; Hall, G.S.; Procop, G.W. Use of partial 16S rRNA gene sequencing for identification of Legionella pneumophila and non-pneumophila Legionella spp. J. Clin. Microbiol., 2007, 45(1), 257-258.
[http://dx.doi.org/10.1128/JCM.01552-06] [PMID: 17108074]
[13]
Krishnaa, P.K.; Priya, V.V. MSA (Multiple sequence alignment) and phylogenetic relationship of 16 s rRNA in bacteria causing infective endocarditis. J. Pharm. Sci. Res., 2017, 9(12), 2444-2446.
[14]
Saitou, N.; Nei, M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 1987, 4(4), 406-425.
[PMID: 3447015]
[15]
Chitte, R.R.; Dey, S. Potent fibrinolytic enzyme from a thermophilic Streptomyces megasporus strain SD5. Lett. Appl. Microbiol., 2000, 31(6), 405-410.
[http://dx.doi.org/10.1046/j.1365-2672.2000.00831.x] [PMID: 11123546]
[16]
Holmström, B. Production of streptokinase in continuous culture. Appl. Microbiol., 1968, 16(1), 73-77.
[http://dx.doi.org/10.1128/AEM.16.1.73-77.1968] [PMID: 4865907]
[17]
Astrup, T.; Müllertz, S. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys., 1952, 40(2), 346-351.
[http://dx.doi.org/10.1016/0003-9861(52)90121-5] [PMID: 12997222]
[18]
Nihalani, D.; Sahni, G. Streptokinase contains two independent plasminogen-binding sites. Biochem. Biophys. Res. Commun., 1995, 217(3), 1245-1254.
[http://dx.doi.org/10.1006/bbrc.1995.2902] [PMID: 8554583]
[19]
Singh, S.J.; Singh, L.R.; Devi, S.K.; Singh, S.S.; Devi, C.B.; Rully, H. Purification and characterization of a thermostable caseinolytic serine protease from the latex of Euphorbia heterophylla L. Protein Pept. Lett., 2015, 22(9), 828-835.
[http://dx.doi.org/10.2174/0929866522666150707114548] [PMID: 26149399]
[20]
Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 1951, 193(1), 265-275.
[PMID: 14907713]
[21]
Hwang, K.J.; Choi, K.H.; Kim, M.J.; Park, C.S.; Cha, J. Purification and characterization of a new fibrinolytic enzyme of Bacillus licheniformis KJ-31, isolated from Korean traditional Jeot-gal. J. Microbiol. Biotechnol., 2007, 17(9), 1469-1476.
[PMID: 18062224]
[22]
Essential Microbiology. 1st ed John Wiley & Sons Ltd publishers: England; , 2005, pp. 172-184.
[23]
D, D.; S, J.N.; S, M.K.; C, S.D. Novel Fibrinolytic protease producing Streptomyces radiopugnans VITSD8 from marine sponges. Mar. Drugs, 2019, 17(3), 164.
[http://dx.doi.org/10.3390/md17030164] [PMID: 30871149]
[24]
Afifah, D.N.; Rustanti, N.; Anjani, G.; Syah, D.; Suhartono, M.T. Proteomics study of extracellular fibrinolytic proteases from Bacillus licheniformis RO3 and Bacillus pumilus 2. g isolated from Indonesian g isolated from Indonesian fermented food. In: In: IOP conference series: Earth and environmental science IOP Publishing; , 2017; 55, p. p. 012025.
[25]
Sivapathasekaran, C.; Mukherjee, S.; Samanta, R.; Sen, R. High-performance liquid chromatography purification of biosurfactant isoforms produced by a marine bacterium. Anal. Bioanal. Chem., 2009, 395(3), 845-854.
[http://dx.doi.org/10.1007/s00216-009-3023-2] [PMID: 19688340]
[26]
Omrane Benmrad, M.; Mechri, S.; Zaraî Jaouadi, N.; Ben Elhoul, M.; Rekik, H.; Sayadi, S.; Bejar, S.; Kechaou, N.; Jaouadi, B. Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest. BMC Biotechnol., 2019, 19(1), 43.
[http://dx.doi.org/10.1186/s12896-019-0536-4] [PMID: 31262286]
[27]
Kim, J.S.; Sapkota, K.; Park, S.E.; Choi, B.S.; Kim, S.; Nguyen, T.H.; Kim, C.S.; Choi, H.S.; Kim, M.K.; Chun, H.S.; Park, Y.; Kim, S.J. A fibrinolytic enzyme from the medicinal mushroom Cordyceps militaris. J. Microbiol., 2006, 44(6), 622-631.
[PMID: 17205040]

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