DOI QR코드

DOI QR Code

Molecular Characterization of Protease Producing Idiomarina Species Isolated from Peruvian Saline Environments

  • Flores-Fernandez, Carol N. (Laboratorio de Biologia Molecular, Facultad de Farmacia y Bioquimica, Universidad Nacional Mayor de San Marcos) ;
  • Chavez-Hidalgo, Elizabeth (Laboratorio de Biologia Molecular, Facultad de Farmacia y Bioquimica, Universidad Nacional Mayor de San Marcos) ;
  • Santos, Marco (Laboratorio de Biologia Molecular, Facultad de Farmacia y Bioquimica, Universidad Nacional Mayor de San Marcos) ;
  • Zavaleta, Amparo I. (Laboratorio de Biologia Molecular, Facultad de Farmacia y Bioquimica, Universidad Nacional Mayor de San Marcos) ;
  • Arahal, David R. (Departamento de Microbiologia y Ecologia, y Coleccion Espanola de Cultivos Tipo, Universidad de Valencia)
  • Received : 2018.07.02
  • Accepted : 2018.09.15
  • Published : 2019.09.28

Abstract

All Idiomarina species are isolated from saline environments; microorganisms in such extreme habitats develop metabolic adaptations and can produce compounds such as proteases with an industrial potential. ARDRA and 16S rRNA gene sequencing are established methods for performing phylogenetic analysis and taxonomic identification. However, 16S-23S ITS is more variable than the 16S rRNA gene within a genus, and is therefore, used as a marker to achieve a more precise identification. In this study, ten protease producing Idiomarina strains isolated from the Peruvian salterns were characterized using biochemical and molecular methods to determine their bacterial diversity and industrial potential. In addition, comparison between the length and nucleotide sequences of a 16S-23S ITS region allowed us to assess the inter and intraspecies variability. Based on the 16S rRNA gene, two species of Idiomarina were identified (I. zobellii and I. fontislapidosi). However, biochemical tests revealed that there were differences between the strains of the same species. Moreover, it was found that the ITS contains two tRNA genes, $tRNA^{Ile(GAT)}$ and $tRNA^{Ala(TGC)}$, which are separated by an ISR of a variable size between strains of I. zobellii. In one strain of I. zobellii (PM21), we found nonconserved nucleotides that were previously not reported in the $tRNA^{Ala}$ gene sequences of Idiomarina spp. Thus, based on the biochemical and molecular characteristics, we can conclude that protease producing Idiomarina strains have industrial potential; only two I. zobellii strains (PM48 and PM72) exhibited the same properties. The differences between the other strains could be explained by the presence of subspecies.

Keywords

Idiomarina;protease;Peruvian saline environments;16S-23S ITS;tRNA

Acknowledgement

Supported by : Consejo Nacional de Ciencia, Tecnologia e Innovacion Tecnologica (CONCYTEC)

References

  1. Leon MJ, Martinez-Checa F, Ventosa A, Sanchez-Porro C. 2015. Idiomarina aquatica sp. nov., a moderately halophilic bacterium isolated from salterns. Int. J. Syst. Evol. Microbiol. 65: 4595-4600. https://doi.org/10.1099/ijsem.0.000619
  2. Hameed A, Lin SY, Lai WA, Shahina M, Liu YC, Hsu YH, et al. 2016. Idiomarina tyrosinivorans sp. nov., isolated from estuarine surface water. Int. J. Syst. Evol. Microbiol. 66: 5384-5391. https://doi.org/10.1099/ijsem.0.001525
  3. Marathe SK, Vashistht MA, Prashanth A, Parveen N, Chakraborty S, Nair SS. 2018. Isolation, partial purification, biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis, Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples. J. Genet. Eng. Biotechnol. 16: 39-46. https://doi.org/10.1016/j.jgeb.2017.10.001
  4. Ali N, Ullah N, Qasim M, Rahman H, Khan SN, Sadiq A, Adnan M. 2016. Molecular characterization and growth optimization of halo-tolerant protease producing Bacillus subtilis Strain BLK-1.5 isolated from salt mines of Karak, Pakistan. Extremophiles 20: 395-402. https://doi.org/10.1007/s00792-016-0830-1
  5. Ibrahim AS, Al-Salamah AA, Elbadawi YB, El-Tayeb MA, Ibrahim SSS. 2015. Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes. Electron. J. Biotechnol. 18: 236-243. https://doi.org/10.1016/j.ejbt.2015.04.001
  6. Rathod MG, Pathak AP. 2014. Wealth from waste: Optimized alkaline protease production from agro-industrial residues by Bacillus alcalophilus LW8 and its biotechnological applications. J. Taibah. Univ. Sci. 8: 307-314. https://doi.org/10.1016/j.jtusci.2014.04.002
  7. Pathak AP, Deshmukh KB. 2012. Alkaline protease production, extraction and characterization from alkaliphilic Bacillus licheniformis KBDL4: a Lonar soda lake isolate. Indian J. Exp. Biol. 50: 569-576.
  8. Jellouli K, Ghorbel-Bellaaj O, Ayed HB, Manni L, Agrebi R, Nasri M. 2011. Alkaline-protease from Bacillus licheniformis MP1: purification, characterization and potential application as a detergent additive and for shrimp waste deproteinization. Process Biochem. 46: 1248-1256. https://doi.org/10.1016/j.procbio.2011.02.012
  9. Essghaier B, Bejji M, Jijakli H, Boudabous A, Sadfi-Zouaoui N. 2009. High salt-tolerant protease from a potential biocontrol agent Bacillus pumilus M3-16. Ann. Microbiol. 59: 553. https://doi.org/10.1007/BF03175145
  10. Yang J, Li J, Hu Y, Li L, Long L, Wang F, et al. 2015. Characterization of a thermophilic hemoglobin-degrading protease from Streptomyces rutgersensis SCSIO 11720 and its application in antibacterial peptides production. Biotechnol. Bioprocess Eng. 20: 79-90. https://doi.org/10.1007/s12257-013-0771-9
  11. Manivasagan P, Venkatesan J, Sivakumar K, Kim SK. 2013. Production, characterization and antioxidant potential of protease from Streptomyces sp. MAB18 using poultry wastes. Biomed. Res. Int. 2013: 1-12.
  12. Chen MH, Sheu SY, Chen C, Wang JT, Chen WM. 2012. Idiomarina aquimaris sp. nov., isolated from the reef-building coral Isopora palifera. Int. J. Syst. Evol. Microbiol. 62: 1536-1542. https://doi.org/10.1099/ijs.0.035592-0
  13. Du J, Lai Q, Liu Y, Du Y, Liu X, Sun F, et al. 2014. Idiomarina atlantica sp. nov., a marine bacterium isolated from the deep sea sediment of the North Atlantic Ocean. Antonie Van Leeuwenhoek 107: 393-401.
  14. Lee JC, Kim YS, Yun BS, Whang KS. 2015. Idiomarina halophila sp. nov., isolated from a solar saltern sediment. Int. J. Syst. Evol. Microbiol. 65: 1268-1273. https://doi.org/10.1099/ijs.0.000094
  15. Kwon SW, Kim BY, Weon HY, Baek YK, Koo BS, Go SJ. 2006. Idiomarina homiensis sp. nov., isolated from seashore sand in Korea. Int. J. Syst. Evol. Microbiol. 56: 2229-2233. https://doi.org/10.1099/ijs.0.64283-0
  16. Poddar A, Lepcha RT, Mukherjee D, Bhattacharyya D, Das SK. 2014. Comparative analysis of 16S rRNA signature sequences of the genus Idiomarina and Idiomarina woesei sp. nov., a novel marine bacterium isolated from the Andaman Sea. Res. Microbiol. 165: 501-507. https://doi.org/10.1016/j.resmic.2014.07.008
  17. Rohban R, Amoozegar MA, Ventosa A. 2009. Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. J. Ind. Microbiol. Biotechnol. 36: 333-340. https://doi.org/10.1007/s10295-008-0500-0
  18. Babavalian H, Amoozegar MA, Pourbabaee AA, Moghaddam MM, Shakeri F. 2013. Isolation and identification of moderately halophilic bacteria producing hydrolytic enzymes from the largest hypersaline playa in Iran. Microbiology 82: 466-474. https://doi.org/10.1134/S0026261713040176
  19. Kumar S, Karan R, Kapoor S, Singh SP, Khare SK. 2012. Screening and isolation of halophilic bacteria producing industrially important enzymes. Braz. J. Microbiol. 43: 1595-1603. https://doi.org/10.1590/S1517-83822012000400044
  20. Sanchez-Porro C, Martin S, Mellado E, Ventosa A. 2003. Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes. J. Appl. Microbiol. 94: 295-300. https://doi.org/10.1046/j.1365-2672.2003.01834.x
  21. El Hadj-Ali N, Agrebi R, Ghorbel-Frikha B, Sellami-Kamoun A, Kanoun S, Nasri M. 2007. Biochemical and molecular characterization of a detergent stable alkaline serine-protease from a newly isolated Bacillus licheniformis NH1. Enzyme Microb. Technol. 40: 515-523. https://doi.org/10.1016/j.enzmictec.2006.05.007
  22. Moreno ML, Perez D, Garcia MT, Mellado E. 2013. Halophilic bacteria as a source of novel hydrolytic enzymes. Life 3: 38-51. https://doi.org/10.3390/life3010038
  23. Ivanova EP, Romanenko LA, Chun J, Matte MH, Matte GR, Mikhailov VV, et al. 2000. Idiomarina gen. nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp. nov. and Idiomarina zobellii sp. nov. Int. J. Syst. Evol. Microbiol. 50: 901-907. https://doi.org/10.1099/00207713-50-2-901
  24. Ivanova EP, Flavier S, Christen R. 2004. Phylogenetic relationships among marine Alteromonas-like proteobacteria: emended description of the family Alteromonadaceae and proposal of Pseudoalteromonadaceae fam. nov., Colwelliaceae fam. nov., Shewanellaceae fam. nov., Moritellaceae fam. nov., Ferrimonadaceae fam. nov., Idiomarinaceae fam. nov. and Psychromonadaceae fam. nov. Int. J. Syst. Evol. Microbiol. 54: 1773-1788. https://doi.org/10.1099/ijs.0.02997-0
  25. Jean WD, Shieh WY, Chiu HH. 2006. Pseudidiomarina taiwanensis gen. nov., sp. nov., a marine bacterium isolated from shallow coastal water of An-Ping Harbour, Taiwan, and emended description of the family Idiomarinaceae. Int. J. Syst. Evol. Microbiol. 56: 899-905. https://doi.org/10.1099/ijs.0.64048-0
  26. Hu ZY, Li Y. 2007. Pseudidiomarina sediminum sp. nov., a marine bacterium isolated from coastal sediments of Luoyuan Bay in China. Int. J. Syst. Evol. Microbiol. 57: 2572-2577. https://doi.org/10.1099/ijs.0.65199-0
  27. Wu YH, Shen YQ, Xu XW, Wang CS, Oren A, Wu M. 2009. Pseudidiomarina donghaiensis sp. nov. and Pseudidiomarina maritima sp. nov., isolated from the East China Sea. Int. J. Syst. Evol. Microbiol. 59: 1321-1325. https://doi.org/10.1099/ijs.0.005702-0
  28. Jean WD, Leu TY, Lee CY, Chu TJ, Lin SY, Shieh WY. 2009. Pseudidiomarina marina sp. nov. and Pseudidiomarina tainanensis sp. nov. and reclassification of Idiomarina homiensis and Idiomarina salinarum as Pseudidiomarina homiensis comb. nov. and Pseudidiomarina salinarum comb. nov., respectively. Int. J. Syst. Evol. Microbiol. 59: 53-59. https://doi.org/10.1099/ijs.0.001180-0
  29. Park SC, Lim CH, Baik KS, Lee KH, Lee JS, Seong CN. 2010. Pseudidiomarina aestuarii sp. nov., a marine bacterium isolated from shallow coastal seawater. Int. J. Syst. Evol. Microbiol. 60: 2071-2075. https://doi.org/10.1099/ijs.0.018051-0
  30. Taborda M, Antunes A, Tiago I, Verissimo A, Nobre MF, da Costa MS. 2009. Description of Idiomarina insulisalsae sp. nov., isolated from the soil of a sea salt evaporation pond, proposal to transfer the species of the genus Pseudidiomarina to the genus Idiomarina and emended description of the genus Idiomarina. Syst. Appl. Microbiol. 32: 371-378. https://doi.org/10.1016/j.syapm.2009.06.005
  31. Wang L, Lai Q, Fu Y, Chen H, Wang W, Wang J, et al. 2011. Idiomarina xiamenensis sp. nov., isolated from surface seawater, and proposal to transfer Pseudidiomarina aestuarii to the genus Idiomarina as Idiomarina aestuarii comb. nov. Int. J. Syst. Evol. Microbiol. 61: 969-973. https://doi.org/10.1099/ijs.0.022970-0
  32. Martinez-Canovas MJ, Bejar V, Martinez-Checa F, Paez R, Quesada E. 2004. Idiomarina fontislapidosi sp. nov. and Idiomarina ramblicola sp. nov., isolated from inland hypersaline habitats in Spain. Int. J. Syst. Evol. Microbiol. 54: 1793-1797. https://doi.org/10.1099/ijs.0.63172-0
  33. Contesini FJ, Melo RR, Sato HH. 2018. An overview of Bacillus proteases: from production to application. Crit. Rev. Biotechnol. 38: 321-334. https://doi.org/10.1080/07388551.2017.1354354
  34. 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. J. Microbiol. Biotechnol. 25: 1944-1953. https://doi.org/10.4014/jmb.1507.07017
  35. Indhuja S, Shiburaj S, Pradeep NS, Thankamani V, Abraham TK. 2012. Isolation and characterization of a feather degrading alkalophilic Streptomyces sp. TBG-S13A5 and its Keratinolytic properties. Agris. Sci. 40: 303-309.
  36. Sivaprakasam S, Dhandapani B, Mahadevan S. 2011. Optimization studies on production of a salt-tolerant protease from Pseudomonas aeruginosa strain BC1 and its application on tannery saline wastewater treatment. Braz. J. Microbiol. 42: 1506-1515. https://doi.org/10.1590/S1517-83822011000400038
  37. Tokajian S, Issa N, Salloum T, Ibrahim J, Farah M. 2016. 16S-23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads. Front Microbiol. 7: 149.
  38. Osorio CR, Collins MD, Romalde JL, Toranzo AE. 2005. Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure. Appl. Environ. Microbiol. 71: 636-645. https://doi.org/10.1128/AEM.71.2.636-645.2005
  39. Conrads G, Claros MC, Citron DM, Tyrrell KL, Merriam V, Goldstein EJ. 2002. 16S-23S rDNA internal transcribed spacer sequences for analysis of the phylogenetic relationships among species of the genus Fusobacterium. Int. J. Syst. Evol. Microbiol. 52: 493-499. https://doi.org/10.1099/00207713-52-2-493
  40. Liguori AP, Warrington SD, Ginther JL, Pearson T, Bowers J, Glass MB, et al. 2011. Diversity of 16S-23S rDNA internal transcribed spacer (ITS) reveals phylogenetic relationships in Burkholderia pseudomallei and its near-neighbors. PLoS One 6: e29323. https://doi.org/10.1371/journal.pone.0029323
  41. Bauer AW, Kirby WMM, Sherris JC, Turck M. 1966. Antibiotic susceptibility testing by a standardized single diffusion method. Am. J. Clin. Pathol. 45: 493-496. https://doi.org/10.1093/ajcp/45.4_ts.493
  42. Hou S, Saw JH, Lee KS, Freitas TA, Belisle C, Kawarabayasi Y, et al. 2004. Genome sequence of the deep-sea ${\gamma}$-proteobacterium Idiomarina loihiensis reveals amino acid fermentation as a source of carbon and energy. Proc. Natl. Acad. Sci. USA 101: 18036-18041. https://doi.org/10.1073/pnas.0407638102
  43. Dodia MS, Joshi RH, Patel RK, Singh SP. 2006. Characterization and stability of extracellular alkaline proteases from halophilic and alkaliphilic bacteria isolated from saline habitat of coastal Gujarat, India. Braz. J. Microbiol. 37: 276-282. https://doi.org/10.1590/S1517-83822006000300015
  44. Pant G, Prakash A, Pavani JVP, Bera S, Deviram GVNS, Kumar A, et al. 2015. Production, optimization and partial purification of protease from Bacillus subtilis. J. Taibah. Univ. Sci. 9: 50-55. https://doi.org/10.1016/j.jtusci.2014.04.010
  45. Singh SK, Tripathi VR, Jain RK, Vikram S, Garg SK. 2010. An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease. Microb. Cell Fact. 59: 1-7.
  46. Suganthi C, Mageswari A, Karthikeyan S, Anbalagan M, Sivakumar A, Gothandam KM. 2013. Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments. Genet. Eng. Biotechnol. J. 11: 47-52. https://doi.org/10.1016/j.jgeb.2013.02.002
  47. Zhou C, Qin H, Chen X, Zhang Y, Xue Y, Ma Y. 2018. A novel alkaline protease from alkaliphilic Idiomarina sp. C9-1 with potential application for eco-friendly enzymatic dehairing in the leather industry. Sci. Rep. 8: 16467. https://doi.org/10.1038/s41598-018-34416-5
  48. Stewart FJ, Cavanaugh CM. 2007. Intragenomic variation and evolution of the internal transcribed spacer of the rRNA operon in bacteria. J. Mol. Evol. 65: 44-67. https://doi.org/10.1007/s00239-006-0235-3
  49. Singh V, Mani I, Chaudhary DK. 2012. Molecular assessment of 16S-23S rDNA internal transcribed spacer length polymorphism of Aeromonas hydrophila. Adv. Microbiol. 2: 72-78. https://doi.org/10.4236/aim.2012.22009
  50. Brettar I, Christen R, Hofle MG. 2003. Idiomarina baltica sp. nov., a marine bacterium with a high optimum growth temperature isolated from surface water of the central Baltic Sea. Int. J. Syst. Evol. Microbiol. 53: 407-413. https://doi.org/10.1099/ijs.0.02399-0
  51. Donachie SP, Hou S, Gregory TS, Malahoff A, Alam M. 2003. Idiomarina loihiensis sp. nov., a halophilic $\gamma$-Proteobacterium from the Lo'ihi submarine volcano, Hawai'i. Int. J. Syst. Evol. Microbiol. 53: 1873-1879. https://doi.org/10.1099/ijs.0.02701-0
  52. Choi DH, Cho BC. 2005. Idiomarina seosinensis sp. nov., isolated from hypersaline water of a solar saltern in Korea. Int. J. Syst. Evol. Microbiol. 55: 379-383. https://doi.org/10.1099/ijs.0.63365-0
  53. Yoon JH, Jung SY, Jung YT, Oh TK. 2007. Idiomarina salinarum sp. nov., isolated from a marine solar saltern in Korea. Int. J. Syst. Evol. Microbiol. 57: 2503-2506. https://doi.org/10.1099/ijs.0.65189-0
  54. Zhang YJ, Zhang XY, Zhao HL, Zhou MY, Li HJ, Gao ZM, et al. 2012. Idiomarina maris sp. nov., a marine bacterium isolated from sediment. Int. J. Syst. Evol Microbiol. 62: 370-375. https://doi.org/10.1099/ijs.0.027896-0
  55. Sitdhipol J, Visessanguan W, Benjakul S, Yukphan P, Tanasupawat S. 2013. Idiomarina piscisalsi sp. nov., from fermented fish (pla-ra) in Thailand. J. Gen. Appl. Microbiol. 59: 385-391. https://doi.org/10.2323/jgam.59.385
  56. Song L, Ren F, Huang Y, Dai X, Zhou Y. 2013. Idiomarina indica sp. nov., isolated from seawater. Int. J. Syst. Evol. Microbiol. 63: 2497-2500. https://doi.org/10.1099/ijs.0.046789-0
  57. Zhong ZP, Liu Y, Liu HC, Wang F, Song L, Liu ZP. 2014. Idiomarina planktonica sp. nov., isolated from a saline lake. Int. J. Syst. Evol. Microbiol. 64: 3411-3416. https://doi.org/10.1099/ijs.0.065938-0