A Comparative Study: Taxonomic Grouping of Alkaline Protease Producing Bacilli


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Polish Journal of Microbiology

Polish Society of Microbiologists

Subject: Microbiology


ISSN: 1733-1331
eISSN: 2544-4646





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VOLUME 66 , ISSUE 1 (March 2017) > List of articles

A Comparative Study: Taxonomic Grouping of Alkaline Protease Producing Bacilli

Nilgun Tekin / Arzu Coleri Cihan / Basar Karaca / Cumhur Cokmus *

Keywords : 16S rRNA gene sequence, alkaline protease, alkaliphilic Bacillus, ARDRA, ITS-PCR, rep-PCR

Citation Information : Polish Journal of Microbiology. Volume 66, Issue 1, Pages 39-56, DOI: https://doi.org/10.5604/17331331.1234992

License : (CC BY-NC-ND 4.0)

Received Date : 05-November-2015 / Accepted: 19-February-2016 / Published Online: 30-March-2017



Alkaline proteases have biotechnological importance due to their activity and stability at alkaline pH. 56 bacteria, capable of growing under alkaline conditions were isolated and their alkaline protease activities were carried out at different parameters to determine their optimum alkaline protease production conditions. Seven isolates were showed higher alkaline protease production capacity than the reference strains. The highest alkaline protease producing isolates (103125 U/g), E114 and C265, were identified as Bacillus licheniformis with 99.4% and Bacillus mojavensis 99.8% based on 16S rRNA gene sequence similarities, respectively. Interestingly, the isolates identified as Bacillus safensis were also found to be high alkaline protease producing strains. Genotypic characterizations of the isolates were also determined by using a wide range of molecular techniques (ARDRA, ITS-PCR, (GTG)5-PCR, BOX-PCR). These different techniques allowed us to differentiate the alkaliphilic isolates and the results were in concurrence with phylogenetic analyses of the 16S rRNA genes. While ITS-PCR provided the highest correlation with 16S rRNA groups, (GTG)5-PCR showed the highest differentiation at species and intra-species level. In this study, each of the biotechnologically valuable alkaline protease producing isolates was grouped into their taxonomic positions with multi-genotypic analyses.

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Arellano-Carbajal F. and J. Olmos-Soto. 2002. Thermostable α-1, 4-and α-1, 6-glucosidase enzymes from Bacillus sp. isolated from a marine environment. World J. Microb. Biot. 18: 791–795.


Cihan A.C. 2013. Taxonomic classification of Anoxybacillus iso-lates from geothermal regions in Turkey by 16S rRNA gene sequences and ARDRA, ITS-PCR, Rep-PCR analyses. Pol. J. Microbiol. 62: 149–163.


Cihan A.C., B. Ozcan, N. Tekin and C. Cokmus. 2011. Phylogenetic diversity of isolates belonging to genera Geobacillus and Aeribacillus isolated from different geothermal regions of Turkey. World J. Microb. Biot. 27: 2683–2696.


Clarridge J.E. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin. Microbiol. Rev. 17: 840–62.


Claus D. and C.W. Berkeley. 1986. The genus Bacillus, pp. 1105– 1139. In: Sneath pHA (eds). Bergey’s Manual of Systematic Bacteriology. Volume 2. Williams, Wilkins, Baltimore.


Daffonchio D, S. Borin, A.D. Consolandi, D. Mora, P.L. Manachini and C. Sorlini. 1998a. 16S–23S rRNA internal transcribed spacers as molecular markers for the species of the 16S rRNA group I of the genus Bacillus. FEMS Microbiol. Lett. 163: 229–236.


Daffonchio D., S. Borin, G. Frova, P.L. Manachini and C. Sorlini. 1998b. PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis. Int. J. Syst. Bacteriol. 48: 107–116.


De Clerck E. and P. De Vos. 2004. Genotypic diversity among Bacil¬lus licheniformis strains from various sources. FEMS Microbiol. Lett. 231: 91–98.


Denizci A.A., D. Kazan, E.C.A Abeln and A. Erarslan. 2004. Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions. J. Appl. Microbiol. 96: 320–327.


Felsenstein J. 1985. Confidence-limits on phylogenies – an approach using the bootstrap. Evolution. 39: 783–791.


Freitas D.B., M.P. Reis, C.I. Lima-Bittencourt, P.S. Costa, P.S. Assis, E. Chartone-Souza and A.M.A. Nascimento. 2008. Geno-typic and phenotypic diversity of Bacillus spp. isolated from steel plant waste. BMC Res. Notes. 1: 92.


Gessesse A. and B.A. Gashe BA. 1997. Production of alkaline pro¬tease by an alkaliphilic bacteria isolated from an alkaline soda lake. Biotechnol. Lett. 19: 479–481.


Haddar A., R. Agrebi, A. Bougatef, N. Hmidet, A. Sellami- Kamoun and M. Nasri. 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.


Horikoshi K. 1999. Alkaliphiles: some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 63: 735–50.


Huang T.-P., D.D.-S. Tzeng, A.C.L. Wong, C.-H. Chen, K.-M. Lu. Y.-H. Lee, W.-D. Huang, B.-F. Hwang and K.-C. Tzeng. 2012. DNA polymorphisms and biocontrol of Bacillus antagonistic to citrus bacterial canker with indication of the interference of phyllosphere biofilms. PLoS ONE. 7: 421–424.


Ito S., T. Kobayashi, K. Ara, K. Ozaki, S. Kawai and Y. Hatada. 1998. Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures. Extremophiles 2: 185–190.


Klappenbach J.A. and J.M. Dunbar. 2000. rRNA operon copy num¬ber reflects ecological strategies of bacteria. Appl. Environ. Microb. 66: 1328–1333


Kumar C.G. and H. Takagi. 1999. Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol. Adv. 17: 561–594


Logan N.A., O. Berge, A.H. Bishop, H.J. Busse, P. De Vos, D. Fritze, M. Heyndrickx, P. Kämpfer, L. Rabinovitch, M.S. Salkinoja- Salonen and others. 2009. Proposed minimal standards for describ¬ing new taxa of aerobic, endospore-forming bacteria. Int. J. Syst. Evol. Microbiol. 59: 2114–2121.


Mora D., M.G. Fortina, G. Nicastro, C. Parini and P.L. Manachini. 1998. Genotypic characterization of thermophilic bacilli: a study on new soil isolates and several reference strains. Res. Microbiol. 149: 711–722.


Nielsen P., D. Fritze and F.G. Priest. 1995. Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species. Micro¬biology 141: 1745–1761.


Niyonzima F.N. and S.S. More. 2014. Concomitant production of detergent compatible enzymes by Bacillus flexus XJU-1. Braz. J. Microbiol. 45: 903–910.


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


Sari E., E. Loğoğlu and A. Öktemer. 2015. Purification and char¬acterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34. Biomed. Chromatogr. 29: 1356–1363.


Stackebrandt E., W. Frederiksen, G.M. Garrity, P.A Grimont, P. Kämpfer, M.C. Maiden, X. Nesme, R. Rosselló-Mora, J. Swings, H.G. Trüper and others. 2002. Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int. J. Syst. Evol. Microbiol. 52: 1043–1047.


Suzuki Y., T. Kishigami and S. Abe. 1976. Production of extra¬cellular alpha-glucosidase by a thermophilic Bacillus species. Appl. Environ. Microbiol. 31: 807–812.


Tamura K., M. Nei and S. Kumar. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA 101: 11030–11035.


Tamura K., J. Dudley, M. Nei and S. Kumar. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596–1599.


Tekin N., A.C. Cihan, Z.S. Takaç, C. Yagci, K. Tunc and C. Cok¬mus. 2012. Alkaline protease production of Bacillus cohnii APT5. Turk. J. Biol. 36: 430–440.


Versalovic J., M. Schneider and F.J. De Bruijn. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Method. Mol. Cell. Biol. 5: 25–40.


White D., R.J. Sharpand and F.G. Priest. 1993. A polyphasic taxo¬nomic study of thermophilic bacilli from a wide geographical area. Antonie Van Leeuwenhoek. 64: 357–386.