Research Article
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Salda ve Van Göllerinden SGTaz Üreten Alkalifilik Bakterilerin İzolasyonu, Tanılanması ve Enzim Üretme Kapasitelerinin Değerlendirilmesi

Year 2019, Volume: 12 Issue: 3, 150 - 158, 15.12.2019

Eda Kabacaoğlu Paakkanen Barçın Karakaş Budak

Abstract

Bu çalışmanın amacı, CGTase pozitif mikroorganizmaların Salda ve Van göllerinden izolasyonudur. Her iki göl de yüksek seviyede bazik (pH> 9,7) özellikte benzersiz ekstrem ortamlardır. Çalışmada SD5, SG2, SG3 ve V3 olarak kodlanan dört bakteri suşu, seçici Horikoshi katı besiyeri üzerinde yetiştirilerek izole edilmiş ve CGTase üreticileri olarak doğrulanmıştır. Mikroskobik inceleme, dört izolatın Gram pozitif çubuk şeklinde bakteriler olduğunu göstermiştir. Suşlar, 16S rDNA bölgesinin DNA dizilerine dayanarak Bacillus cinsi bakteriler olarak tanılanmıştır. Bunlardan sadece V3 ve SG2 izolatları tür seviyesinde, sırasıyla B. agaradhaerens ve B. patagoniensis olarak tanılanabilmiştir. Suşların büyüme ve CGTase üretim kapasiteleri çalkalamalı inkübatör koşullarında değerlendirilmiştir. Tüm izolatların, 24 saatlik inkübasyon sonrasında hücre dışı ortamda 6-8 U/mL CGTase üretebildiği tespit edilmiştir. Bacillus sp. SG3, Bacillus sp. SD5 ve B. agaradhaerens V3 suşlarının büyüme eğrileri 24-30 saat içerisinde durağan faza ulaşmış ancak 168 saate kadar uzatılmış fermentasyon süresince B. patagoniensis SG2’nin yavaş ancak istikrarlı şekilde çoğaldığı ve CGTase aktivitesinin yükselerek devam ettiği gözlemlenmiştir. Hücredışı CGTase salgılama kapasitesi olduğu doğrulanan bu dört bakteri suşu gelecekte CGTase'in endüstriyel ölçekte üretilebilmesi için yürütülecek araştırmalarda potansiyel gen kaynakları olarak değerlendirilebilecektir.

Keywords

Bacillus sp. bakteriyel tanılama siklodekstrin glikosiltransferaz alkalifilik

References

  • Tonkova, A. (1998). Bacterial cyclodextrin glucanotransferase. Enzyme and Microbial Technology, 22(8), 678–686. https://doi:10.1016/S0141-0229(97)00263-9
  • Van der Veen, B. A., Uitdehaag, J. C. M., Dijkstra, B. W., & Dijkhuizen, L. (2000). Engineering of cyclodextrin glycosyltransferase reaction and product specifity. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1543(336), 336–360. https://doi:10.1016/S0167-4838(00)00233-8
  • Del Valle, E. M. M. (2004). Cyclodextrins and their uses: a review. Process Biochemistry, 39(9), 1033–1046. https://doi:10.1016/S0032-9592(03)00258-9
  • Szejtli, J. (2013). Cyclodextrin technology - topics in inclusion science (Vol. 1). Netherlands: Springer Science & Business Media. https://doi:10.1007/978-94-015-7797-7
  • Biwer, A., Antranikian, G., & Heinzle, E. (2002). Enzymatic production of cyclodextrins. Applied Microbiology and Biotechnology, 59(6), 609–617. https://doi:10.1007/s00253-002-1057-x
  • Han, R., Li, J., Shin, H.-D., Chen, R. R., Du, G., Liu, L., & Chen, J. (2014). Recent advances in discovery, heterologous expression, and molecular engineering of cyclodextrin glycosyltransferase for versatile applications. Biotechnology Advances, 32(2), 415–428. https://doi:10.1016/j.biotechadv.2013.12.004
  • Atanasova, N., Petrova, P., Ivanova, V., Yankov, D., Vassileva, A., & Tonkova, A. (2008). Isolation of novel alkaliphilic bacillus strains for cyclodextrin glucanotransferase production. Applied Biochemistry and Biotechnology, 149(2), 155–167. https://doi:10.1007/s12010-007-8128-5
  • Horikoshi, K. (2011). Introduction and history of alkaliphiles. In K. Horikoshi (Ed.), Extremophiles handbook (pp. 19–26). Japan: Springer. https://doi:10.1007/978-4-431-53898-1_1.1
  • Russel, M. J., Ingham, J. K., Zedef, V., Maktav, D., Sunar, F., Hall, A. J., & Fallick, A. E. (1999). Search for signs of ancient life on Mars: expectations from hydromagnesite microbialites, Salda Lake, Turkey. Journal of the Geological Society, 156(5), 869–888. https://doi:10.1144/gsjgs.156.5.0869
  • Berber, İ., & Yenidünya, E. (2005). Identification of alkaliphilic Bacillus species isolated from Lake Van and its surroundings by computerized analysis of extracellular protein profiles. Turkish Journal of Biology, 29(3), 181–188.
  • Kempe, S., Kazmierczak, J., Landmann, G., Konuk, T., Reimer, A., & Lipp, A. (1991). Largest known microbialites discovered in Lake Van, Turkey. Nature, 349(6310), 605–608. https://doi.org/10.1038/349605a0
  • Berber, İ., & Berber, Ş. (2006). Numerical analysis of SDS-PAGE protein patterns of facultative alkaliphilic Bacillus species isolated from lake Van, Turkey. Fresenius Environmental Bulletin, 15(5), 409–416.
  • Ozcan, B., Cokmus, C., Coleri, A., & Caliskan, M. (2006). Characterization of extremely halophilic Archaea isolated from saline environment in different parts of Turkey. Microbiology, 75(6), 739–746. https://doi:10.1134/s002626170606018x
  • Poyraz, N., & Mutlu, M. B. (2017). Alkaliphilic bacterial diversity of Lake Van/Turkey. Biological Diversity and Conservation, 10(1), 92–103.
  • Aygan, A., & Arikan, B. (2008). A new halo-alkaliphilic, thermostable endoglucanase from moderately halophilic Bacillus sp. C14 isolated from Van Soda Lake. International Journal of Agriculture & Biology, 10(4), 369–374.
  • Aygan, A., Arikan, B., Korkmaz, H., Dinçer, S., & Çolak, Ö. (2008). Highly thermostable and alkaline α-amylase from a halotolerant-alkaliphilic Bacillus sp. AB68. Brazilian Journal of Microbiology, 39(3), 547–553. https://doi:10.1590/S1517-83822008000300027
  • Park, C. S., Park, K. H., & Kim, S. H. (1989). Screening method for glucanotransferase using. Agricultural and Biological Chemistry, 53(4), 1167–1169.
  • Kaneko, T., Kato, T., Nakamura, N., & Horikoshi, K. (1987). Spectrophotometric determination of cyclization activity of β-cyclodextrin-forming cyclomaltodextrin glucanotransferase. Journal of the Japanese Society of Starch Science, 34(1), 45–48.
  • Lane, D. J. (1991) 16S/23S rRNA sequencing. In E. Stackebrandt & M.Goodfellow (Eds.), Nucleic acid techniques in bacterial systematics (pp. 115–175). Chichester, United Kingdom: John Wiley and Sons Ltd.).
  • Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389–3402.
  • Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30(12), 2725–2729. https://doi:10.1093/molbev/mst197
  • Fraser, G., Hughes G. (1999) Swarming motility. Current Opinion in Microbiology, 2(6): 630-635. https://doi:10.1016/S1369-5274(99)00033-8
  • Kabacaoğlu, E., & Karakaş Budak, B. (2017). Heterologous expression of β-γ-type cyclodextrin glycosyltransferase of newly isolated alkaliphilic Bacillus sp. SD5 in Pichia pastoris. Starch/Staerke, 69(9–10), 1600365. https://doi:10.1002/star.201600365
  • Gomes, A. C. S. M., Dos Santos, S. R., Ribeiro, M. C., Cravo, P. V. L., Vieira, D. G., De Souza, K. M. C., & Amaral, A. C. (2018). Is there still room to explore cyclodextrin glycosyltransferase-producers in Brazilian biodiversity? Anais Da Academia Brasileira de Ciências, 90(2), 1473–1480. https://doi:10.1590/0001-3765201820170670
  • Hao, J.-H., Huang, L.-P., Chen, X., Sun, J.-J., Liu, J.-Z., wang, W., & Sun, M. (2017). Identification, cloning and expression analysis of an alpha-CGTase produced by stain Y112. Protein Expression and Purification, 140, 8–15. https://doi:10.1016/J.PEP.2017.07.015
  • Abdelnasser, S. S. I., Mohamed, A. E. T., & Ali, A. A. S. (2010). Characterization of immobilized alkaline cyclodextringlycosyltransferase from a newly isolated Bacillus agaradhaerens KSU-A11. African Journal of Biotechnology, 9(44), 7550–7559. https://doi:10.5897/AJB10.644
  • Martins, R. F., & Hatti-Kaul, R. (2002). A new cyclodextrin glycosyltransferase from an alkaliphilic Bacillus agaradhaerens isolate: purification and characterisation. Enzyme and Microbial Technology, 30(1), 116–124. https://doi:10.1016/S0141-0229(01)00461-6

Isolation, Identification and Evaluation of Enzyme Production Capacity of CGTase Producing Bacteria from Lakes Salda and Van/Turkey

Year 2019, Volume: 12 Issue: 3, 150 - 158, 15.12.2019

Eda Kabacaoğlu Paakkanen Barçın Karakaş Budak

Abstract

The aim of this study was isolation of CGTase positive microorganisms from lakes Salda and Van, both being extreme environments unique in character with high alkalinity pH>9.7. Four bacterial strains designated as SD5, SG2, SG3 and V3 were isolated by cultivation on selective Horikoshi agar and confirmed as CGTase producers. Microscopic inspection showed the four isolates to be Gram positive rods. Strains were identified as Bacillus based on DNA sequences of 16S rDNA region. Only the V3 and SG2 isolates could be resolved at species level identified as B. agaradhaerens and B. patagoniensis, respectively. Growth and CGTase production capacity of the strains were evaluated under shake flask conditions. All isolates achieved extracellular CGTase levels of 6-8 U/mL within 24 h of incubation. Growth curves of Bacillus sp. SG3, Bacillus sp. SD5, B. agaradhaerens V3 showed the bacteria to reach a plateau phase within 24-30 h however, extended fermentation up to 168 h showed B. patagoniensis SG2 to continue the growth cycle until the end of this period with higher CGTase activity observed. These four strains isolated which have thus been confirmed as CGTase positive bacteria can potentially be exploited as genetic sources for the synthesis of CGTase in industrial scale.

Keywords

Bacillus sp. bacterial identification cyclodextrin glycosyltransferase alkaliphilic

References

  • Tonkova, A. (1998). Bacterial cyclodextrin glucanotransferase. Enzyme and Microbial Technology, 22(8), 678–686. https://doi:10.1016/S0141-0229(97)00263-9
  • Van der Veen, B. A., Uitdehaag, J. C. M., Dijkstra, B. W., & Dijkhuizen, L. (2000). Engineering of cyclodextrin glycosyltransferase reaction and product specifity. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1543(336), 336–360. https://doi:10.1016/S0167-4838(00)00233-8
  • Del Valle, E. M. M. (2004). Cyclodextrins and their uses: a review. Process Biochemistry, 39(9), 1033–1046. https://doi:10.1016/S0032-9592(03)00258-9
  • Szejtli, J. (2013). Cyclodextrin technology - topics in inclusion science (Vol. 1). Netherlands: Springer Science & Business Media. https://doi:10.1007/978-94-015-7797-7
  • Biwer, A., Antranikian, G., & Heinzle, E. (2002). Enzymatic production of cyclodextrins. Applied Microbiology and Biotechnology, 59(6), 609–617. https://doi:10.1007/s00253-002-1057-x
  • Han, R., Li, J., Shin, H.-D., Chen, R. R., Du, G., Liu, L., & Chen, J. (2014). Recent advances in discovery, heterologous expression, and molecular engineering of cyclodextrin glycosyltransferase for versatile applications. Biotechnology Advances, 32(2), 415–428. https://doi:10.1016/j.biotechadv.2013.12.004
  • Atanasova, N., Petrova, P., Ivanova, V., Yankov, D., Vassileva, A., & Tonkova, A. (2008). Isolation of novel alkaliphilic bacillus strains for cyclodextrin glucanotransferase production. Applied Biochemistry and Biotechnology, 149(2), 155–167. https://doi:10.1007/s12010-007-8128-5
  • Horikoshi, K. (2011). Introduction and history of alkaliphiles. In K. Horikoshi (Ed.), Extremophiles handbook (pp. 19–26). Japan: Springer. https://doi:10.1007/978-4-431-53898-1_1.1
  • Russel, M. J., Ingham, J. K., Zedef, V., Maktav, D., Sunar, F., Hall, A. J., & Fallick, A. E. (1999). Search for signs of ancient life on Mars: expectations from hydromagnesite microbialites, Salda Lake, Turkey. Journal of the Geological Society, 156(5), 869–888. https://doi:10.1144/gsjgs.156.5.0869
  • Berber, İ., & Yenidünya, E. (2005). Identification of alkaliphilic Bacillus species isolated from Lake Van and its surroundings by computerized analysis of extracellular protein profiles. Turkish Journal of Biology, 29(3), 181–188.
  • Kempe, S., Kazmierczak, J., Landmann, G., Konuk, T., Reimer, A., & Lipp, A. (1991). Largest known microbialites discovered in Lake Van, Turkey. Nature, 349(6310), 605–608. https://doi.org/10.1038/349605a0
  • Berber, İ., & Berber, Ş. (2006). Numerical analysis of SDS-PAGE protein patterns of facultative alkaliphilic Bacillus species isolated from lake Van, Turkey. Fresenius Environmental Bulletin, 15(5), 409–416.
  • Ozcan, B., Cokmus, C., Coleri, A., & Caliskan, M. (2006). Characterization of extremely halophilic Archaea isolated from saline environment in different parts of Turkey. Microbiology, 75(6), 739–746. https://doi:10.1134/s002626170606018x
  • Poyraz, N., & Mutlu, M. B. (2017). Alkaliphilic bacterial diversity of Lake Van/Turkey. Biological Diversity and Conservation, 10(1), 92–103.
  • Aygan, A., & Arikan, B. (2008). A new halo-alkaliphilic, thermostable endoglucanase from moderately halophilic Bacillus sp. C14 isolated from Van Soda Lake. International Journal of Agriculture & Biology, 10(4), 369–374.
  • Aygan, A., Arikan, B., Korkmaz, H., Dinçer, S., & Çolak, Ö. (2008). Highly thermostable and alkaline α-amylase from a halotolerant-alkaliphilic Bacillus sp. AB68. Brazilian Journal of Microbiology, 39(3), 547–553. https://doi:10.1590/S1517-83822008000300027
  • Park, C. S., Park, K. H., & Kim, S. H. (1989). Screening method for glucanotransferase using. Agricultural and Biological Chemistry, 53(4), 1167–1169.
  • Kaneko, T., Kato, T., Nakamura, N., & Horikoshi, K. (1987). Spectrophotometric determination of cyclization activity of β-cyclodextrin-forming cyclomaltodextrin glucanotransferase. Journal of the Japanese Society of Starch Science, 34(1), 45–48.
  • Lane, D. J. (1991) 16S/23S rRNA sequencing. In E. Stackebrandt & M.Goodfellow (Eds.), Nucleic acid techniques in bacterial systematics (pp. 115–175). Chichester, United Kingdom: John Wiley and Sons Ltd.).
  • Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389–3402.
  • Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30(12), 2725–2729. https://doi:10.1093/molbev/mst197
  • Fraser, G., Hughes G. (1999) Swarming motility. Current Opinion in Microbiology, 2(6): 630-635. https://doi:10.1016/S1369-5274(99)00033-8
  • Kabacaoğlu, E., & Karakaş Budak, B. (2017). Heterologous expression of β-γ-type cyclodextrin glycosyltransferase of newly isolated alkaliphilic Bacillus sp. SD5 in Pichia pastoris. Starch/Staerke, 69(9–10), 1600365. https://doi:10.1002/star.201600365
  • Gomes, A. C. S. M., Dos Santos, S. R., Ribeiro, M. C., Cravo, P. V. L., Vieira, D. G., De Souza, K. M. C., & Amaral, A. C. (2018). Is there still room to explore cyclodextrin glycosyltransferase-producers in Brazilian biodiversity? Anais Da Academia Brasileira de Ciências, 90(2), 1473–1480. https://doi:10.1590/0001-3765201820170670
  • Hao, J.-H., Huang, L.-P., Chen, X., Sun, J.-J., Liu, J.-Z., wang, W., & Sun, M. (2017). Identification, cloning and expression analysis of an alpha-CGTase produced by stain Y112. Protein Expression and Purification, 140, 8–15. https://doi:10.1016/J.PEP.2017.07.015
  • Abdelnasser, S. S. I., Mohamed, A. E. T., & Ali, A. A. S. (2010). Characterization of immobilized alkaline cyclodextringlycosyltransferase from a newly isolated Bacillus agaradhaerens KSU-A11. African Journal of Biotechnology, 9(44), 7550–7559. https://doi:10.5897/AJB10.644
  • Martins, R. F., & Hatti-Kaul, R. (2002). A new cyclodextrin glycosyltransferase from an alkaliphilic Bacillus agaradhaerens isolate: purification and characterisation. Enzyme and Microbial Technology, 30(1), 116–124. https://doi:10.1016/S0141-0229(01)00461-6
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Details

Primary Language English
Subjects Conservation and Biodiversity
Journal Section Research Articles
Authors

Eda Kabacaoğlu Paakkanen This is me

Barçın Karakaş Budak

Publication Date December 15, 2019
Submission Date April 6, 2019
Acceptance Date December 15, 2019
Published in Issue Year 2019 Volume: 12 Issue: 3

Cite

APA Kabacaoğlu Paakkanen, E., & Karakaş Budak, B. (2019). Isolation, Identification and Evaluation of Enzyme Production Capacity of CGTase Producing Bacteria from Lakes Salda and Van/Turkey. Biological Diversity and Conservation, 12(3), 150-158.
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