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Research article

Bioactive Compounds and Chemical Biology

Purification and Characterization of Thermostable Agarase from Bacillus sp. BI-3, a Thermophilic Bacterium Isolated from Hot Spring

Jiang Li 1, 2*, Yujie Sha 1, 2, Dewi Seswita-Zilda 3, Qiushi Hu 1, 2 and Peiqing He 1, 2

1The First Institute of Oceanography, SOA, Qingdao 266061, China, 2Key Laboratory of Marine Bioactive Substances, SOA, Qingdao 266061, China, 2Research Center for Marine and Fisheries Product Processing and Biotechnology, Agency for Marine and Fisheries Research and Development, Ministry of Marine and Fisheries Affairs, Jakarta 40115, Indonesia

Received: August 20, 2013; Accepted: September 24, 2013

J. Microbiol. Biotechnol. 2014; 24(1): 19-25

Published January 28, 2014 https://doi.org/10.4014/jmb.1308.08055

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

An extracellular agarase was purified from Bacillus sp. BI-3, a thermophilic agar-degrading bacterium isolated from a hot spring in Indonesia. The purified agarase revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 58 kDa. The optimum pH and temperature of the agarase were 6.4 and 70oC, respectively. The activity of the agarase was stable at high temperatures, and more than 50% activity was retained at 80oC for 15 min. Furthermore, the enzyme was stable in the pH range of 5.8–8.0, and more than 60% of the residual activity was retained. Significant activation of the agarase was observed in the presence of K+, Na+, Ca2+, Mg2+, and Sr2+; on the other hand, Ba2+, Zn2+, Cu2+, Mn2+, Co2+, Fe2+, and EDTA inhibited or inactivated the enzyme activity. The components of the hydrolytic product analyzed by thin-layer chromatography showed that the agarase mainly produced neoagarobiose. This study is the first to present evidence of agarolytic activity in aerobic thermophilic bacteria.

Keywords

Hot spring bacteria, Thermostable agarase, Purification, Characterization

References

  1. Agbo JAC, Moss MO. 1979. The isolation and characterization of agarolytic bacteria from a lowland river. J. Gen. Microbiol. 115: 355-368.
    CrossRef
  2. Aoki T, Araki T, Kitamikado M. 1990. Purification and characterization of a novel beta-agarase from Vibrio sp. AP-2. Eur. J. Biochem. 187: 461-465.
    Pubmed CrossRef
  3. Araki T, Hayakawa M, Zhang L, Karita S, Morishita T. 1998. Purification and characterization of agarases from a marine bacterium, Vibrio sp. PO-303. J. Marine Biotechnol. 6: 260-265.
    Pubmed
  4. Bannikova GE, Lopatin SA, Varlamov VP, Kuznetsov BB, Kozina IV, Miroshnichenko ML, et al. 2008. The thermophilic bacteria hydrolyzing agar: characterization of thermostable agarase. Appl. Biochem. Microbiol. 45: 366-371.
    CrossRef
  5. Dong J, Tamaru Y, Araki T. 2007. Molecular cloning, expression, and characterization of a beta-agarase gene, agaD, from a marine bacterium, Vibrio sp. strain PO-303. Biosci. Biotechnol. Biochem. 71: 38-46.
    Pubmed CrossRef
  6. Feng ZH, Peng L, Chen M, Li MY. 2012. Rhodococcus sp. Q5, a novel agarolytic bacterium isolated from printing and dyeing wastewater. Folia Microbiol. 57: 379-386.
    Pubmed CrossRef
  7. Fu XT, Lin H, Kim SW. 2008. Purification and characterization of a novel β-agarase, AgaA34, from Agarivorans albus YKW-34. Appl. Microbiol. Biotechnol. 78: 265-273.
    Pubmed CrossRef
  8. Helbert W, Michel G, Barbeyron T. 2005. The endo-betaagarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours. Biochem. J. 385: 703713.
  9. Huber H, Stetter KO. 1998. Hyperthermophiles and their possible potential in biotechnology. J. Biotechnol. 64: 39-52.
    CrossRef
  10. Jonnadula R, Ghadi SC. 2011. Purification and characterization of β-agarase from seaweed decomposing bacterium Microbulbifer sp. strain CMC-5. Biotechnol. Bioprocess Eng. 16: 513-519.
    CrossRef
  11. Khambhaty Y, Mody K, Jha B. 2008. Purification, characterization and application of a novel extracellular agarase from a marine Bacillus megaterium. Biotechnol. Bioprocess Eng. 13: 584-591.
    CrossRef
  12. Kobayashi R, Takisada M, Suzuki T, Kirimura K, Usami S. 1997. Neoagarobiose as a novel moisturizer with whitening effect. Biosci. Biotechnol. Biochem. 61: 162-163.
    Pubmed CrossRef
  13. Kristjansson JK. 1989. Thermophilic organisms as sources of thermostable enzymes. Trends Biotechnol. 7: 349-353.
    CrossRef
  14. Ladenstein R, Antranikian G. 1988. Proteins from hyperthermophiles: stability and enzymatic catalysis close to the boiling point of water. Adv. Biochem. Eng. Biotechnol. 61:37-85.
  15. Lee DG, Park GT, Kim NY, Lee EJ, Jang MK, Shin YG, et al. 2006. Cloning, expression, and characterization of a glycoside hydrolase family 50 β-agarase from a marine Agarivorans isolate. Biotechnol. Lett. 28: 1925-1932.
    Pubmed CrossRef
  16. Leon O, Quintana L, Peruzzo G, Slebe JC. 1992. Purification and properties of an extracellular agarase from Alteromonas sp. strain C-1. Appl. Environ. Microbiol. 58: 4060-4063.
    Pubmed
  17. Long M, Yu Z, Xu X. 2010. A novel beta-agarase with high pH stability from marine Agarivorans sp. LQ48. Mar. Biotechnol. (NY) 12: 62-69.
    Pubmed CrossRef
  18. Lowe SE, Jain MK, Zeikus JG. 1993. Biology, ecology, and biotechnological applications of anaerobic bacteria in temperature, pH salinity or substrate. Microbiol. Rev. 57: 451-509.
    Pubmed
  19. Malmqvist M. 1978. Purification and characterization of two different agarose degrading enzymes. Biochim. Biophys. Acta 537: 31-43.
    CrossRef
  20. Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428.
    CrossRef
  21. Namsaraev ZB, Gorlenko VM, Namsaraev BB, Barkhutova DD. 2006. Microbial Communities of Alkaline Hydrotherms. Russian Academy of Sciences, Novosibirsk, Russia.
  22. Ohta Y, Hatada Y, Miyazaki M, Nogi Y, Ito S, Horikoshi K. 2005. Purification and characterization of a novel β-agarase from a Thalassomonas sp. Curr. Microbiol. 50: 212-216.
    Pubmed CrossRef
  23. Ohta Y, Hatada Y, Nogi Y, Li Z, Ito S, Horikoshi K. 2004. Cloning, expression, and characterization of a glycoside hydrolase family 86 beta agarase from a deep-sea Microbulbifer-like isolate. Appl. Microbiol. Biotechnol. 66: 266-275.
    Pubmed CrossRef
  24. Shi YL, Lu XZ, Yu WG. 2008. A new β-agarase from marine bacterium Janthinobacterium sp. SY12. World J. Microbiol. Biotechnol. 24: 2659-2664.
    CrossRef
  25. Sugano Y, Terada I, Arita M, Noma M, Matsumoto T. 1993. Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107. Appl. Environ. Microbiol. 59: 1549-1554.
    Pubmed
  26. Sunna A, Moracci M, Rossi M, Antranikian G. 1997. Glycosyl hydrolases from hyperthermophiles. Extremophiles 1: 2-13.
    Pubmed CrossRef
  27. Suzuki H, Sawai Y, Suzuki T, Kawai K. 2003. Purification and characterization of an extracellular β-agarase from Bacillus sp. MK03. J. Biosci. Bioeng. 93: 456-463.
  28. Van der Meulen HJ, Harder W. 1975. Production and characterization of the agarase of Cytophaga flevensis. Antonie Van Leeuwenhoek 41: 431-447.
    Pubmed CrossRef
  29. Wang J, Mou H, Jiang X, Guan H. 2006. Characterization of a novel β-agarase from marine Alteromonas sp. SY37-12 and its degrading products. Appl. Microbiol. Biotechnol. 71: 833839.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2014; 24(1): 19-25

Published online January 28, 2014 https://doi.org/10.4014/jmb.1308.08055

Copyright © The Korean Society for Microbiology and Biotechnology.

Purification and Characterization of Thermostable Agarase from Bacillus sp. BI-3, a Thermophilic Bacterium Isolated from Hot Spring

Jiang Li 1, 2*, Yujie Sha 1, 2, Dewi Seswita-Zilda 3, Qiushi Hu 1, 2 and Peiqing He 1, 2

1The First Institute of Oceanography, SOA, Qingdao 266061, China, 2Key Laboratory of Marine Bioactive Substances, SOA, Qingdao 266061, China, 2Research Center for Marine and Fisheries Product Processing and Biotechnology, Agency for Marine and Fisheries Research and Development, Ministry of Marine and Fisheries Affairs, Jakarta 40115, Indonesia

Received: August 20, 2013; Accepted: September 24, 2013

Abstract

An extracellular agarase was purified from Bacillus sp. BI-3, a thermophilic agar-degrading
bacterium isolated from a hot spring in Indonesia. The purified agarase revealed a single band
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular
mass of 58 kDa. The optimum pH and temperature of the agarase were 6.4 and 70oC,
respectively. The activity of the agarase was stable at high temperatures, and more than 50%
activity was retained at 80oC for 15 min. Furthermore, the enzyme was stable in the pH range
of 5.8–8.0, and more than 60% of the residual activity was retained. Significant activation of
the agarase was observed in the presence of K+, Na+, Ca2+, Mg2+, and Sr2+; on the other hand,
Ba2+, Zn2+, Cu2+, Mn2+, Co2+, Fe2+, and EDTA inhibited or inactivated the enzyme activity. The
components of the hydrolytic product analyzed by thin-layer chromatography showed that
the agarase mainly produced neoagarobiose. This study is the first to present evidence of
agarolytic activity in aerobic thermophilic bacteria.

Keywords: Hot spring bacteria, Thermostable agarase, Purification, Characterization

References

  1. Agbo JAC, Moss MO. 1979. The isolation and characterization of agarolytic bacteria from a lowland river. J. Gen. Microbiol. 115: 355-368.
    CrossRef
  2. Aoki T, Araki T, Kitamikado M. 1990. Purification and characterization of a novel beta-agarase from Vibrio sp. AP-2. Eur. J. Biochem. 187: 461-465.
    Pubmed CrossRef
  3. Araki T, Hayakawa M, Zhang L, Karita S, Morishita T. 1998. Purification and characterization of agarases from a marine bacterium, Vibrio sp. PO-303. J. Marine Biotechnol. 6: 260-265.
    Pubmed
  4. Bannikova GE, Lopatin SA, Varlamov VP, Kuznetsov BB, Kozina IV, Miroshnichenko ML, et al. 2008. The thermophilic bacteria hydrolyzing agar: characterization of thermostable agarase. Appl. Biochem. Microbiol. 45: 366-371.
    CrossRef
  5. Dong J, Tamaru Y, Araki T. 2007. Molecular cloning, expression, and characterization of a beta-agarase gene, agaD, from a marine bacterium, Vibrio sp. strain PO-303. Biosci. Biotechnol. Biochem. 71: 38-46.
    Pubmed CrossRef
  6. Feng ZH, Peng L, Chen M, Li MY. 2012. Rhodococcus sp. Q5, a novel agarolytic bacterium isolated from printing and dyeing wastewater. Folia Microbiol. 57: 379-386.
    Pubmed CrossRef
  7. Fu XT, Lin H, Kim SW. 2008. Purification and characterization of a novel β-agarase, AgaA34, from Agarivorans albus YKW-34. Appl. Microbiol. Biotechnol. 78: 265-273.
    Pubmed CrossRef
  8. Helbert W, Michel G, Barbeyron T. 2005. The endo-betaagarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours. Biochem. J. 385: 703713.
  9. Huber H, Stetter KO. 1998. Hyperthermophiles and their possible potential in biotechnology. J. Biotechnol. 64: 39-52.
    CrossRef
  10. Jonnadula R, Ghadi SC. 2011. Purification and characterization of β-agarase from seaweed decomposing bacterium Microbulbifer sp. strain CMC-5. Biotechnol. Bioprocess Eng. 16: 513-519.
    CrossRef
  11. Khambhaty Y, Mody K, Jha B. 2008. Purification, characterization and application of a novel extracellular agarase from a marine Bacillus megaterium. Biotechnol. Bioprocess Eng. 13: 584-591.
    CrossRef
  12. Kobayashi R, Takisada M, Suzuki T, Kirimura K, Usami S. 1997. Neoagarobiose as a novel moisturizer with whitening effect. Biosci. Biotechnol. Biochem. 61: 162-163.
    Pubmed CrossRef
  13. Kristjansson JK. 1989. Thermophilic organisms as sources of thermostable enzymes. Trends Biotechnol. 7: 349-353.
    CrossRef
  14. Ladenstein R, Antranikian G. 1988. Proteins from hyperthermophiles: stability and enzymatic catalysis close to the boiling point of water. Adv. Biochem. Eng. Biotechnol. 61:37-85.
  15. Lee DG, Park GT, Kim NY, Lee EJ, Jang MK, Shin YG, et al. 2006. Cloning, expression, and characterization of a glycoside hydrolase family 50 β-agarase from a marine Agarivorans isolate. Biotechnol. Lett. 28: 1925-1932.
    Pubmed CrossRef
  16. Leon O, Quintana L, Peruzzo G, Slebe JC. 1992. Purification and properties of an extracellular agarase from Alteromonas sp. strain C-1. Appl. Environ. Microbiol. 58: 4060-4063.
    Pubmed
  17. Long M, Yu Z, Xu X. 2010. A novel beta-agarase with high pH stability from marine Agarivorans sp. LQ48. Mar. Biotechnol. (NY) 12: 62-69.
    Pubmed CrossRef
  18. Lowe SE, Jain MK, Zeikus JG. 1993. Biology, ecology, and biotechnological applications of anaerobic bacteria in temperature, pH salinity or substrate. Microbiol. Rev. 57: 451-509.
    Pubmed
  19. Malmqvist M. 1978. Purification and characterization of two different agarose degrading enzymes. Biochim. Biophys. Acta 537: 31-43.
    CrossRef
  20. Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428.
    CrossRef
  21. Namsaraev ZB, Gorlenko VM, Namsaraev BB, Barkhutova DD. 2006. Microbial Communities of Alkaline Hydrotherms. Russian Academy of Sciences, Novosibirsk, Russia.
  22. Ohta Y, Hatada Y, Miyazaki M, Nogi Y, Ito S, Horikoshi K. 2005. Purification and characterization of a novel β-agarase from a Thalassomonas sp. Curr. Microbiol. 50: 212-216.
    Pubmed CrossRef
  23. Ohta Y, Hatada Y, Nogi Y, Li Z, Ito S, Horikoshi K. 2004. Cloning, expression, and characterization of a glycoside hydrolase family 86 beta agarase from a deep-sea Microbulbifer-like isolate. Appl. Microbiol. Biotechnol. 66: 266-275.
    Pubmed CrossRef
  24. Shi YL, Lu XZ, Yu WG. 2008. A new β-agarase from marine bacterium Janthinobacterium sp. SY12. World J. Microbiol. Biotechnol. 24: 2659-2664.
    CrossRef
  25. Sugano Y, Terada I, Arita M, Noma M, Matsumoto T. 1993. Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107. Appl. Environ. Microbiol. 59: 1549-1554.
    Pubmed
  26. Sunna A, Moracci M, Rossi M, Antranikian G. 1997. Glycosyl hydrolases from hyperthermophiles. Extremophiles 1: 2-13.
    Pubmed CrossRef
  27. Suzuki H, Sawai Y, Suzuki T, Kawai K. 2003. Purification and characterization of an extracellular β-agarase from Bacillus sp. MK03. J. Biosci. Bioeng. 93: 456-463.
  28. Van der Meulen HJ, Harder W. 1975. Production and characterization of the agarase of Cytophaga flevensis. Antonie Van Leeuwenhoek 41: 431-447.
    Pubmed CrossRef
  29. Wang J, Mou H, Jiang X, Guan H. 2006. Characterization of a novel β-agarase from marine Alteromonas sp. SY37-12 and its degrading products. Appl. Microbiol. Biotechnol. 71: 833839.
    Pubmed CrossRef