Advertisement

Microbial Ecology

, Volume 75, Issue 2, pp 387–399 | Cite as

Screening and Characterization of Phytases from Bacteria Isolated from Chilean Hydrothermal Environments

  • Milko A. JorqueraEmail author
  • Stefanie Gabler
  • Nitza G. Inostroza
  • Jacquelinne J. Acuña
  • Marco A. Campos
  • Daniel Menezes-Blackburn
  • Ralf Greiner
Environmental Microbiology

Abstract

Phytases are enzymes involved in organic phosphorus cycling in nature and widely used as feed additives in animal diets. Thermal tolerance is a desired property of phytases. The objectives of this study were to screen and characterize bacterial phytases from Chilean hydrothermal environments. In this study, 60% (30 of 63) of screened thermophilic (60 °C) isolates showed phytase activity in crude protein extracts. The characterization of phytase from two selected isolates (9B and 15C) revealed that both isolates produce phytases with a pH optimum at 5.0. The temperature optimum for phytate dephosphorylation was determined to be 60 and 50 °C for the phytases from the isolates 9B and 15C, respectively. Interestingly, the phytase from the isolate 15C showed a residual activity of 46% after incubation at 90 °C for 20 min. The stepwise dephosphorylation of phytate by protein extracts of the isolates 9B and 15C was verified by HLPC analysis. Finally, the isolates 9B and 15C were identified by partial sequencing of the 16S rRNA gene as members of the genera Bacillus and Geobacillus, respectively.

Keywords

Bacillus Geobacillus Geyser Hot spring Phytate Thermophile 

Notes

Acknowledgements

This study was supported by cooperative project (code PCCI 1-2011) funded by The National Commission for Scientific and Technological Research (CONICYT, Chile) and Deutscher Akademischer Austauschdienst German (DAAD, Germany). This work was also partial financed with funds from CONICYT-PCCI (code USA2013-0010) and The National Fund for Scientific and Technological Development (FONDECYT, Chile; projects no. 1120505 and 1160302). J.J. Acuña acknowledges FONDECYT programs (projects no. 3140620 and 11160112).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Balaban NP, Suleimanova AD, Valeeva LR, Shakirov EV, Sharipova MR (2016) Structural characteristics and catalytic mechanism of Bacillus β-propeller phytases. Biochemistry 81:785–793PubMedGoogle Scholar
  2. 2.
    Borgi MA, Boudebbouze S, Mkaouar H, Maguin E, Rhimi M (2015) Bacillus phytases: current status and future prospects. Bioengineered 6:233–236CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Boukhris I, Farhat-Khemakhem A, Blibech M, Bouchaala K, Chouayekh H (2015) Characterization of an extremely salt-tolerant and thermostable phytase from Bacillus amyloliquefaciens US573. Int J Biol Macromol 80:581–587CrossRefPubMedGoogle Scholar
  4. 4.
    Choi KY, Noh DO, Cho SH, Lee HK, Suh HJ, Chung S-H (1999) Isolation of a phytase-producing Bacillus sp. KHU-10 and its phytase production. J Microbiol Biotechnol 9:223–226Google Scholar
  5. 5.
    Choi YM, Suh HJ, Kim JM (2001) Purification and properties of extracellular phytase from Bacillus sp. KHU-10. J Protein Chem 20:287–292CrossRefPubMedGoogle Scholar
  6. 6.
    Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16s ribosomal RNA. Nucleic Acids Res 17:7843–7853CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Greiner R (2007) Phytate-degrading enzymes: regulation of synthesis in microorganisms and plants. In: Turner BL, Richardson AE, Mullaney EJ (eds) Inositol phosphates: linking agriculture and the environment/secondary phytate-degrading enzymes: regulation of synthesis in microorganisms and plants. CAB International, Oxfordshire, pp. 78–96CrossRefGoogle Scholar
  8. 8.
    Greiner R, Konietzny U, Jany KD (1993) Purification and characterization of two phytases from Escherichia coli. Arch Biochem Biophys 303:107–113CrossRefPubMedGoogle Scholar
  9. 9.
    Gulati HK, Chadha BS, Saini HS (2007) Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil. J Ind Microbiol Biotechnol 34:91–98CrossRefPubMedGoogle Scholar
  10. 10.
    Huang H, Luo H, Yang P, Meng K, Wang Y, Yuan T, Bai Y, Yao B (2006) A novel phytase with preferable characteristics from Yersinia intermedia. Biochem Bioph Res Co 350:884–889CrossRefGoogle Scholar
  11. 11.
    Huang H, Shi P, Wang Y, Luo H, Shao N, Wang G, Yang P, Yao B (2009) Diversity of beta−propeller phytase genes in the intestinal contents of grass carp provides insight into the release of major phosphorus from phytate in nature. Appl Environ Microb 75:1508–1516CrossRefGoogle Scholar
  12. 12.
    Huang H, Zhang R, Fu D, Luo J, Li Z, Luo H, Shi P, Yang P, Diao Q, Yao B (2011) Diversity, abundance and characterization of ruminal cysteine phytases suggest their important role in phytate degradation. Environ Microbiol 13:747–757CrossRefPubMedGoogle Scholar
  13. 13.
    Jain J, Sapna, Singh B (2016) Characteristics and biotechnological applications of bacterial phytases. Process Biochem 51:159–169CrossRefGoogle Scholar
  14. 14.
    Jorquera MA, Crowley DE, Marschner P, Greiner R, Fernández MT, Romero D, Menezes-Blackburn D, Mora ML (2011) Identification of β-propeller phytase-encoding genes in culturable Paenibacillus and Bacillus spp. from the rhizosphere of pasture plants on volcanic soils. FEMS Microb Ecol 75:163–172CrossRefGoogle Scholar
  15. 15.
    Jorquera MA, Inostroza NG, Lagos LM, Barra PJ, Marileo LG, Rilling JI, Campos DC, Crowley DE, Richardson AE, Mora ML (2014) Bacterial community structure and detection of putative plant growth-promoting rhizobacteria associated with plants grown in Chilean agro-ecosystems and undisturbed ecosystems. Biol Fertil Soils 50:1141–1153CrossRefGoogle Scholar
  16. 16.
    Kim YO, Lee JK, Kim HK, Yu JH, Oh TK (1998) Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli. FEMS Microbiol Lett 162:185–191CrossRefPubMedGoogle Scholar
  17. 17.
    Kumar V, Yadav AN, Verma P, Sangwan P, Saxena A, Kumar K, Singh B (2017) β-Propeller phytases: diversity, catalytic attributes, current developments and potential biotechnological applications. Int J Biol Macromol 98:595–609CrossRefPubMedGoogle Scholar
  18. 18.
    Lim BL, Yeung P, Cheng C, Hill JE (2007) Distribution and diversity of phytate-mineralizing bacteria. The ISME J 1:321–330PubMedGoogle Scholar
  19. 19.
    Mandviwala T, Khire J (2000) Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation. J Ind Microbiol Biotech 24:237–243CrossRefGoogle Scholar
  20. 20.
    Marchesi JR, Sato T, Weightman AJ, Martin TA, Fry JC, Hiom SJ, Wade WG (1998) Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl Environ Microb 64:795–799Google Scholar
  21. 21.
    Menezes-Blackburn D, Greiner R (2015) Enzymes used in animal feed: leading technologies and forthcoming developments, In: Cirillo G, Gianfranco–Spizzirri G & Lemma F (eds.) Functional Polymers in Food Science, Scrivener Publishing LLC, 47–73 ppGoogle Scholar
  22. 22.
    Menezes-Blackburn D, Jorquera MA, Greiner R, Gianfreda L, Mora ML (2013) Phytases and phytase-labile organic phosphorus in manures and soils. Crit Rev Environ Sci Technol 43:916–954CrossRefGoogle Scholar
  23. 23.
    Menezes-Blackburn D, Gabler S, Greiner R (2015) Performance of seven commercial phytases in an in vitro simulation of poultry digestive tract. J Agric Food Chem 63:6142–6149CrossRefPubMedGoogle Scholar
  24. 24.
    Nakashima BA, Mcallister TA, Sharma R, Selinger LB (2007) Diversity of phytases in the rumen. Microb Ecol 53:82–88CrossRefPubMedGoogle Scholar
  25. 25.
    Nam S-J, Kim Y-O, Ko T-K, Kang J-K, Chun K-H, Auh J-H, Lee C-S, Lee I-K, Park S, Oh B-C (2014) Molecular and biochemical characteristics of β-propeller phytase from marine Pseudomonas sp. BS10-3 and its potential application for animal feed additives. J Microbiol Biotechnol 24:1413–1420CrossRefPubMedGoogle Scholar
  26. 26.
    Oh B-C, Kim ME, Yun B-S, Choi W-C, Park S-C, Bae S-C, Oh T-K (2006) Ca2+-inositol phosphate chelation mediates the substrate specificity of β-propeller phytase. Biochemistry 45:9531–9539CrossRefPubMedGoogle Scholar
  27. 27.
    Parhamfar M, Badoei-Dalfard A, Khaleghi M, Hassanshahian M (2015) Purification and characterization of an acidic, thermophilic phytase from a newly isolated Geobacillus stearothermophilus strain DM12. Prog Biol Sci 5:61–73Google Scholar
  28. 28.
    Powar VK, Jagannathan V (1982) Purification and properties of phytate-specific phosphatase from Bacillus subtilis. J Bacteriol 151:1102–1108PubMedPubMedCentralGoogle Scholar
  29. 29.
    Rebello S, Jose L, Sindhu R, Aneesh EM (2017) Molecular advancements in the development of thermostable phytases. Appl Microbiol Biotechnol 101:2677–2689CrossRefPubMedGoogle Scholar
  30. 30.
    Sajidan WR, Sari EN, Ratriyanto A, Weldekiros H, Greiner R (2015) Phytase-producing bacteria from extreme regions in Indonesia. Braz Arch Biol Technol 58:711–717CrossRefGoogle Scholar
  31. 31.
    Sandberg AS, Ahderinne R (1986) HPLC method for determination of inositol tri-, tetra-, penta-, and hexaphosphates in foods and intestinal contents. J Food Sci 51:547–550CrossRefGoogle Scholar
  32. 32.
    Sato VS, Jorge JA, Guimarães LH (2016) Characterization of a thermotolerant phytase produced by Rhizopus microsporus var. microsporus biofilm on an inert support using sugarcane bagasse as carbon source. Appl Biochem Biotechnol 179:610–624CrossRefPubMedGoogle Scholar
  33. 33.
    Shimizu M (1992) Purification and characterization of phytase from Bacillus subtilis (Natto) N-77. Biosci Biotechnol Biochem 56:1266–1269CrossRefGoogle Scholar
  34. 34.
    Tye A, Siu F, Leung T, Lim B (2002) Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis. Appl Microbiol Biot 59:190–197CrossRefGoogle Scholar
  35. 35.
    Yu P, Chen Y (2013) Purification and characterization of a novel neutral and heat-tolerant phytase from a newly isolated strain Bacillus nealsonii ZJ0702. BMC Biotechnol 13:78CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Zhang R, Yang P, Huang H, Shi P, Yuan T, Yao B (2011) Two types of phytases (histidine acid phytase and beta-propeller phytase) in Serratia sp. TN49 from the gut of Batocera horsfieldi (Coleoptera) larvae. Curr Microbiol 63:408–415CrossRefPubMedGoogle Scholar
  37. 37.
    Zhang R, Yang P, Huang H, Yuan T, Shi P, Meng K, Yao B (2011) Molecular and biochemical characterization of a new alkaline β-propeller phytase from the insect symbiotic bacterium Janthinobacterium sp. TN115. Appl Microbiol Biotechnol 92:317–325CrossRefPubMedGoogle Scholar
  38. 38.
    Zhou JR, Erdman Jr JW (1995) Phytic acid in health and disease. Crit Rev Food Sci Nutr 35:495–508CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Milko A. Jorquera
    • 1
    • 2
    Email author
  • Stefanie Gabler
    • 3
  • Nitza G. Inostroza
    • 1
    • 2
  • Jacquelinne J. Acuña
    • 1
    • 2
  • Marco A. Campos
    • 1
    • 2
  • Daniel Menezes-Blackburn
    • 3
    • 4
  • Ralf Greiner
    • 3
  1. 1.Applied Microbial Ecology Laboratory, Department of Chemical Sciences and Natural ResourcesUniversidad de La FronteraTemucoChile
  2. 2.Scientific and Technological Bioresource NucleusUniversidad de La FronteraTemucoChile
  3. 3.Department of Food Technology and Bioprocess Engineering, Federal Research Institute of Nutrition and FoodMax Rubner–InstitutKarlsruheGermany
  4. 4.Lancaster Environment CentreLancaster UniversityLancasterUK

Personalised recommendations