A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

  • Thi Thuy Tran
  • Gashaw Mamo
  • Bo Mattiasson
  • Rajni Hatti-Kaul
Original Paper

Abstract

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100°C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20–30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed–batch cultivation, glucose concentration was maintained at 2–3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.

Keywords

Alkaline phytase Bacillus sp. Fed-batch cultivation Protein secretion 

References

  1. 1.
    Åkesson M, Hagander P, Axelsson P (2001) Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding. Biotechnol Bioeng 73:223–230CrossRefPubMedGoogle Scholar
  2. 2.
    Cheryan M (1980) Phytic acid interaction in food systems. CRC Crit Rev Food Sci Nutr 13:297–335CrossRefGoogle Scholar
  3. 3.
    Choi YM, Suh HJ, Kim JM (2001) Purification and properties of extracellular phytase from Bacillus sp. KHU-10. J. Protein Chem 20:287–292CrossRefGoogle Scholar
  4. 4.
    Fu S, Sun J, Qian L, Li Z (2008) Bacillus phytases: present scenario and future perspectives. Appl Biochem Biotechnol 151:1–8CrossRefPubMedGoogle Scholar
  5. 5.
    Golovan SP, Meidinger RG, Ajakaiye A, Cottrill M, Weiderkehr MZ, Barney D, Plante C, Pollard JW, Fan MZ, Hayes MA, Laursen J, Hjorth JP, Hacker RR, Phillips JP, Forsberg CW (2001) Pigs expressing salivary phytase produce low-phosphorus manure. Nature Biotech 19:741–745CrossRefGoogle Scholar
  6. 6.
    Gombert AK, Kilikian BV (1998) Recombinant gene expression in Escherichia coli cultivation using lactose as inducer. J Biotechnol 60:47–54CrossRefPubMedGoogle Scholar
  7. 7.
    Gulati HK, Chadha BS, Saini HS (2007) Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil. J Indust Microbiol Biotechnol 34:91–98Google Scholar
  8. 8.
    Hara A, Ebina S, Kondo A, Funagua T (1985) A new type of phytase from Typha latifolia L. Agric Biol Chem 49:3539–3544Google Scholar
  9. 9.
    Holm T, Arvidson S, Lindholm B, Pavlu B (1970) Enzyme laboratory-scale production. Process Biochem 5:62–66Google Scholar
  10. 10.
    Horn U, Strittmatter W, Krebber A, Knupfer U, Kujau M, Wenderoth R, Muller K, Matzku S, Pluckthun A, Riesenberg D (1996) High volumetric yield of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth condition. Appl Microbiol Biotechnol 46:524–532CrossRefPubMedGoogle Scholar
  11. 11.
    Huber RE, Kurz G, Wallenfels K (1976) A quantitation of the factors which affect the hydrolase and transgalactosidase activities of ß-galactosidase (E. coli) on lactose. Biochem 15:1994–2001CrossRefGoogle Scholar
  12. 12.
    Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N, Apajalahti J (1998) Isolation, characterization, molecular gene cloning and sequencing of novel phytase from Bacillus subtilis. Appl Environ Microbiol 64:2079–2085PubMedGoogle Scholar
  13. 13.
    Kerovuo J, Von Weymarn N, Povelainen M, Auer S, Miasnikov A (2000) A new efficient expression system for Bacillus and its application to production of recombinant phytase. Biotech Lett 22:1311–1317CrossRefGoogle Scholar
  14. 14.
    Kim YO, Kim HK, Bae KS, Yu JH, Oh TK (1998) Purification and properties of a thermostable phytase from Bacillus sp. DS11. Enzyme Microb Technol 22:2–7CrossRefGoogle Scholar
  15. 15.
    Kim YO, Kim HK, Lee JK, Yu JH, Oh TK (1998) Cloning of the thermostable phytase gene (phy) from Bacillus DS11 and its over expression in Escherichia coli. FEMS Microbiol Lett 162:185–191CrossRefPubMedGoogle Scholar
  16. 16.
    Kim DH, Oh BC, Choi WC, Lee JK, Oh TK (1999) Enzymatic evaluation of Bacillus amyloliquefaciens phytase as a feed additive. Biotech Lett 21:925–927CrossRefGoogle Scholar
  17. 17.
    Kim YO, Lee JK, Oh BC, Oh TK (1999) High-level expression of a recombinant thermostable phytase in Bacillus subtilis. Biosci Biotechnol Biochem 63:2205–2207CrossRefGoogle Scholar
  18. 18.
    Kleist S, Miksch G, Hitzmann B, Arndt M, Friehs K, Flaschel E (2003) Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies. Appl Microbiol Biotechnol 61:456–462PubMedGoogle Scholar
  19. 19.
    Lassen SF, Breinholt J, Ostergaard PR, Brugger R, Bischoff A, Wyss M, Fuglsang CC (2001) Expression, gene cloning, and characterization of five novel phytase from four Basidomycete fungi: Peniophora lycii, Agrocybe pediades a Ceriporia sp., and Trametes pubescens. Appl Environ Microbiol 67:4701–4707CrossRefPubMedGoogle Scholar
  20. 20.
    Lopez HW, Leenhardt F, Coudray C, Remesy C (2002) Mineral and phytic acid interactions: is it a real problem for human nutrition? Int J Food Sci Tech 37:727–739CrossRefGoogle Scholar
  21. 21.
    Maddaiah VT, Kurnick AA, Reid BL (1964) Phytic acid studies. Proc Soc Exp Biol Med 115:391–393PubMedGoogle Scholar
  22. 22.
    Maenz DD, Engele-Schaan CM, Newkirk RW, Classen HL (1999) The effect of minerals and mineral chelators on the formation of phytase-resistant and phytase-susceptible forms of phytic acid in solution and in a slurry of canola meal. Anim Feed Sci Technol 81:177–192CrossRefGoogle Scholar
  23. 23.
    Mallin MA (2000) Impact of industrial animal production on river and estuaries. Am Sci (January–February) 88:26–73Google Scholar
  24. 24.
    Miksch G, Kleist S, Frieh K, Flaschel E (2002) Overexpression of phytase from Escherichia coli and its extracellular production in bioreactors. Appl Microbiol Biotechnol 59:685–694CrossRefPubMedGoogle Scholar
  25. 25.
    Muller-Hill B, Rickenberg HV, Wallenfels K (1964) Specificity of the induction of the enzymes of the lac operon in Escherichia coli. J Mol Biol 10:303–318CrossRefGoogle Scholar
  26. 26.
    Ni Y, Chen R (2009) Extracellular recombinant protein production from Escherichia coli. Biotechnol Lett 31:1661–1670CrossRefPubMedGoogle Scholar
  27. 27.
    Nolan KB, Duffin PA, Mc Weeny DJ (1987) Effect of phytate on mineral bioavailability. In vitro studies on Mg2+, Ca2+, Fe3+, Cu2+ and Zn2+ (also Cd2+) solubilities in the presence of phytate. J Sci Food Agric 40:79–85CrossRefGoogle Scholar
  28. 28.
    Oh BC, Choi WC, Park S, Kim YO, Oh TK (2004) Biochemical properties and substrate specificities of alkaline and histidine acid phytase. Appl Microbiol Biotechnol 63:362–372CrossRefPubMedGoogle Scholar
  29. 29.
    Pages JM, Anba J, Lazdunski C (1987) Conditions leading to secretion of a normally periplasmic protein in Escherichia coli. J Bacteriol 169:1386–1390PubMedGoogle Scholar
  30. 30.
    Purva V, Uttam CB (2004) Production studies and catalytic properties of phytases (myo-inositolhexakisphosphate phosphohydrolase): an overview. Enzyme Micob Technol 35:3–4CrossRefGoogle Scholar
  31. 31.
    Ramchuran SO, Nordberg Karlsson E, Velut S, De Mare L, Hagander P, Holst O (2002) Production of heterologous thermostable glycoside hydrolases and presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3). Appl Microbiol Biotechnol 60:408–416CrossRefPubMedGoogle Scholar
  32. 32.
    Ramchuran SO, Nordberg Karlsson E, Holst O (2003) Effect of post induction, nutrient feed composition and use of lactose as inducer during production of thermostable xylanase in Escherichia coli. J Biosci Bioeng 99:477–484CrossRefGoogle Scholar
  33. 33.
    Rao DECS, Rao KV, Reddy VD (2008) Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodies. J Appl Microbiol 105:1128–1137CrossRefPubMedGoogle Scholar
  34. 34.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  35. 35.
    Scott JJ, Loewus FA (1986) A calcium-activated phytase from pollen of Lilium longiflorum. Plant Physiol 82:333–335CrossRefPubMedGoogle Scholar
  36. 36.
    Shah P, Bhavsar K, Soni SK, Khire JM (2009) Strain improvement and up scaling of phytase production by Aspergillus niger NCIM 563 under submerged fermentation conditions. J Ind Microbiol Biotechnol 36:373–380CrossRefPubMedGoogle Scholar
  37. 37.
    Shimizu M (1992) Purification and characterization of phytase from Bacillus subtillis (nato) N-77. Biosci Biotech Biochem 56:1266–1269CrossRefGoogle Scholar
  38. 38.
    Simon O, Igbasan F (2002) In vitro properties of phytase from various microbial origins. Inter J Food Sci Tech 37:813–822CrossRefGoogle Scholar
  39. 39.
    Stahl CH, Wilson DB, Lei XG (2003) Comparison of extracellular Escherichia coli AppA phytase expressed in Streptomyces lividans and Pichia pastoris. Biotech Lett 25:827–831CrossRefGoogle Scholar
  40. 40.
    Sunitha K, Lee JK, Oh TK (1999) Optimization of medium components for phytase production by E. coli using response surface methodology. Bioproc Eng 21:477–481Google Scholar
  41. 41.
    Suominen I, Karp M, Lahde M, Kopio A, Glumoff T, Meyer P, Mantsala P (1987) Extracellular production of cloned α-amylase by Escherichia coli. Gene 61:165–176CrossRefPubMedGoogle Scholar
  42. 42.
    Viitanen MI, Vasala A, Neubauer P, Alatossava T (2003) Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli. Microb Cell Fact 2:2CrossRefPubMedGoogle Scholar
  43. 43.
    Vohra A, Satyanarayana T (2004) A cost-effective molasses medium for enhanced cell-bound phytase production by Pichia anomala. J Appl Microbiol 97:471–476CrossRefPubMedGoogle Scholar
  44. 44.
    Vuolanto A, Von Weymarn N, Kerovuo J, Ojamo H, Leisola M (2001) Phytase production by high cell density culture of recombinant Bacillus subtilis. Biotech Lett 23:761–766CrossRefGoogle Scholar
  45. 45.
    Wyss M, Brugger R, Kronenberger A, Remy R, Fimbel R, Oesterhelt G, Lehmann M, Van Loon AP (1999) Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl Environ Microbiol 65:367–373PubMedGoogle Scholar
  46. 46.
    Yamabhai M, Emrat S, Sukasem S, Pesatcha P, Jaruseranee N, Buranabanyat B (2008) Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems. J Biotechnol 133:50–57CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2009

Authors and Affiliations

  • Thi Thuy Tran
    • 1
    • 2
  • Gashaw Mamo
    • 1
  • Bo Mattiasson
    • 1
  • Rajni Hatti-Kaul
    • 1
  1. 1.Department of BiotechnologyLund UniversityLundSweden
  2. 2.Biotechnology and Microbiology DepartmentHanoi National University of EducationHanoiVietnam

Personalised recommendations