Skip to main content
SpringerLink
Log in

Effect of metals ions on thermostable alkaline phytase from Bacillus subtilis YCJS isolated from soybean rhizosphere soil

  • Original Article
  • Published:
Annals of Microbiology Aims and scope Submit manuscript

Abstract

A Bacillus sp.YCJS strain showing phytase activity was isolated, and the phytase-encoding gene was cloned and expressed in Escherichia coli. The 1,149-bp full-length gene encoded a 26-residue putative signal peptide and a 356-residue mature protein. The molecular weight was estimated to be 47.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified recombinant enzyme phy(ycE) from E. coli exhibited a specific activity of 14 U mg-1 protein. The optimum pH and temperature were 6.0 and 50 °C, respectively. The thermal stability of phy(ycE) was drastically improved in the presence of calcium ions (Ca2+). Fluorescence analysis results indicated that compared with phy(ycE) without added Ca2+, phy(ycE) in the presence of Ca2+ was more stable and the melting temperature improved from 47.8 to 62.4 °C. Circular dichroism spectrometric analysis revealed that the loss of enzymatic activity was most likely due to a conformational change, as the circular dichroism spectra of the holoenzyme and metal-depleted enzyme were significantly different. Compared with the Ca2+-reactivated enzyme, the La3+-reactivated enzyme did not undergo a significant recovery with respect to its conformation. The aromatic-sensitized terbium (Tb3+) fluorescence results indicated that five Tb3+ could bind to each molecule of phy(ycE) and that there were two high-affinity and three low-affinity binding sites.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Asryants RA, Duszenkova IV, Nagradova NK (1985) Determination of Sepharose-bound protein with Coomassie brilliant blue G-250. Anal Biochem 151(2):571–574

    Article  CAS  PubMed  Google Scholar 

  • Bohn L, Meyer AS, Rasmussen SK (2008) Phytate: impact on environment and human nutrition. A challenge for molecular breeding. J Zhejiang Univ Sci B 9(3):165–191

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Evans CH (1990) Biochemistry of the lanthanides. Plenum Press, New York

  • Farhat A, Chouayekh H, Ben Farhat M, Bouchaala K, Bejar S (2008) Gene cloning and characterization of a thermostable phytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme. Mol Biotechnol 40(2):127–135

    Article  CAS  PubMed  Google Scholar 

  • 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(1):91–98

    Article  CAS  PubMed  Google Scholar 

  • Ha NC, Oh BC, Shin S, Kim HJ, Oh TK, Kim YO, Choi KY, Oh BH (2000) Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states. Nat Struct Biol 7(2):147–153

    Article  CAS  PubMed  Google Scholar 

  • Haefner S, Knietsch A, Scholten E, Braun J, Lohscheidt M, Zelder O (2005) Biotechnological production and applications of phytases. Appl Microbiol Biotechnol 68(5):588–597

    Article  CAS  PubMed  Google Scholar 

  • Harland BF, Morris ER (1983) Phytate: a good or a bad food component. Nutr Res Rev 15:733–754

    Article  Google Scholar 

  • Hogue CW, MacManus JP, Banville D, Szabo AG (1992) Comparison of terbium (III) luminescence enhancement in mutants of EF hand calcium binding proteins. J Biol Chem 267(19):13340–13347

    CAS  PubMed  Google Scholar 

  • Holman WI (1943) A new technique for the determination of phosphorus by the molybdenum blue method. Biochem J 37(2):256–259

    CAS  PubMed Central  PubMed  Google Scholar 

  • Howson SJ, Davis RP (1983) Production of phytate-hydrolysing enzyme by some fungi enzyme. Microbiol Technol 5:377–382

    Article  CAS  Google Scholar 

  • Hu J, Jia X, Li Q, Yang X, Wang K (2004) Binding of La3+ to calmodulin and its effects on the interaction between calmodulin and calmodulin binding peptide, polistes mastoparan. Biochemistry 43(10):2688–2698

    Article  CAS  PubMed  Google Scholar 

  • Kellenberger E, Rodrigo J, Muller P, Rognan D (2004) Comparative evaluation of eight docking tools for docking and virtual screening accuracy. Proteins 57(2):225–242

    Article  CAS  PubMed  Google Scholar 

  • Kemme PA, Radcliffe JS, Jongbloed AW, Mroz Z (1997) The effects of sow parity on digestibility of proximate components and minerals during lactation as influenced by diet and microbial phytase supplementation. J Anim Sci 75(8):2147–2153

    CAS  PubMed  Google Scholar 

  • Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N, Apajalahti J (1998) Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl Environ Microbiol 64(6):2079–2085

    CAS  PubMed Central  PubMed  Google Scholar 

  • Kerovuo J, Lappalainen I, Reinikainen T (2000) The metal dependence of Bacillus subtilis phytase. Biochem Biophys Res Commun 268(2):365–369

    Article  CAS  PubMed  Google Scholar 

  • Kies AK, De Jonge LH, Kemme PA, Jongbloed AW (2006) Interaction between protein, phytate, and microbial phytase. In vitro studies. J Agric Food Chem 54(5):1753–1758

    Article  CAS  PubMed  Google Scholar 

  • 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(1):185–191

    Article  CAS  PubMed  Google Scholar 

  • Lei XG, Stahl CH (2001) Biotechnological development of effective phytases for mineral nutrition and environmental protection. Appl Microbiol Biotechnol 57(4):474–481

    Article  CAS  PubMed  Google Scholar 

  • Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666

    Article  CAS  Google Scholar 

  • Liu J, Bollinger DW, Ledoux DR, Ellersieck MR, Veum TL (1997) Soaking increases the efficacy of supplemental microbial phytase in a low-phosphorus corn-soybean meal diet for growing pigs. J Anim Sci 75(5):1292–1298

    CAS  PubMed  Google Scholar 

  • Makarewicz O, Dubrac S, Msadek T, Borriss R (2006) Dual role of the PhoP approximately P response regulator: Bacillus amyloliquefaciens FZB45 phytase gene transcription is directed by positive and negative interactions with the phyC promoter. J Bacteriol 188(19):6953–6965

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Oh BC, Chang BS, Park KH, Ha NC, Kim HK, Oh BH, Oh TK (2001) Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11. Biochemistry 40(32):9669–9676

    Article  CAS  PubMed  Google Scholar 

  • Oh BC, Choi WC, Park S, Kim YO, Oh TK (2004) Biochemical properties and substrate specificities of alkaline and histidine acid phytases. Appl Microbiol Biotechnol 63(4):362–372

    Article  CAS  PubMed  Google Scholar 

  • Rao DE, 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(4):1128–1137

    Article  CAS  PubMed  Google Scholar 

  • Reddy NR, Sathe SK, Salunkhe DK (1982) Phytates in legumes and cereals. Adv Food Res 28:1–92

    Article  CAS  PubMed  Google Scholar 

  • Shim JH, Oh BC (2012) Characterization and application of calcium-dependent beta-propeller phytase from Bacillus amyloliquefaciens DS11. J Agric Food Chem 60:7532–7537

    Article  CAS  PubMed  Google Scholar 

  • Shin S, Ha NC, Oh BC, Oh TK, Oh BH (2001) Enzyme mechanism and catalytic property of beta propeller phytase. Structure 9(9):851–858

    Article  CAS  PubMed  Google Scholar 

  • Sudhindra NM, Minaz AG, Indira DM, Ram SS (2004) Biological and clinical aspects of lanthanide coordination compounds. Bioinorg Chem Appl 2(3–4):155–192

    Google Scholar 

  • Tomschy A, Tessier M, Wyss M, Brugger R, Broger C, Schnoebelen L, van Loon AP, Pasamontes L (2000) Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure. Protein Sci Publ Protein Soc 9(7):1304–1311

    Article  CAS  Google Scholar 

  • Tran TT, Mamo G, Mattiasson B, Hatti-Kaul R (2010) A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli. J Ind Microbiol Biotechnol 37(3):279–287

    Article  CAS  PubMed  Google Scholar 

  • Tran TT, Hashim SO, Gaber Y, Mamo G, Mattiasson B, Hatti-Kaul R (2011) Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability. J Inorg Biochem 105(7):1000–1007

    Article  CAS  PubMed  Google Scholar 

  • Wang ZJ, Ren LX, Zhao YQ, Li GT, Liang AH, Yang BS (2007) Metal ions-induced conformational change of P23 by using TNS as fluorescence probe. Spectrochim Acta A Mol Biomol Spectrosc 66(4–5):1323–1326

    Article  PubMed  Google Scholar 

  • Wang ZJ, Ren LX, Zhao YQ, Li GT, Duan L, Liang AH, Yang BS (2008) Investigation on the binding of TNS to centrin, an EF-hand protein. Spectrochim Acta A Mol Biomol Spectrosc 70(4):892–897

    Article  PubMed  Google Scholar 

  • Wyss M, Pasamontes L, Remy R, Kohler J, Kusznir E, Gadient M, Muller F, van Loon A (1998) Comparison of the thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase, and A. niger PH 2.5 acid phosphatase. Appl Environ Microbiol 64(11):4446–4451

    CAS  PubMed Central  PubMed  Google Scholar 

  • Zeng YF, Ko TP, Lai HL, Cheng YS, Wu TH, Ma Y, Chen CC, Yang CS, Cheng KJ, Huang CH, Guo RT, Liu JR (2011) Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate. J Mol Biol 409(2):214–224

    Article  CAS  PubMed  Google Scholar 

  • Zhang R, Yang P, Huang H, Yuan T, Shi P, Meng K, Yao B (2011) Molecular and biochemical characterization of a new alkaline beta-propeller phytase from the insect symbiotic bacterium Janthinobacterium sp. TN115. Appl Microbiol Biotechnol 92(2):317–325

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the “National Natural Science Foundation of China” (No. 31071924, 31272100), the “Natural Science Foundation of Shanxi Province” (2010011040–1), and the “Shanxi Scholarship Council of China”. We are grateful to Prof. Daxiong Han from Medical College Xiamen University for software support.

Author information

Authors and Affiliations

  1. Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, 92 Wucheng Rd, Taiyuan, 030006, People’s Republic of China

    Ming-Ze Yao, Wen-Liang Lu, Wei Wang, Yue-Jun Fu & Ai-Hua Liang

  2. Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, 92 Wucheng Rd, Taiyuan, 030006, People’s Republic of China

    Ting-Gui Chen & Bin-Sheng Yang

Authors
  1. Ming-Ze Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wen-Liang Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ting-Gui Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yue-Jun Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bin-Sheng Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ai-Hua Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ai-Hua Liang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yao, MZ., Lu, WL., Chen, TG. et al. Effect of metals ions on thermostable alkaline phytase from Bacillus subtilis YCJS isolated from soybean rhizosphere soil. Ann Microbiol 64, 1123–1131 (2014). https://doi.org/10.1007/s13213-013-0751-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13213-013-0751-5

Keywords

Search

Navigation