Abstract
Poly-γ-glutamic acid (PGA) was easily phosphorylated by direct addition of phosphorylating agents into the culture medium of Bacillus subtilis (natto). Tetrapolyphosphate salt was the most incorporated into PGA molecules of all used reagents. Phosphorylation occurred at the α-carboxyl side chains of PGA molecule. The amounts of bound phosphate to PGA were dependent on the amounts of added phosphorylating agent. In low molecular weight regions of less than 100 kDa, a cross-linked peak was observed in the phosphorylated PGAs, whereas their peaks at approximately 1000 kDa shifted to a higher molecular weight due to the bound phosphate. The PGA derivatives had a wide range in viscosity up to 15/1000 to 15 times when compared to the native PGA, depending on the degree of phosphorylation (DP) in the PGA derivatives. The PGA with low DP had a high viscosity due to the unfolding conformation whereas highly phosphorylated PGA had aggregation with low viscosity. Heat treatment at 80 °C after the addition of phosphate salt elicited a novel collagen-like helix structure. These observations show that phosphorylation is an effective way to diversify the physicochemical properties of PGA.
Graphical Abstract
Similar content being viewed by others
References
Ahmed GAR, Khorshid FAR, Kumosani TA (2009) FT-IR spectroscopy as a tool for identification of apoptosis-induced structural changes in A549 cells treated PM701. Int J Nano Biomater 2:396–408
Akagi T, Matsusaki M, Akashi M (2010) Pharmaceutical and medical applications of poly-gamma-glutamic acid. In: Hamano Y (ed) Amino-acid homopolymers occuring in nature, microbiology monographs 15. Springer-Verlag, Berlin, pp 119–153
Ashiuchi M, Nawa C, Kamei T, Song JJ, Hong SP, Sung MH, Soda K, Yagi T, Misano H (2001) Physiological and biochemical characteristics of poly γ-glutamate synthetase complex of Bacillus subtilis. Eur J Biochem 268:5321–5328
Blanch H (2008) The kinetics of aggregation of poly-glutamic acid based polypeptides. Biophys Chem 136:74–86
Cardamone M, Puri NK (1992) Spectrofluorimetric assessment of the surface hydrophobicity of proteins. Biochem J 282:589–593
Cejas MA, Kinney WA, Chen C, Vinter JG, Almond JHR, Balss KM, Maryanoff CA, Schmidt U, Breslav M, Mahan A, Lacy E, Maryanoff BE (2008) Thrombogenic collagen-mimetic peptides: self-assembly of triple helix-based fibrils driven by hydrophobic interactions. Proc Natl Acad Sci USA 105:8513–8518
Filip Z, Herrmann S, Kubat J (2004) FT-IR spectroscopic characteristics of differently cultivated Bacillus subtilis. Microbiol Res 159:257–262
Goto A, Kunioka M (1992) Biosynthesis and hydrolysis of poly (gamma-glutamic acid) from Bacillus subtilis IFO3335. Biosci Biotechnol Biochem 156:1031–1035
Greenfield NJ (2006) Using circular dichronism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc 1:2527–2535
Grimshaw CE, Huang S, Hanstein CG, Strauch MA, Burbulys D, Wang L, Hoch JA, Whiteley JM (1998) Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis. Biochemistry 37:1365–1375
Holmgren SK, Taylor KM, Bretscher LE, Raines RT (1998) Code for collagen’s stability deciphered. Nature 392:666–667
Hsieh CY, Tsai SP, Wang DM, Chang YN, Hsieh HJ (2005) Preparation of γ-PGA/chitosan composite tissue engineering matrices. Biomaterials 26:5617–5623
Johnson LN, Barford D (1993) The effects of phosphorylation on the structure and function of proteins. Annu Rev Biophys Biomol Struct 22:199–232
Joseph MH, Davies P (1983) Electrochemical activity of o-phthalaldehyde -mercaptoethanol derivatives of amino acids: application to high-performance liquid chromatographic determination of amino acids in plasma and other biological materials. J Chromatogr B Biomed Sci Appl 277:125–136
Kajiyama T, Kuroishi M, Takayanagi M (1975) The mechanisms of the viscoelastic crystalline absorption of polyglutamic acid ester. J Macromol Sci 11:121–150
Kambourova M, Tangney M, Priest FG (2001) Regulation of polyglutamic acid synthesis by glutamate in Bacillus licheniformis and Bacillus subtilis. Appl Environ Microbiol 67:1004–1007
Katewa SD, Katyare SS (2003) A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays. Anal Biochem 323:180–187
Kimura K, Tranan LSP, Uchida I, Itoh Y (2004) Characterization of Bacillus subtilis γ-glutamyltransferase and its involvement in the degradation of capsule poly-γ-glutamate. Microbiology 150:4115–4123
Kongklom N, Chuensangjun C, Pechyen C, Sirisansaneeyakul S (2012) Production of poly-γ-glutamic acid by Bacillus licheniformis: synthesis and characterization. J Metal Mater Miner 22:7–11
Krebs MRH, Domike KR, Donald AM (2009) Protein aggregation: more than just fibrils. Biochem Soc Trans 37:682–686
Kurita O, Sago T, Umetani K, Kokean Y, Yamaoka C, Takahashi N, Iwamoto H (2017) Feasible protein aggregation of phosphorylated poly-γ-glutamic acid derivative from Bacillus subtilis (natto). Int J Biol Macromol 103:484–492
Matsusaki M, Serizawa T, Kishida A, Endo T, Akashi M (2002) Novel functional biodegradable polymer: syntheesis and anticoagulant activity of poly (γ-glutamic acid) sulfonate (γ-PGA-sulfonate). Bioconjug Chem 13:23–28
Meuzelaar H, Tros M, Viga AH, van Dijk CN, Vreede J, Woutersen S (2014) Solvent-exposed salt bridges influence the kinetics of α-helix folding and unfolding. J Phys Chem Lett 5:900–904
Moraes LP, Brito PN, Alergre RM (2013) The exsisting studies on biosynthesis of poly (γ-glutamic acid) by fermentation. Food Pub Health 3:28–36
Nishi Y, Uchiyama S, Doi M, Nishiuchi Y, Nakazawa T, Ohkubo T, Kobayashi Y (2005) Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix. Biochemistry 44:6034–6042
Ogunleye A, Bhat A, Irorere VU, Hill D, Williams C, Radecka I (2015) Poly-γ-glutamic acid: production, properties and applications. Microbiology 161:1–17
Semisotnov GV, Rodionova NA, Razgulyaev OI, Uversky VN, Gripaś AF, Gilmanshin RI (1991) Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31:119–128
Shih IL, Van YT (2001) The production of poly-(γ-glutamic acid) from microorganisms and its various applications. Biores Technol 79:207–225
Suzuki T, Tahara Y (2003) Characterization of the Bacillus subtilis ywtD gene, whose product is involved in γ-polyglutamic acid degradation. J Bacteriol 185:2379–2382
Takano K, Tsuchimori K, Yamagata Y, Yutani K (2000) Contribution of salt bridges near the surface of a protein to the conformational stability. Biochemistry 39:12375–12381
Tiffany ML, Krimm S (1968) New chain conformation of poly (glutamic acid) and poly lysine. Biopolymers 6:1379–1382
Timpl R, Martin GR, Bruckner P, Wick G, Wiedemann H (1978) Nature of the collagenous protein in a tumor basement membrane. Eur J Biochem 84:43–52
Wei X, Tian G, Ji Z, Chen S (2015) A new strategy for enhancement of poly-γ-glutamic acid production by multiple physicochemical stresses in Bacillus licheniformis. J Chem Technol Biotechnol 90:709–713
Zhu Y, Huang W, Lee SSK, Xu W (2005) Crystal structure of a polyphosphate kinase and its implications for polyphosphate synthesis. EMBO Rep 6:681–687
Acknowledgements
We are thankful to Shaun O’Brien for comments on the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kurita, O., Umetani, K., Kokean, Y. et al. Regulatory phosphorylation of poly-γ-glutamic acid with phosphate salts in the culture of Bacillus subtilis (natto). World J Microbiol Biotechnol 34, 60 (2018). https://doi.org/10.1007/s11274-018-2443-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-018-2443-6