Physicochemical Properties of Poly-γ-glutamic Acid Produced by a Novel Bacillus subtilis HA Isolated from Cheonggukjang

  • Seo, Ji-Hyun (The Center for Traditional Microorganism Resources (TMR), Keimyung University) ;
  • Kim, Chan-Shick (Faculty of Biotechnology, College of Applied Life Sciences, Cheju National University) ;
  • Lee, Sam-Pin (Department of Food Science and Technology, Keimyung University)
  • Published : 2008.12.31


A novel bacterium isolated from Cheonggukjang was identified as a glutamate-dependent Bacillus subtilis HA with 98.3% similarity to Bacillus subtilis Z99104. Optimization of poly-$\gamma$-glutamic acid ($\gamma$-PGA) production by modulating fermentation factors including carbon sources, nitrogen sources, inorganic salts and fermentation time was investigated. Optimum culture broth for $\gamma$-PGA production consisted of 3% glutamate, 3% glucose and various salts, resulting in the PGA production of 22.5 g/L by shaking culture for 72 hr at $37^{\circ}C$. Average molecular weight of $\gamma$-PGA was determined to be 1,220 kDa through MALLS analysis. The $\gamma$-PGA solution showed a typical pseudoplastic flow behavior, and a great decrease in consistency below pH 6.0 regardless of the same molecular weight of $\gamma$-PGA. The molecular weights of isolated $\gamma$-PGA were drastically decreased by heat treatment in various acidic conditions, resulting in different hydrolysis of $\gamma$-PGA. The consistency of $\gamma$-PGA solution was greatly decreased with increase heating time in acidic conditions.


  1. Shih IL, Van YT. 2001. The production of poly(${\gamma}$-glutamic acid) from microorganisms and its various applications. Bioresour Technol 79: 207-225
  2. Thorne CB, Gomez CG, Noyes HE, Husewright RD. 1954. Production of glutamyl polypeptide by Bacillus subtilis. J Bacteriol 68: 307-315
  3. Troy FA. 1973. Chemistry and biosynthesis of poly(${\gamma}$-glutamyl) capsule in Bacillus licheniformis: properties of the membrane-mediated biosynthetic reaction. J Biol Chem 248: 305-315
  4. Liang HF, Yand TF, Huang CT, Chen MC, Sung HW. 2005. Preparation of nanoparticles composed of poly(${\gamma}$-glutamic acid)-poly(lactide) block copolymers and evaluations of their uptake by HepG2 cells. J Control Release 105: 213-225
  5. Richard A, Margaritis A. 2001. Poly(glutamic acid) for biomedical applications. Crit Rev Biotechnol 21: 219-232
  6. Otani Y, Tabata Y, Ikada Y. 1996. Rapidly curable biological glue composed of gelatin and poly(l-glutamic acid). Biomaterials 17: 1387-1391
  7. Sekine T, Nakamura T, Shimizu Y, Ueda H, Matsumoto K, Takimoto Y, Kiyotani T. 2000. A new type of surgical adhesive made from porcine collagen and polyglutamic acid. J Biomed Mater Res 35: 305-310<305::AID-JBM18>3.0.CO;2-B
  8. Borbely M, Nagasaki Y, Borbely J, Fan K, Bhogle A, Sevoian M. 1994. Biosynthesis and chemical modification of poly(${\gamma}$-glutamic acid). Polym Bull 32: 127-132
  9. Park YJ, Liang J, Yang Z, Yang VC. 2001. Controlled release of clot-dissolving tissue-type plasmmogen activator from a poly(L-glutamic acid) semi-interpenetrating polymer network hydrogel. J Control Relese 74: 243-247
  10. Ashiuchi M, Kamei T, Baek DH, Shin SY, Sung MH, Soda K, Yagi T, Misono H. 2001. Isolation of Bacillus subtilis (chungkookjang), a poly-${\gamma}$-glutamate producer with high genetic mompetence. Appl Microbiol Biotechnol 57: 764-769
  11. Inatsu Y, Kimura K, Itoh Y. 2002. Characterization of Bacillus subtilis strains isolated form fermented soybean foods in Southeast Asia: Comparison with B. subtilis (natto) starter strains. Jpn Agric Res Q 36: 169-175
  12. Oh SM, Jang EK, Seo JH, Ryu MJ, Lee SP. 2007. Characterization of the ${\gamma}$-polyglutamic acid produced from the solid-state fermentation of soybean milk cake using Bacillus sp. Food Sci Biotechnol 16: 509-514
  13. Akagi T, Kaneko T, Kida T, Akashi M. 2005. Preparation and characterization of biodegradable nanoparticles based on poly(${\gamma}$-glutamic acid) with ${\iota}$-phenylalanine as a protein carrier. J Control Release 108: 226-236
  14. Ye H, Jin L, Hu R, Yi Z, Li J, Wu Y, Xi X, Wu Z. 2006. Poly(r,L-glutamic acid)-cisplatin conjugate effectively inhibits human breast tumor xenografted in nude mice. Biomaterials 27: 5958-5965
  15. Seo JH, Lee SP. 2004. Optimization of the production of fibrinolytic enzyme from Bacillus firmus NA-1 in fermented soybeans. J Food Sci Nutr 9: 14-20
  16. Goto A, Kunioka M. 1992. Biosynthesis and hydrolysis of poly(${\gamma}$-glutamic acid) from Bacillus subtilis IFO3335. Biosci Biotechnol Biochem 56: 1031-1035
  17. Thompson JD, Higgins DG, Gibson TJ, Clustal W. 1994. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalies and weight matrix choice. Nucleic Acids Res 22: 4673-4680
  18. Choi JI, Lee SY. 2004. High level production of supra molecular weight poly(3-hydroxybutyrate) by metabolically engineered Escherichia coli. Biotechnol Bioprocess Eng 9: 196-200
  19. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254
  20. McNeil B, Harvey LM. 1993. Viscous fermentation products. Crit Rev Biotechnol 13: 275-304
  21. You S, Fiedorwicz M, Lim ST. 1999. Molecular characterization of wheat amylopectins by multiangle laser light scattering analysis. Cereal Chem 96: 116-121
  22. Hong X, Min J, Hui L, Dingqiang L, Pingkai O. 2005. Efficient production of poly(γ-glutamic acid) by newly isolated Bacillus subtilis NX-2. Proc Biochem 40: 519-523
  23. Kelessidis VC, Maglione R. 2006. Modeling rhelogical behavior of bentonite suspensions as Casson and Robertson-Stiff fluids using Newtonian and true shear rates in Couette viscometry. Powder Technol 168: 134-147
  24. Ito Y, Tanaka T, Ohmachi T, Asada Y. 1996. Glutamic acid independent production of poly(${\gamma}$-glutamic acid) by Bacillus subtilis TAM-4. Biosci Biotechnol Biochem 60: 1239-1242
  25. Cromwick AM, Gross RA. 1995. Effect of manganese (II) on Bacillus lichniformis ATCC9945A physiology and ${\gamma}$-poly(glutamic acid) formation. Int J Biol Macromol 16: 265-275
  26. Leonard CG, Housewright RD, Throne CB. 1958. Effect of some metallic ions on glutamyl polypeptide synthesis by Bacillus subtilis. J Bacteriol 76: 499-503
  27. Suzuki T, Tahara Y. 2003. Characterization of the Bacillus subtilis ywtD gene, whose product is involved in ${\gamma}$-polyglutamic acid degradation. J Bacteriol 185: 2379-2382
  28. Kubota H, Matsunobu T, Uotani K, Takebe H, Satoh A, Tanaka T, Taniguchi M. 1993. Production of poly(${\gamma}$-glutamic acid) by Bacillus subtilis F-2-01. Biosci Biotechnol Biochem 57: 1212-1213
  29. Kunioka M, Goto A. 1994. Biosynthesis of poly(${\gamma}$-glutamic acid) from l-glutamic acid, citric acid, and ammonium sulfate in Bacillus subtilis IFO 3335. Appl Microbiol Biotechnol 40: 867-872
  30. Kunioka M, Furusawa K. 1997. Poly(${\gamma}$-glutamic acid) hydrogel prepared form microbial poly(${\gamma}$-glutamic acid) and alkane diamine with water-soluble carbodiimide. J Appl Polym Sci 65: 1889-1893<1889::AID-APP5>3.0.CO;2-B
  31. Scolnik Y, Portnaya I, Cogan U, Tal S, Haimovitz R, Fridkin M, Elitzur AC, Deamer DW, Shinitzky M. 2006. Subtle differences in structural transitions between poly-land poly-d-amino acids of equal length in water. Phys Chem Chem Phys 8: 333-339
  32. Zanuy D, Aleman C, Munoz-Guerra S. 1998. On the helical conformation of un-ionized poly(${\gamma}$-D-glutamic acid). Int J Biol Macromol 23: 175-184

Cited by

  1. An injectable collagen/poly(γ-glutamic acid) hydrogel as a scaffold of stem cells and α-lipoic acid for enhanced protection against renal dysfunction vol.5, pp.2, 2017,
  2. Production of Carrot Pomace Fortified with Mucilage, Fibrinolytic Enzyme and Probiotics by Solid-state Fermentation Using the Mixed Culture of Bacillus subtilis and Leuconostoc mesenteroides vol.14, pp.4, 2009,
  3. Optimized Production of GABA and γ-PGA in a Turmeric and Roasted Soybean Mixture Co-fermented by Bacillus subtilis and Lactobacillus plantarum vol.22, pp.2, 2016,
  4. Novel bioconversion of sodium glutamate to γ-poly-glutamic acid and γ-amino butyric acid in a mixed fermentation using Bacillus subtilis HA and Lactobacillus plantarum K154 vol.23, pp.5, 2014,
  5. Fortification of Bioactive Compounds in Roasted Wheat Bran by Solid-State Fermentation Using Bacillus subtilis HA vol.23, pp.3, 2017,
  6. Evaluation of Radical Scavenging Activity and Physical Properties of Textured Vegetable Protein Fermented by Solid Culture with Bacillus subtilis HA According to Fermentation Time vol.39, pp.6, 2010,
  7. Physicochemical and functional properties of roasted soybean flour, barley, and carrot juice mixture fermented by solid-state fermentation using Bacillus subtilis HA vol.20, pp.6, 2011,
  8. Influences of Culture Medium Components on the Production Poly (γ-Glutamic Acid) by Bacillus subtilis GS-2 Isolated Chungkookjang vol.25, pp.3, 2012,
  9. Anti-obesity Effects of Black Bean Chungkugjang Extract in 3T3-L1 Adipocytes and Obese Mice Induced by High Fat Diet vol.40, pp.9, 2011,
  10. Fortification of Mucilage and GABA in Hovenia dulcis Extract by Co-fermentation with Bacillus subtilis HA and Lactobacillus plantarum EJ2014 vol.24, pp.2, 2018,