Overproduction of a γ-glutamyltranspeptidase from Bacillus amyloliquefaciens in Bacillus subtilis through medium optimization

배지최적화를 통한 재조합 바실러스 서브틸리스에서 바실러스 아밀로리퀴파시엔스 유래 γ-글루타밀펩타이드전달효소의 대량생산

  • Received : 2017.07.31
  • Accepted : 2017.08.14
  • Published : 2017.12.31


${\gamma}$-Glutamyltranspeptidase (GGT, EC transfers ${\gamma}$-glutamyl moiety from glutamine to amino acids or peptides and hydrolyzes glutamine to glutamate and ammonia. In order to overproduce ${\gamma}$-glutamyltranspeptidase from Bacillus amyloliquefaciens (BAGGT), the encoding gene was cloned and expressed in Bacillus subtilis. The productivity of BAGGT in Bacillus subtilis was improved by 42-fold by using a dual-promoter system that was generated by combining promoters from B. subtilis ${\alpha}$-amylase and BAGGT genes. Through optimization of medium composition by Plackett-Burman design and central composition design, BAGGT was produced at $18.3{\times}10^7U/L$ of culture in the optimized medium. Compared to previously used Luria-Bertani medium, the optimized culture medium (15 g/L molasses, 60 g/L corn steep liquor, 6 g/L yeast extract, 4 g/L NaCl, 6 g/L $K_2HPO_4$, and 2 g/L $KH_2PO_4$), resulted in a 4.3-fold increase in production of BAGGT.


Bacillus subtilis;dual-promoter system;${\gamma}$-glutamyltranspeptidase;medium optimization


Supported by : (주)농심 중앙연구소, 농촌진흥청


  1. Hillmann H, Behr J, Ehrmann MA, Vogel RF, Hofmann T. Formation of kokumi-enhancing ${\gamma}$-glutamyl dipeptides in parmesan cheese by means of ${\gamma}$-glutamyltransferase activity and stable isotope double-labeling studies. J. Agr. Food Chem. 64: 1784-1793 (2016)
  2. Zhao CJ, Gnzle MG. Synthesis of taste-active ${\gamma}$-glutamyl dipeptides during sourdough fermentation by Lactobacillus reuteri. J. Agr. Food Chem. 64: 7561-7568 (2016)
  3. Wang Q, Min C, Zhu F, Xin Y, Zhang S, Luo L, Yin Z. Production of bioactive ${\gamma}$-glutamyl transpeptidase in Escherichia coli using SUMO fusion partner and application of the recombinant enzyme to L-theanine synthesis. Curr. Microbiol. 62: 1535-1541 (2011)
  4. Mu W, Zhang T, Jiang B. An overview of biological production of L-theanine. Biotechnol. Adv. 33: 335-342 (2015)
  5. Shuai Y, Zhang T, Mu W, Jiang B. Purification and characterization of ${\gamma}$-glutamyltranspeptidase from Bacillus subtilis SK11. 004. J. Agr. Food Chem. 59: 6233-6238 (2011)
  6. Vermeulen N, Gnzle MG, Vogel RF. Glutamine deamidation by cerealassociated lactic acid bacteria. J. Appl. Microbiol. 103: 1197-1205 (2007)
  7. Pariza MW, Johnson EA. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul. Toxicol. Pharmacol. 33: 173-186 (2001)
  8. Schumann W. Production of recombinant proteins in Bacillus subtilis. Adv. Appl. Microbiol. 62: 137-190 (2007)
  9. Nguyen HD, Nguyen QA, Ferreira RC, Ferreira L, Tran LT, Schumann W. Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid 54: 241-248 (2005)
  10. Chen PT, Chiang CJ, Chao YP. Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis. Biotechnol. Prog. 23: 808-813 (2007)
  11. Inoue M. Glutathionists in the battlefield of gamma-glutamyl cycle. Arch. Biochem. Biophys. 595: 61-63 (2016)
  12. Suzuki H, Kumagai H. Autocatalytic processing of ${\gamma}$-glutamyltranspeptidase. J. Biol. Chem. 277: 43536-43543 (2002)
  13. Castellano I, Merlino A, Rossi M, La Cara F. Biochemical and structural properties of ${\gamma}$-glutamyl transpeptidase from Geobacillus thermodenitrificans: An enzyme specialized in hydrolase activity. Biochimie 92: 464-474 (2010)
  14. Okada T, Suzuki H, Wada K, Kumagai H, Fukuyama K. Crystal structure of the ${\gamma}$-glutamyltranspeptidase precursor protein from Escherichia coli. Structural changes upon autocatalytic processing and implications for the maturation mechanism. J. Biol. Chem. 282: 2433-2439 (2007)
  15. Minami H, Suzuki H, Kumagai H. Salt-tolerant ${\gamma}$-glutamyltranspeptidase from Bacillus subtilis 168 with glutaminase activity. Enzyme Microb. Technol. 32: 431-438 (2003)
  16. Kang HK, Jang JH, Shim JH, Park JT, Kim YW, Park KH. Efficient constitutive expression of thermostable 4-${\alpha}$-glucanotransferase in Bacillus subtilis using dual promoters. World J. Microbiol. Biotechnol. 26: 1915-1918 (2010)
  17. Choi CH, Kim SH, Jang JH, Park JT, Shim JH, Kim YW, Park KH. Enzymatic synthesis of glycosylated puerarin using maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis. J. Sci. Food Agric. 90: 1179-1184 (2010)
  18. Zhang K, Su L, Duan X, Liu L, Wu J. High-level extracellular protein production in Bacillus subtilis using an optimized dualpromoter expression system. Microb. Cell Fact. 16: 32 (2017)
  19. Keskin Gundogdu T, Deniz I, Caliskan G, Sahin ES, Azbar N. Experimental design methods for bioengineering applications. Crit. Rev. Biotechnol. 36: 368-388 (2016)
  20. Plackett RL, Burman JP. The design of optimum multifactorial experiments. Biometrika 33: 305-325 (1946)
  21. Singh V, Haque S, Niwas R, Srivastava A, Pasupuleti M, Tripathi CK. Strategies for fermentation medium optimization: An in-depth review. Front Microbiol. 7: 2087 (2017)
  22. Ahn JH. Characterization of ${\gamma}$-glutamyltransferase in Bacillus amyloliquefaciens. MS thesis, Korea University, Seoul, Korea (2013)
  23. Kim MS, Jang JH, Kim YW. Overproduction of a thermostable 4-${\alpha}$-glucanotransferase by codon optimization at N-terminus region. J. Sci. Food Agric. 93: 2683-2690 (2013)
  24. Sadaie Y, Kada T. Formation of competent Bacillus subtilis cells. J. Bacteriol. 153: 813-821 (1983)
  25. Yamazaki H, Ohmura K, Nakayama A, Takeichi Y, Otozai K, Yamasaki M, Tamura G, Yamane K. ${\alpha}$-Amylase genes (amyR2 and amy$E^+$) from an ${\alpha}$-amylase-hyperproducing Bacillus subtilis strain: molecular cloning and nucleotide sequences. H. Bacteriol. 156: 327-337 (1983)
  26. Li L, Ma Y. Effects of metal ions on growth, ${\beta}$-oxidation system, and thioesterase activity of Lactococcus lactis. J. Dairy Sci. 97: 1-8 (2014)