Applied Microbiology and Biotechnology

, Volume 87, Issue 6, pp 2087–2096 | Cite as

Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis

  • Keiichi Yokoyama
  • Hiroe Utsumi
  • Takefumi Nakamura
  • Daisuke Ogaya
  • Nobuhisa Shimba
  • Eiichiro Suzuki
  • Seiichi Taguchi
Biotechnologically Relevant Enzymes and Proteins


Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. Two enzyme engineering approaches were applied to improve MTG activity. One is a novel method of rational mutagenesis, called water-accessible surface hot-space region-oriented mutagenesis (WASH-ROM). One hundred and fifty-one point mutations were selected at 40 residues, bearing high solvent-accessibility surface area, within a 15 Å space from the active site Cys64. Among them, 32 mutants showed higher specific activity than the wild type. The other is a random mutagenesis of the whole region of the MTG gene, coupled with a new plate assay screening system, using Corynebacterium Expression System CORYNEX®. This in vivo system allowed us to readily distinguish the change in enzymatic activity by monitoring the intensity of enzymatic reaction-derived color zones surrounding recombinant cells. From the library of 24,000 mutants, ten were finally selected as beneficial mutants exhibiting higher specific activity than the wild type. Furthermore, we found that Ser199Ala mutant with additional N-terminal tetrapeptide showed the highest specific activity (1.7 times higher than the wild type). These various beneficial positions leading to increased specific activity of MTG were identified to achieve further enzyme improvements.


Transglutaminase Streptomyces CORYNEX® Screening Solvent-accessibility Specific activity 



We thank Dr. Kashiwagi and Dr. Nio for helpful discussions. We also thank Dr. Onoe and Ms. Tagami for technical assistance. This work was done by in part of the finance from the Global Center of Excellence Program (Project No. B01: Catalysis as the Basis for Innovation in Materials Science) from the Ministry of Education, Culture, Sports, Science, and Technology-Japan. Pacific Edit reviewed the manuscript prior to submission.


  1. Ando H, Adachi M, Umeda K, Matsuura A, Nonaka M, Uchio R, Tanaka H, Motoki M (1989) Purification and characterization of a novel transglutaminase derived from microorganisms. Agric Biol Chem 53:2613–2617Google Scholar
  2. Beninati S, Bergamini CM, Piacentini M (2009) An overview of the first 50 years of transglutaminase research. Amino Acids 36:591–598CrossRefGoogle Scholar
  3. Date M, Yokoyama K, Umezawa Y, Matsui H, Kikuchi Y (2003) Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum. Appl Environ Microbiol 69:3011–3014CrossRefGoogle Scholar
  4. Eisenhaber F, Lijnzaad P, Argos P, Sander C, Scharf M (1995) The double cubic lattice method: efficient approaches to numerical integration of surface area and volume and to dot surface contouring of molecular assemblies. J Comp Chem 16:273–284CrossRefGoogle Scholar
  5. Fontana A, Spolaore B, Mero A, Veronese FM (2008) Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase. Adv Drug Deliv Rev 60:13–28CrossRefGoogle Scholar
  6. Ichinose A, Hendrickson LE, Fujikawa K, Davie EW (1986) Amino acid sequence of the subunit of human factor XIII. Biochemistry 25:6900–6906CrossRefGoogle Scholar
  7. Ikura K, Nasu T, Yokota H, Tsuchiya Y, Sasaki R, Chiba H (1988) Amino acid sequence of guinea pig liver transglutaminase from its cDNA sequence. Biochemistry 27:2898–2905CrossRefGoogle Scholar
  8. Kawajiri H, Ide H, Motoki M, Shimonishi Y (1993) Primary structure of microbial transglutaminase from Streptoverticillium sp. strain s-8112. J Biol Chem 268:11565–11572Google Scholar
  9. Kashiwagi T, Yokoyama K, Ishikawa K, Ono K, Ejima D, Matui H, Suzuki E (2002) Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J Biol Chem 277:44252–44260CrossRefGoogle Scholar
  10. Kikuchi Y, Date M, Yokoyama K, Umezawa Y, Matsui H (2003) Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-like protease from Streptomyces albogriseolus. Appl Environ Microbiol 69:358–366CrossRefGoogle Scholar
  11. Kuraishi C, Sakamoto J, Soeda T (1996) The usefulness of transglutaminase for food processing. In: Takeoka GR, Teranishi R, Williams PJ, Kobayashi A (eds) Biotechnology for improved foods and flavors, ACS Symposium Series 637. American Chemical Society, Washington, DC, pp 29–38CrossRefGoogle Scholar
  12. Liebl W, Bayerl A, Schein B, Stillner U, Schleifer KH (1989) High efficiency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol Lett 53:299–303CrossRefGoogle Scholar
  13. Marx CK, Hertel TC, Pietzsch M (2008) Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants. J Biotechnol 136:156–162CrossRefGoogle Scholar
  14. Motoki M, Seguro K (1998) Transglutaminase and its use for food processing. Trends Food Sci Technol 9:204–210CrossRefGoogle Scholar
  15. Nonaka M, Tanaka H, Okiyama A, Motoki M, Ando H, Umeda K, Matsuura A (1989) Polymerization of several proteins by calcium-independent transglutaminase derived from microorganisms. Agric Biol Chem 53:2619–2623Google Scholar
  16. Pasternack R, Dorsch S, Otterbach JT, Robenek IR, Wolf S, Fuchsbauer HL (1998) Bacterial pro-transglutaminase from Streptoverticillium mobaraense: purification, characterization and sequence of zymogen. Eur J Biochem 257:570–576CrossRefGoogle Scholar
  17. Sakamoto H, Nonaka M, Motoki M (1993) Calcium-independent transglutaminase derived from a microorganism: its characteristic and capability in protein crosslinking and gel formation. Food Hydrocoll 8:383–386Google Scholar
  18. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  19. Tagami U, Shimba N, Nakamura M, Yokoyama K, Suzuki E, Hirokawa T (2009) Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis. Protein Eng Des Sel 22:747–752CrossRefGoogle Scholar
  20. Taguchi S, Tsuge T (2008) Natural polyester-related proteins: structure, function, evolution and engineering. In Lutz S, Bornschuer UT (eds) Protein engineering handbook. Wiley, Weinheim, pp 877–914Google Scholar
  21. Taguchi S, Ozaki A, Momose H (1998) Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system. Appl Environ Microbiol 64:492–495Google Scholar
  22. Thacher SM (1989) Purification of keratinocyte transglutaminase and its expression during squamous differentiation. J Invest Dermatol 92:578–584Google Scholar
  23. Umezawa Y, Yokoyama K, Kikuchi Y, Date M, Ito K, Yoshimoto T, Matsui H (2004) Novel Prolyl Tri/Tetra-Peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning. J Biochem 136:293–300CrossRefGoogle Scholar
  24. Washizu K, Ando K, Koikeda S, Hirose S, Matsuura A, Takagi H, Motoki M, Takeuchi K (1994) Molecular cloning of the gene for microbail transglutaminase from streptoverticillium and its expression in Streptomyces lividans. Biosci Biotech Biochem 58:82–87CrossRefGoogle Scholar
  25. Yasueda H, Nakanishi K, Kumazawa Y, Nagase K, Motoki M, Matui H (1995) Tissue-type transglutaminase from red sea bream (Pagrus major) sequence analysis of the cDNA and functional expression in Escherichia coli. Eur J Biochem 232:411–419CrossRefGoogle Scholar
  26. Yokoyama K, Nakamura N, Saguaro K, Kubota K (2000) Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form. Biosci Biotechnol Biochem 64:1263–1270CrossRefGoogle Scholar
  27. Yokoyama K, Ono K, Ohtsuka T, Nakamura N, Seguro K, Ejima D (2002) In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence. Protein Expr Purif 26:329–335CrossRefGoogle Scholar
  28. Yokoyama K, Nio N, Kikuchi Y (2004) Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol 64:447–454CrossRefGoogle Scholar
  29. Zhu Y, Tramper J (2008) Novel applications for microbial transglutaminase beyond food processing. Trends Biotechnol 26:559–565CrossRefGoogle Scholar
  30. Zotzel J, Keller P, Fuchsbauer HL (2003a) Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease. Eur J Biochem 270:3214–3222CrossRefGoogle Scholar
  31. Zotzel J, Pasternack R, Pelzer C, Ziegert D, Mainusch M, Fuchsbauer HL (2003b) Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step. Eur J Biochem 270:4149–4155CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Keiichi Yokoyama
    • 1
  • Hiroe Utsumi
    • 2
  • Takefumi Nakamura
    • 1
  • Daisuke Ogaya
    • 2
  • Nobuhisa Shimba
    • 1
  • Eiichiro Suzuki
    • 1
  • Seiichi Taguchi
    • 2
  1. 1.Institute of Life Sciences, Ajinomoto Co., IncKawasaki-shiJapan
  2. 2.Division of Biotechnology and Macromolecular Chemistry, Graduate School of EngineeringHokkaido UniversitySapporoJapan

Personalised recommendations