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Applied Microbiology and Biotechnology

, Volume 85, Issue 3, pp 553–561 | Cite as

Delayed supplementation of glycine enhances extracellular secretion of the recombinant α-cyclodextrin glycosyltransferase in Escherichia coli

  • Zhaofeng Li
  • Zhengbiao Gu
  • Miao Wang
  • Guocheng Du
  • Jing WuEmail author
  • Jian ChenEmail author
Biotechnological Products and Process Engineering

Abstract

The targeting of recombinant proteins for secretion to the culture medium of Escherichia coli presents significant advantages over cytoplasmic or periplasmic expression. However, a major barrier is inadequate secretion across two cell membranes. In the present study, we attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine could promote extracellular secretion of the recombinant α-CGTase for which one potential mechanism might be the increase in membrane permeability. However, further analysis indicated that glycine supplementation resulted in impaired cell growth, which adversely affected overall recombinant protein production. Significantly, delayed supplementation of glycine could control cell growth impairment exerted by glycine. As a result, if the supplementation of 1% glycine was optimally carried out at the middle of the exponential growth phase, the α-CGTase activity in the culture medium reached 28.5 U/ml at 44 h of culture, which was 11-fold higher than that of the culture in regular terrific broth medium and 1.2-fold higher than that of the culture supplemented with 1% glycine at the beginning of culture.

Keywords

Cyclodextrin glycosyltransferase Paenibacillus macerans Extracellular secretion Glycine Escherichia coli Delayed supplementation 

Notes

Acknowledgments

This work was supported financially by the Natural Science Foundation of Jiangsu Province (BK2007019), the National Outstanding Youth Foundation of China (20625619), the National High-tech Research and Development Program of China (863 Program; 2006AA10Z335), Research Program of State Key Laboratory of Food Science and Technology (SKLF-MB-200802), Program of Innovation Team of Jiangnan University (2008CXTD01), and the Graduate Student Creative Research Program of Jiangsu Province in 2008 (CX08B_127Z).

References

  1. Aristidou AA, Yu P, San KY (1993) Effects of glycine supplement on protein production and release in recombinant Escherichia coli. Biotechnol Lett 15:331–336CrossRefGoogle Scholar
  2. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421CrossRefGoogle Scholar
  3. Blight MA, Chervaux C, Holland IB (1994) Protein secretion pathway in Escherichia coli. Curr Opin Biotechnol 5:468–474CrossRefGoogle Scholar
  4. Chang HY, Irwin PM, Nikolov ZL (1998) Effects of mutations in the starch-binding domain of Bacillus macerans cyclodextrin glycosyltransferase. J Biotechnol 65:191–202CrossRefGoogle Scholar
  5. Chen J, Wu J, Li ZF, Li B, Cheng CC (2008) Cloning and expression of the gene encoding α-cyclodextrin glycosyltransferase. CN Patent 200810024162.3Google Scholar
  6. Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64:625–635CrossRefGoogle Scholar
  7. Eriksson M, Nielsen PE, Good L (2002) Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli. J Biol Chem 277:7144–7147CrossRefGoogle Scholar
  8. Hammes W, Schleifer KH, Kandler O (1973) Mode of action of glycine on the biosynthesis of peptidoglycan. J Bacteriol 116:1029–1053Google Scholar
  9. Jana S, Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67:289–298CrossRefGoogle Scholar
  10. Jang KH, Seo JW, Song KB, Kim CH, Rhee SK (1999) Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli. Bioprocess Biosyst Eng 21:453–458Google Scholar
  11. Jeang CL, Wung CH, Chang BY, Yeh SS, Lour DW (1999) Characterization of the Bacillus macerans cyclodextrin glucanotransferase overexpressed in Escherichia coli. Proc Natl Sci Counc Repub China B 23:62–68Google Scholar
  12. Jeang CL, Lin DG, Hsieh SH (2005) Characterization of cyclodextrin glycosyltransferase of the same gene expressed from Bacillus macerans, Bacillus subtilis, and Escherichia coli. J Agric Food Chem 53:6301–6304CrossRefGoogle Scholar
  13. Kaderbhai N, Karim A, Hankey W, Jenkins G, Venning J, Kaderbhai MA (1997) Glycine-induced extracellular secretion of a recombinant cytochrome expressed in Escherichia coli. Biotechnol Appl Biochem 25(Pt1):53–61Google Scholar
  14. Kato C, Kudo T, Watanabe K, Horikoshi K (1983) Extracellular production of Bacillus penicillinase by Escherichia coli carrying pEAP2. Appl Microbiol Biotechnol 18:339–343CrossRefGoogle Scholar
  15. Kim CI, Kim MD, Park YC, Han NS, Seo JH (2000) Refolding of Bacillus macerans cyclodextrin glucanotransferase expressed as inclusion bodies in recombinant Escherichia coli. J Microbiol Biotechnol 10:632–637Google Scholar
  16. Kim SG, Kweon DH, Lee DH, Park YC, Seo JH (2005) Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli. Protein Expr Purif 41:426–432CrossRefGoogle Scholar
  17. Koebnik R, Locher KP, Van Gelder P (2000) Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol Microbiol 37:239–253CrossRefGoogle Scholar
  18. Lehrer R, Barton A, Ganz T (1988) Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry. J Immunol Methods 108:153–158CrossRefGoogle Scholar
  19. Lejeune A, Sakaguchi K, Imanaka T (1989) A spectrophotometric assay for the cyclization activity of cyclomaltohexaose (α-cyclodextrin) glucanotransferase. Anal Biochem 181:6–11CrossRefGoogle Scholar
  20. Li ZF, Wang M, Wang F, Gu ZB, Du GC, Wu J, Chen J (2007) γ-Cyclodextrin: a review on enzymatic production and applications. Appl Microbiol Biotechnol 77:245–255CrossRefGoogle Scholar
  21. Li ZF, Zhang JY, Wang M, Gu ZB, Du GC, Li JK, Wu J, Chen J (2009) Mutations at subsite −3 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhancing alpha-cyclodextrin specificity. Appl Microbiol Biotechnol 83:483–490CrossRefGoogle Scholar
  22. Loh B, Grant C, Hancock REW (1984) Use of the fluorescent probe 1-n-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa. Antimicrob Agents Chemother 26:546–551Google Scholar
  23. Lucas-Abellan C, Gabaldon-Hernandez JA, Penalva J, Fortea MI, Nunez-Delicado E (2008) Preparation and characterization of the inclusion complex of chlorpyrifos in cyclodextrins to improve insecticide formulations. J Agric Food Chem 56:8081–8085CrossRefGoogle Scholar
  24. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538Google Scholar
  25. Manting EH, Driessen AJ (2000) Escherichia coli translocase: the unravelling of a molecular machine. Mol Microbiol 37:226–238CrossRefGoogle Scholar
  26. Mergulhao FJ, Summers DK, Monteiro GA (2005) Recombinant protein secretion in Escherichia coli. Biotechnol Adv 23:177–202CrossRefGoogle Scholar
  27. Mourtzinos I, Salta F, Yannakopoulou K, Chiou A, Karathanos VT (2007) Encapsulation of olive leaf extract in β-cyclodextrin. J Agric Food Chem 55:8088–8094CrossRefGoogle Scholar
  28. Rinas U, Hoffmann F (2004) Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli. Biotechnol Prog 20:679–687CrossRefGoogle Scholar
  29. Saenger W (1980) Cyclodextrin inclusion compounds in research and industry. Angew Chem 19:344–362CrossRefGoogle Scholar
  30. Sanwal BD (1970) Regulatory mechanisms involving nicotinamide adenine nucleotides as allosteric effectors III. Control of glucose 6-phosphate dehydrogenase. J Biol Chem 245:1626–1631Google Scholar
  31. Shin HD, Chen RR (2008) Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant. Biotechnol Bioeng 101:1288–1296CrossRefGoogle Scholar
  32. Shokri A, Sanden AM, Larsson G (2003) Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli. Appl Microbiol Biotechnol 60:654–664Google Scholar
  33. Sorensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115:113–128CrossRefGoogle Scholar
  34. Sugamata Y, Shiba T (2005) Improved secretory production of recombinant proteins by random mutagenesis of hlyB, an α-hemolysin transporter from Escherichia coli. Appl Environ Microbiol 71:656–662CrossRefGoogle Scholar
  35. Szente L, Szejtli J (2004) Cyclodextrins as food ingredients. Trends Food Sci Technol 15:137–142CrossRefGoogle Scholar
  36. Tang JB, Yang HM, Song SL, Zhu P, Ji AG (2008) Effect of glycine and Triton X-100 on secretion and expression of ZZ-EGFP fusion protein. Food Chem 108:657–662CrossRefGoogle Scholar
  37. Yang J, Moyana T, MacKenzie S, Xia Q, Xiang J (1998) One hundred seventy-fold increase in excretion of an FV fragment-tumor necrosis factor alpha fusion protein (sFV/TNF-α) from Escherichia coli caused by the synergistic effects of glycine and Triton X-100. Appl Environ Microbiol 64:2869–2874Google Scholar
  38. Yu P, Aristidou AA, San KY (1991) Synergistic effect of glycine and bacteriocin release protein in the release of periplasmic protein in recombinant E. coli. Biotechnol Lett 13:311–316CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiPeople’s Republic of China
  2. 2.School of Food Science and TechnologyJiangnan UniversityWuxiPeople’s Republic of China
  3. 3.School of Biotechnology and the Key Laboratory of Industrial Biotechnology, Ministry of EducationJiangnan UniversityWuxiPeople’s Republic of China

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