Skip to main content
SpringerLink
Log in

Biophysical Characterization of a Recombinant α-Amylase from Thermophilic Bacillus sp. strain TS-23

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Environmental variables can significantly influence the folding and stability of a protein molecule. In the present study, the biophysical properties of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC) were characterized in detail by glutaraldehyde cross-linking, analytical ultracentrifugation, and various spectroscopic techniques. With cross-linking experiment and analytical ultracentrifuge, we demonstrated that the oligomeric state of BACΔNC in solution is monomeric. Far-UV circular dichroism analysis revealed that the secondary structures of BACΔNC were significantly altered in the presence of various metal ions and SDS, whereas acetone and ethanol had no detrimental effect on folding of the enzyme. BACΔNC was inactive and unstable at extreme pH conditions. Thermal unfolding of the enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.2 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]0.5, N–U, at 1.14 M. BACΔNC was active at the concentrations of urea below 6 M, but it experienced an irreversible unfolding by >8 M denaturant. Taken together, this work lays a foundation for the future structural studies with Bacillus sp. TS-23 α-amylase, a typical member of glycoside hydrolases family 13.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

GH:

Glycoside hydrolase

SBD:

Starch-binding domain

BACΔNC:

Bacillus sp. TS-23 α-amylase without signal sequence and SBD

LB:

Luria-Bertani

Ni2+-NTA:

Ni2+-nitrilotriacetate

IPTG:

Isopropyl-β-thiogalactopyranoside

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

AUC:

Analytical ultracentrifugation

GdnHCl:

Guanidine hydrochloride

CD:

Circular dichroism

λmax :

Wavelength maximum

AEW:

Average emission wavelength

BlGGT:

Bacillus licheniformis γ-glutamyltranspeptidase

References

  1. Benkovic SJ, Hammes GG, Hammes-Schiffer S (2008) Biochemistry 47:3317–3321

    Article  CAS  Google Scholar 

  2. Burgess BR, Dobson RCJ, Bailey MF, Atkinson SC, Griffin MDW, Jameson GB, Parker MW, Gerrard JA, Perugini MA (2008) J Biol Chem 283:27598–27603

    Article  CAS  Google Scholar 

  3. Brown PH, Schuck P (2006) Biophys J 90:4651–4661

    Article  CAS  Google Scholar 

  4. Cansu S, Doruker P (2008) Biochemistry 47:1358–1368

    Article  CAS  Google Scholar 

  5. Chakrabarti P, Saha RP, Mukherjee D (2009) Biochim Biophys Acta 1784–1141

  6. Chakraborty S, Khopade A, Kokarc C, Mahadik K, Chopade B (2009) J Mol Catal B Enzym 58:17–23

    Article  CAS  Google Scholar 

  7. Chang HP, Liang WC, Lyu RC, Chi MC, Wang TF, Su KL, Hung HC, Lin LL (2010) Biochemistry-Moscow 75:919–929

    Article  CAS  Google Scholar 

  8. Chen YH, Chuang LY, Lo HF, Hu HY, Wu TJ, Lin LL, Chi MC (2010) Ann Microbiol 60:307–315

    Article  CAS  Google Scholar 

  9. Christiansen C, Abou Hachem M, Janeček Š, Viksǿ-Nielsen A, Blennow A, Svensson B (2009) FEBS J 276:5006–5029

    Article  CAS  Google Scholar 

  10. Dengra-Pozo J, Martinez-Rodriguez S, Contreras LM, Prieto J, Andujar-Sanchez M, Clemente-Jimenez JM, Las Heras-Vazquez FJ, Rodriguez-Vico F, Neira JL (2009) Biopolymers 91:757–772

    Article  CAS  Google Scholar 

  11. Dong G, Vieille C, Savchenko A, Zeikus JG (1997) Appl Environ Microbiol 63:3569–3576

    CAS  Google Scholar 

  12. Duy C, Fitter J (2005) J Biol Chem 280:37360–37365

    Article  CAS  Google Scholar 

  13. Fitter J, Herrmann R, Dencher NA, Blume A, Hauss T (2001) Biochemistry 40:10723–10731

    Article  CAS  Google Scholar 

  14. Freire E, van Osdol WW, Mayorga OL, Sanchez-Ruiz JM (1990) Annu Rev Biophys Biophys Chem 19:159–188

    Article  CAS  Google Scholar 

  15. Fritzsche HB, Schwede T, Schulz GE (2003) Eur J Biochem 270:2332–2341

    Article  CAS  Google Scholar 

  16. Fukada H, Takahashi K, Sturtevant JM (1987) Biochemistry 26:4063–4068

    Article  CAS  Google Scholar 

  17. Fukushima T, Mizuki T, Echigo A, Inoue A, Usami R (2005) Extremophiles 9:85–89

    Article  CAS  Google Scholar 

  18. Galisteo ML, Mateo PL, Sanchez-Ruiz JM (1991) Biochemistry 30:2061–2066

    Article  CAS  Google Scholar 

  19. Griffin MD, Dobson RC, Pearce FG, Antonio L, Whitten AE, Liew CK, Mackay JP, Trewhella J, Jameson GB, Perugini MA, Gerrard JA (2008) J Mol Biol 380:691–703

    Article  CAS  Google Scholar 

  20. Gruber CW, Cemazar M, Mechler A, Martin LL, Crail DJ (2009) Peptide Sci 92:35–43

    Article  CAS  Google Scholar 

  21. Gupta R, Gigras P, Mohapatra H, Goswam VK, Chauhan B (2003) Process Biochem 38:1599–1616

    Article  CAS  Google Scholar 

  22. He B, Zhang Y, Zhang T, Wang HR, Zhou HM (1995) J Protein Chem 14:349–357

    Article  CAS  Google Scholar 

  23. Hensley P (1996) Structure 4:367–373

    Article  CAS  Google Scholar 

  24. Janeček Š (1997) Prog Biophys Mol Biol 67:67–97

    Article  Google Scholar 

  25. Kamitori S, Abe A, Ohtaki A, Kaji A, Tonozuka T, Sakano Y (2002) J Mol Biol 318:443–453

    Article  CAS  Google Scholar 

  26. Kim JW, Kim YH, Lee HS, Yang SJ, Kim YW, Lee MH, Kim JW, Seo NS, Park CS, Park KH (2007) Biochim Biophys Acta 1774:661–669

    CAS  Google Scholar 

  27. Kiran KK, Chandra TS (2008) Appl Microbiol Biotechnol 77:1023–1031

    Article  CAS  Google Scholar 

  28. Kuriki T, Imanaka T (1999) J Biosci Bioeng 87:557–565

    Article  CAS  Google Scholar 

  29. Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Kluwer Academic/Plenum Publishers, New York

    Google Scholar 

  30. Laue TM, Statford WF (1999) Annu Rev Biophys Biomol Struct 28:75–100

    Article  CAS  Google Scholar 

  31. Lepock JR, Ritchie KP, Kolios MC, Rodahl AM, Heinz KA, Kruuv J (1992) Biochemistry 31:12706–12712

    Article  CAS  Google Scholar 

  32. Lin LL, Tsau MR, Chu WS (1997) J Appl Microbiol 82:325–334

    Article  CAS  Google Scholar 

  33. Lo FH, Lin LL, Chen SL, Hsu WH, Chang CT (2001) Process Biochem 36:743–750

    Article  CAS  Google Scholar 

  34. Lo HF, Lin LL, Chiang WY, Chi MC, Hsu WH, Chang CT (2002) Arch Microbiol 178:115–123

    Article  CAS  Google Scholar 

  35. Lo FH, Lin LL, Li CC, Hsu WH, Chang CT (2001) Curr Microbiol 43:170–175

    Article  CAS  Google Scholar 

  36. Loveridge EJ, Rodriguez RJ, Swanwick RS, Allemann RK (2009) Biochemistry 48:5822–5933

    Article  CAS  Google Scholar 

  37. Lunn FA, MacLeod TJ, Bearne SL (2008) Biochem J 412:113–121

    Article  CAS  Google Scholar 

  38. MacGregor EA, Janeček Š, Svensson B (2001) Biochim Biophys Acta 1546:1–20

    Article  CAS  Google Scholar 

  39. Machovic M, Janeček Š (2006) Cell Mol Life Sci 63:2710–2724

    Article  CAS  Google Scholar 

  40. Makhatadze GI, Privalov PL (1992) J Mol Biol 226:491–505

    Article  CAS  Google Scholar 

  41. Miller GL (1959) Anal Chem 31:426–428

    Article  CAS  Google Scholar 

  42. Monera OD, Kay CM, Hodges RS (1994) Protein Sci 3:1984–1991

    Article  CAS  Google Scholar 

  43. Nakagawa Y, Saburi W, Takada M, Hatada Y, Horikoshi K (2008) Biochim Biophys Acta 1784:2004–2011

    CAS  Google Scholar 

  44. Nakajima R, Imanaka T, Aiba S (1985) J Bacteriol 163:401–406

    CAS  Google Scholar 

  45. Nandi PK, Robinson DR (1984) Biochemistry 23:6661–6668

    Article  CAS  Google Scholar 

  46. Nazmi AR, Reinisch T, Hinz HJ (2006) Arch Biochem Biophys 453:18–25

    Article  CAS  Google Scholar 

  47. Pace CN (1990) Trends Biotechnol 8:93–98

    Article  CAS  Google Scholar 

  48. Plaza del Pino IM, Ibarra-Molero B, Sachez-Ruiz JM (2000) Proteins 40:58–70

    Article  CAS  Google Scholar 

  49. Royer CA (2006) Chem Rev 106:1769–1784

    Article  CAS  Google Scholar 

  50. Royer CA, Mann CJ, Matthews CR (1995) Protein Sci 2:1844–1852

    Article  Google Scholar 

  51. Sai Kumar RS, Singh SA, Appu Rao AG (2009) Biochimie 91:548–557

    Article  CAS  Google Scholar 

  52. Sanchez-Ruiz JM (1992) Biophys J 61:921–935

    Article  CAS  Google Scholar 

  53. Schellman JA (2002) Biophys Chem 96:91–101

    Article  CAS  Google Scholar 

  54. Schuck P (2000) Biophys J 78:1600–1619

    Article  Google Scholar 

  55. Shafiei M, Ziaee AA, Amoozegar MA (2010) J Ind Microbiol Biotechnol doi:10.1007/s10295-010-0770-1

  56. Shriver JW, Edmondson SP (2009) Method Mol Biol 499:57–82

    Google Scholar 

  57. Southall SM, Simpson PJ, Gilbert HJ, Williamson G, Willamson MP (1999) FEBS Lett 447:58–60

    Article  CAS  Google Scholar 

  58. Tello-Solis SR, Hernandez-Arana A (1995) Biochem J 311:969–974

    CAS  Google Scholar 

  59. Tomazic SJ, Klibanov AM (1988) J Biol Chem 263:3086–3091

    CAS  Google Scholar 

  60. van der Maarel M, van der Veen B, Uitdehaag J, Leemhuis H, Dijkhuizen L (2002) J Biotechnol 94:137–155

    Article  Google Scholar 

  61. Vecchio PD, Granziano G, Granata V, Barone G, Mandrich L, Rossi M, Manco G (2002) Biochem J 367:857–863

    Article  Google Scholar 

  62. Vihinen N, Mäntsälä P (1990) Biochem Biophys Res Commun 166:61–65

    Article  CAS  Google Scholar 

  63. Violet M, Meunier JC (1989) Biochem J 263:665–670

    CAS  Google Scholar 

  64. Vogl T, Jatzke C, Hinz HJ, Benz J, Huber R (1997) Biochemistry 36:1657–1668

    Article  CAS  Google Scholar 

  65. Wang ZF, Huang MQ, Zou XM, Zhou HM (1995) Biochim Biophys Acta 1251:109–114

    Article  Google Scholar 

  66. White MF, Fothergill-Gilmore LA, Kelly SM, Price NC (1993) Biochem J 291:479–483

    CAS  Google Scholar 

  67. Yuuki T, Nomura T, Tezuka H, Tsuboi A, Yamagata H, Tsukagoshi N, Udaka S (1985) J Biochem (Tokyo) 98:1147–1156

    CAS  Google Scholar 

  68. Singh AR, Joshi S, Arya R, Kayastha AM, Saxena JK (2010) Eur Biophys J 39:289–297

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful to Dr. Hui-Chih Hung for providing the necessary facilities to carry out the AUC experiment. This work was supported by a research grant (NSC 97-2628-B-415-001-MY3) from National Science Council of Taiwan.

Author information

Authors and Affiliations

  1. Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi County, 60004, Taiwan

    Meng-Chun Chi, Tai-Jung Wu & Long-Liu Lin

  2. Department of Aquatic Biosciences, National Chiayi University, 300 Syuefu Road, Chiayi County, 60004, Taiwan

    Tzu-Ting Chuang

  3. Department of Food Science and Technology, Hungkuang University, Shalu, Taichung, 433, Taiwan

    Hsiang-Ling Chen & Huei-Fen Lo

Authors
  1. Meng-Chun Chi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tai-Jung Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tzu-Ting Chuang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hsiang-Ling Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Huei-Fen Lo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Long-Liu Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Long-Liu Lin.

Additional information

Meng-Chun Chi and Tai-Jung Wu contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chi, MC., Wu, TJ., Chuang, TT. et al. Biophysical Characterization of a Recombinant α-Amylase from Thermophilic Bacillus sp. strain TS-23. Protein J 29, 572–582 (2010). https://doi.org/10.1007/s10930-010-9287-8

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-010-9287-8

Keywords

Search

Navigation