Skip to main content
SpringerLink
Log in

Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity

  • Original Paper
  • Published:
Journal of Industrial Microbiology and Biotechnology

Abstract

Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase. The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography. The purified enzyme retained LAP activity and adsorbed raw starch. LAPsbd was stable at 70°C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition. Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k cat value.

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. 1a, b
Fig. 2
Fig. 3a, b
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Chang HY, Irwin PM, Nikolov ZL (1998) Effects of mutations in the starch-binding domain of Bacillus macerans cyclodextrin glycosyltransferase. J Biotechnol 65:191–202

    Article  CAS  PubMed  Google Scholar 

  2. Chen LJ, Ford C, Nikolov Z (1991) Adsorption to starch of a β-galactosidase fusion protein containing the starch-binding region of Aspergillus glucoamylase. Gene 99:121–126

    Article  CAS  PubMed  Google Scholar 

  3. Dalmia B, Schütte K, Nikolv Z (1995) Domain E of Bacillus macerans cyclodextrin glucanotransferase: an independent starch-binding domain. Biotechnol Bioeng 47:575–584

    CAS  Google Scholar 

  4. Gonzales T, Robert-Baudouy J (1996) Bacterial aminopeptidases: properties and functions. FEMS Microbiol Rev 18:319–344

    Article  CAS  PubMed  Google Scholar 

  5. Hellman J, Mäntsälä P (1992) Construction of an Escherichia coli export-affinity vector for expression and purification of foreign proteins by fusion to cyclomaltodextrin glucanotransferase. J Biotechnol 23:19–34

    Article  CAS  PubMed  Google Scholar 

  6. Juge N, Sógaard M, Chaix JC, Martin-Eauclaire MF, Svensson B, Marchis-Mouren G, Guo XJ (1993) Comparison of barley malt α-amylase isozymes 1 and 2: construction of cDNA hybrids by in vivo recombination and their expression in yeast. Gene 130:159–166

    Article  CAS  PubMed  Google Scholar 

  7. Kamphuis J, Meijer EM, Boesten WHJ, Broxterman QB, KapteinB, Hermes HFM, Schoemaker HE (1992) Production of natural and synthetic L- and D-amino acids by aminopeptidases and amino amidases. In: Rozzell JD, Wagner F (eds) Biocatalytic production of amino acids and derivatives. Wiley, New York, pp 178–206

  8. Kuo LY, Hwang GY, Lai YJ, Yang SL, Lin LL (2003) Overexpression, purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus. Curr Microbiol 47:40–45

    Article  CAS  PubMed  Google Scholar 

  9. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685

    Google Scholar 

  10. Lin LL, Hsu WH, Chu WS (1997) A gene encoding for an α-amylase from thermophilic Bacillus sp. strain TS-23 and its expression in Escherichia coli. J Appl Microbiol 82:325–334

    Article  CAS  PubMed  Google Scholar 

  11. Lin LL, Lo HF, Chen JN, Ku KL, Hsu WH (2002) Isolation of a recombinant Bacillus sp. TS-23 α-amylase by adsorption-elution on raw starch. Starch/Stärke 54:338–342

    Google Scholar 

  12. Lin LL, Lo HF, Chi MC, Ku KL (2003) Functional expression of the raw starch-binding domain of Bacillus sp. strain TS-23 α-amylase in recombinant Escherichia coli. Starch/Stärke 55:197–202

    Google Scholar 

  13. Lo HF, Lin LL, Chiang WY, Chi MC, Hsu WH, Chang CT (2002) Deletion analysis of the C-terminal region of the α-amylase of Bacillus sp. strain TS-23. Arch Microbiol 178:115–123

    Article  CAS  PubMed  Google Scholar 

  14. Manchenko GP (1994) Handbook of detection of enzymes on electrophoretic gels. CRC Press, London, pp 3.4.11.1

  15. Moraes LMP, Astolfi-filho S, Oliver SG (1995) Development of yeast strains for the efficient utilization of starch: evaluation of constructs that express α-amylase and glucoamylase separately or as bifunctional fusion proteins. Appl Microbiol Biotechnol 43:1067–1076

    PubMed  Google Scholar 

  16. Mori H, Tanizawa K, Fukui T (1993) A chimeric α-glucan phosphorylase of plant type L and H isozymes: functional role of 78-residue insertion in type L isozyme. J Biol Chem 268:5574–5581

    CAS  PubMed  Google Scholar 

  17. Motoshima H, Minagawa E, Tsukasaki F, Kaminogawa S (1997) Cloning of genes of the aminopeptidase T family from Thermus thermophilus HB8 and Bacillus stearothermophilus NCIB8924: apparent similarity to the leucyl aminopeptidase family. Biosci Biotechnol Biochem 61:1710–1717

    CAS  PubMed  Google Scholar 

  18. Nakano YJ, Kuramitsu HK (1992) Mechanism of Streptococcus mutans glucosyltransferase: hybrid-enzyme analysis. J Bacteriol 174:5639–5646

    CAS  PubMed  Google Scholar 

  19. Ohdan K, Kuriki T, Takata H, Okada H (2000) Cloning of cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. A2-5a and analysis of the raw starch-binding domain. Appl Microbiol Biotechnol 53:430–434

    Article  CAS  PubMed  Google Scholar 

  20. Rao MB, Tanksale AM, Ghatge MS, Desphade VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635

    CAS  PubMed  Google Scholar 

  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 17.2–17.44

    Google Scholar 

  22. Sauer J, Christensen T, Frandsen TP, Mirgorodskaya E, McGuire KA, Driguez H, Roepstorff P, Sigurskjold BW, Svensson B (2001) Stability and function of interdomain linker variants of glucoamylase I from Aspergillus niger. Biochemistry 40:9336–9346

    Article  CAS  PubMed  Google Scholar 

  23. Shibuya I, Tamura G, Shima H, Ishikawa T, Hara S (1992) Construction of an α-amylase-glucoamylase fusion gene and its expression in Saccharomyces cerevisiae. Biosci Biotechnol Biochem 56:884–889

    CAS  PubMed  Google Scholar 

  24. Stoll E, Weder HG, Zuber H (1976) Aminopeptidase II from Bacillus stearothermophilus. Biochim Biophys Acta 438:212–220

    Article  CAS  Google Scholar 

  25. Sugimoto M, Ohta T, Kawai F (2003) Change in maltose- and soluble starch-hydrolyzing activities of chimeric α-glucosidases of Mucor javanicus and Aspergillus oryzae. Biochim Biophys Acta 1645:1–5

    Article  CAS  PubMed  Google Scholar 

  26. Terashima M, Hosono M, Katoh S (1997) Functional roles of protein domains on rice α-amylase activity. Appl Microbiol Biotechnol 47:364–367

    Article  CAS  PubMed  Google Scholar 

  27. Toldrá F, Aristoy AC, Flores M (2000) Contribution of muscle aminopeptidases to flavor development in dry-cured ham. Food Res Int 33:181–185

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by a research grant (NSC 92-2313-B-415-005) from the National Science Council of the Republic of China.

Author information

Authors and Affiliations

  1. Department of Applied Chemistry, National Chiayi University, 300 University Road, 60083, Chiayi, Taiwan

    Yu-Wen Hua, Meng-Chun Chi & Long-Liu Lin

  2. Department of Food and Nutrition, Hungkuang University, Taichung, Taiwan

    Huei-Fen Lo

  3. Institute of Molecular Biology, National Chung Hsing University, 402-27, Taichung, Taiwan

    Wen-Hwei Hsu

Authors
  1. Yu-Wen Hua
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Meng-Chun Chi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huei-Fen Lo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wen-Hwei Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Long-Liu Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Long-Liu Lin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hua, YW., Chi, MC., Lo, HF. et al. Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity. J IND MICROBIOL BIOTECHNOL 31, 273–277 (2004). https://doi.org/10.1007/s10295-004-0146-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-004-0146-5

Keywords

Search

Navigation