Volume 5 Supplement 1

The 4th Recombinant Protein Production Meeting: a comparative view on host physiology

Open Access

Heterologous overexpression of a halophilic α-amylase

  • Vanesa Bautista1,
  • Julia Esclapez1,
  • Rosa Ma Martínez-Espinosa1,
  • Francisco Pérez-Pomares1,
  • Mónica Camacho1 and
  • Ma José Bonete1
Microbial Cell Factories20065(Suppl 1):P13

DOI: 10.1186/1475-2859-5-S1-P13

Published: 10 October 2006

Background

Extracellular hydrolytic enzymes such as α-amylases are widely used in diverse applications in different industrial areas. α-amylase (EC 3.2.1.1) is an important endo-type carbohydrase that hydrolyzes α-1,4 glycosidic linkages of D-glucose oligomers and polymers. This enzyme has been found in organisms of the three Domains, being a key enzyme of carbohydrate metabolism. Haloferax mediterranei is an extremely halophilic Archaea that requires high salt concentrations to grow. This microorganism is able to grow in a minimal medium with ammonium acetate as the only source of carbon and nitrogen.H. mediterranei shows α-amylase extracellular activity when grows in this minimal medium in the presence of starch. The main role of this enzyme is the starch metabolism in the extracellular medium, so a lot of microorganisms depend on amylases for survival [1].

Results

The extracellular halophilic H. mediterranei α-amylase has been purified to electrophoretic homogeneity [2]. The enzyme has been digested in the presence of trypsin to analyse the resulting peptides using the technique of nanoelectrospray LC/MS. From the obtained peptides and by sequence homology with other α-amylases, the H. mediterranei α-amylase gene has been isolated and sequenced, using a library of genomic DNA from H. mediterranei in bacteriophage lambda EMBL3. The molecular mass estimated from the deduced amino acid sequence was similar to the calculated by SDS-PAGE. This enzyme is rich in acidic amino acids with a 16% Asp and Glu content which is also the case in other halophilic enzymes [3]. The PCR product for α-amylase was ligated in the plasmid pSTBlue1 and cloned into E. coli NovaBlue cells. The insert was digested and subcloned in expression vector pET3a in E. coli NovaBlue cells, and introduced in the E. coli BL21(DE3) cells for expression. The recombinant protein was mainly obtained as inclusion bodies, although a small amount of protein was presented in the cytoplasmic soluble fraction (Figure 1). The protein obtained as inclusion bodies was refolded by solubilisation in 8 M urea followed by dilution into a high salt concentration buffer in the presence of calcium (Figure 2), and the protein of the soluble fraction was obtained in active state.
Figure 1

SDS-PAGE for expression cell fractions.(a) Lane 1: molecular weight standards; Lanes 3 and 4:E. coli BL21(DE3) containing pET3a; Lanes 2 and 5: E. coli BL21(DE3) containing pET3a-Amy; Lanes 2 and 3: total cell proteins; Lanes 4 and 5: soluble cytoplasmic fractions.(b) Lane 1: molecular weight standards; Lanes 2 and 3: inclusion bodies E. coli BL21(DE3) containing pET3a-Amy with different concentrations.

Figure 2

(a) Effect of NaCl concentration on the folding efficiency of α-amylase inclusion bodies dissolved in 8 M urea. Refolding buffer: 20 mM Tris-HCl pH 7.4 with salt. (b) Effect of calcium concentration on the folding efficiency of α-amylase inclusion bodies dissolved in 8 M urea. Refolding buffer: 20 mM Tris-HCl pH 7.4, 3 M NaCl and calcium.

Furthermore, four genes have been sequenced by primer walking, which match up with four proteins belonging to ABC maltose transport system.

Conclusion

The α-amylase gene from H. mediterranei has been cloned, sequenced and overexpressed. The recombinant protein has been obtained in large amounts and refolded with a high efficiency.

Authors’ Affiliations

(1)
Department of Biochemistry and Molecular Biology, University of Alicante

References

  1. Jones RA, Jermiin LS, Easteal S, Patel BKC, Beacham IR: Amylase and 16S rRNA genes from a hyperthermophilic archaebacterium. J Appl Microbiol. 1999, 86: 93-107. 10.1046/j.1365-2672.1999.00642.x.View ArticleGoogle Scholar
  2. Pérez-Pomares F, Bautista V, Ferrer J, Pire C, Marhuenda-Egea FC, Bonete MJ: Alpha-amylase activity from the halophilic archaeon Haloferax mediterranei . Extremophiles. 2003, 7 (4): 299-306. 10.1007/s00792-003-0327-6.View ArticleGoogle Scholar
  3. Pire C, Esclapez J, Ferrer J, Bonete MJ: Heterologous overexpression of glucose dehydrogenase from the halophilic archaeon Haloferax mediterranei, an enzyme of the medium chain dehydrogenase family. FEMS Microbiol Lett. 2001, 200: 221-227. 10.1111/j.1574-6968.2001.tb10719.x.View ArticleGoogle Scholar

Copyright

© Bautista et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

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