Abstract
A high level expression of thermostable α-amylase gene from Bacillus licheniformis in Escherichia coli was obtained. The recombinant enzyme was mainly produced in the form of insoluble aggregates. The enzyme was solubilized without using denaturing agents and purified to homogeneity in a single step by ion exchange chromatography. The enzyme was purified 138-fold with a final yield of 349 %; the specific activity of the purified enzyme was 1343 U/mg.
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References
Botterman J., Zabeau M.: High-level production of the EcoRI endonuclease under the control of the pL promoter of bacteriophage lambda. Gene37, 229–239 (1985).
Bradford M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal.Biochem. 72, 248–254 (1976).
Cheng Y.S.: Increased cell buoyant densities of protein overproducing Escherichia coli cells. Biochem.Biophys.Res.Commun. 111, 104–111 (1983).
Clark E.D.: Protein refolding for industrial processes. Curr.Opin.Biotechnol. 12, 202–207 (2001).
Crabb W.D., Mitchinson C.: Enzymes involved in the processing of starch to sugars. Trends Biotechnol. 15, 349–352 (1997).
Dong G., Vieille C., Savchenko A., Zeikus J.G.: Cloning, sequencing, and expression of the gene encoding extracellular α-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl.Environ.Microbiol. 63, 3569–3576 (1997).
Gribskov M., Burgess R.R.: Overexpression and purification of the σ subunit of Escherichia coli RNA polymerase. Gene26, 109–118 (1983).
Hockney R.C.: Recent developments in heterologous protein production in Escherichia coli. Trends Biotechnol. 12, 456–463 (1994).
Jorgensen S., Vorgias C.E., Antranikian G.: Cloning, sequencing, characterization, and expression of an extracellular α-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis. J.Biol.Chem. 272, 16335–16342 (1997).
Kilara A., Desai M.: Enzymes, pp. 661–706, in A.L. Branen, P.M. Davidson, S. Salminen, J.H. Thorngate III (Eds): Food Additives. Marcel Dekker Inc., New York 2002.
Lin L.L., Hsu W.H.: Lactose-induced expression of Bacillus sp. TS-23 amylase gene in Escherichia coli regulated by a T7 promoter. Lett.Appl.Microbiol. 24, 365–368 (1997).
Linden A., Niehaus F., Antranikian G.: Single-step purification of a recombinant thermostable α-amylase after solubilization of the enzyme from insoluble aggregates. J.Chromatogr. 737, 253–259 (2000).
van der Maarel M.J., van der Veen B., Uitdehaag J.C., Leemhuis H., Dijkhuizen L.: Properties and applications of starch-converting enzymes of the α-amylase family. J.Biotechnol. 94, 137–155 (2002).
Marco J.L., Bataus L.A., Valência F.F., Ulhoa C.J., Astolfi-Filho S., Felix C.R.: Purification and characterization of a truncated Bacillus subtilis α-amylase produced by Escherichia coli. Appl.Microbiol.Biotechnol. 44, 746–752 (1996).
Marston F.A.: The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem.J. 240, 1–12 (1986).
Park C.S., Chang C.C., Kim J.Y., Ogrydziak D.M., Ryu D.D.: Expression, secretion, and processing of rice α-amylase in the yeast Yarrowia lipolytica. J.Biol.Chem. 272, 6876–6881 (1997).
Prouty W.F., Karnovsky M.J., Goldberg A.L.: Degradation of abnormal proteins in Escherichia coli. Formation of protein inclusions in cells exposed to amino acid analogs. J.Biol.Chem. 250, 1112–1122 (1975).
Rashid N., Morikawa M., Imanaka T.: An abnormally acidic TATA-binding protein from a hyperthermophilic archaeon. Gene166, 139–143 (1995).
Rashid N., Shimada Y., Ezaki S., Atomi H., Imanaka T.: Low-temperature lipase from a psychrotrophic Pseudomonas sp. strain KB700A. Appl.Environ.Microbiol.67, 4064–4069 (2001).
Rashid N., Cornista J., Ezaki S., Fukui T., Atomi H., Imanaka T.: Characterization of an archaeal cyclodextrin glucanotransferase with a novel C-terminal domain. J.Bacteriol. 184, 777–784 (2002).
Rashid N., Farooq A., Haque I., Akhtar M.: Insoluble but enzymatically active α-amylase from Bacillus licheniformis. Biologia64, 660–663 (2009).
Riegal E.R., Bissinger H.G.: Industrial fermentation: principles, processes and products, pp. 963–1045 in J.A. Kent (Eds): Riegal’s Handbook of Industrial Chemistry. Kluwer Academic-Plenum Publishers, New York 2003.
Rudolph R., Lilie H.: In vitro folding of inclusion body proteins. FASEB J. 10, 49–56 (1996).
Schoemaker J.M., Brasnett A.H., Marston F.A.: Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies. EMBO J. 4, 775–780 (1985)
Schofield L.R., Patchett M.L., Parker E.J.: Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Pyrococcus furiosus. Protein Expr.Purif. 34, 17–27 (2004).
Sibakov M.: High expression of Bacillus licheniformis α-amylase with a Bacillus secretion vector. Eur.J.Biochem. 155, 577–581 (1986).
Speed M.A., Wang D.I.C., King J.: Specific aggregation of partially folded polypeptide chains: the molecular basis of inclusion body composition. Nat.Biotechnol. 14, 1283–1287 (1996).
Suominen I., Meyer P., Tilgmann C., Glumoff T., Glumoff V., Käpylä J., Mäntsälä P.: Effects of signal peptide mutations on processing of Bacillus stearothermophilus α-amylase in Escherichia coli. Microbiology141, 649–654 (1995).
Thomas J.G., Baneyx F.: Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli. Protein Expr.Purif. 11, 289–296 (1997).
Wang L., Zhou Q., Chen H., Chu Z., Lu J., Zhang Y., Yang S.: Efficient solubilization, purification of recombinant extracellular α-amylase from Pyrococcus furiosus expressed as inclusion bodies in Escherichia coli. J.Ind.Microbiol.Biotechnol. 34, 187–192 (2007).
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Rashid, N., Ahmed, N., Saleem Haider, M. et al. Effective solubilization and single-step purification of Bacillus licheniformis α-amylase from insoluble aggregates. Folia Microbiol 55, 133–136 (2010). https://doi.org/10.1007/s12223-010-0020-y
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DOI: https://doi.org/10.1007/s12223-010-0020-y