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Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis

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Summary

The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli. Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an Mr of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region. A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene. The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance. The B. stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - σH). The glgB promoter was utilized in B. subtilis exclusively in the stationary phase, and only transcribed at low levels in B. subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B. subtilis. In an expression vector, the B. stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E. coli as well as in B. subtilis, with optimal branching activity at 53° C.

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Author notes

  1. G. Beldman

    Present address: Department of Food Science, Agricultural University Wageningen, P.O. box 8129, NL-6700, EV Wageningen, The Netherlands

Authors and Affiliations

  1. Department of Genetics, Center of Biological Sciences, Kerklaan 30, NL-9751, NN Haren, The Netherlands

    J. A. K. W. Kiel, J. M. Boels & G. Venema

  2. Netherlands Institute for Carbohydrate Research (NIKO-TNO), Rouaanstraat 27, NL-9723, CC Groningen, The Netherlands

    G. Beldman

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  1. J. A. K. W. Kiel
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  2. J. M. Boels
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  3. G. Beldman
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  4. G. Venema
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Kiel, J.A.K.W., Boels, J.M., Beldman, G. et al. Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis . Molec. Gen. Genet. 230, 136–144 (1991). https://doi.org/10.1007/BF00290661

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