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Applied Microbiology and Biotechnology

, Volume 27, Issue 1, pp 64–71 | Cite as

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

  • Pauli Kallio
  • Airi Palva
  • Ilkka Palva
Applied Genetics and Regulation

Summary

The α-amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the α-amylase gene as two randomly located copies. These strains produced α-amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same α-amylase gene. With the plasmid system, however, the rate of the α-amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded α-amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight α-amylase gene copies per one genome and producing up to eightfold higher α-amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the α-amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.

Keywords

Bacillus Bacillus Subtilis Antibiotic Concentration Integration Procedure Antibiotic Selection 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag 1987

Authors and Affiliations

  • Pauli Kallio
    • 1
  • Airi Palva
    • 1
  • Ilkka Palva
    • 1
  1. 1.Recombinant DNA LaboratoryUniversity of HelsinkiHelsinkiFinland

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