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


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.


Bacillus Bacillus Subtilis Antibiotic Concentration Integration Procedure Antibiotic Selection 
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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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