Cloning of the Maltose Phosphorylase Gene from Bacillus sp. Strain RK-1 and Efficient Production of the Cloned Gene and the Trehalose Phosphorylase Gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis.

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  • Cloning of the Maltose Phosphorylase Gene from<i>Bacillus</i>sp. Strain RK-1 and Efficient Production of the Cloned Gene and the Trehalose Phosphorylase Gene from…
  • Cloning of the maltose phosphorylase from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

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Abstract

  The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens α-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.<br>

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