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  • Title: Expression and secretion of Bacillus polymyxa neopullulanase in Saccharomyces cerevisiae.
    Author: Yebra MJ, Blasco A, Sanz P.
    Journal: FEMS Microbiol Lett; 1999 Jan 01; 170(1):41-9. PubMed ID: 9919651.
    Abstract:
    We have isolated the gene encoding the neopullulanase enzyme from Bacillus polymyxa CECT 155. It consists of an open reading frame of 1545 bp that could code for a protein of 515 amino acids. This open reading frame was expressed in Bacillus subtilis and the corresponding transformants produced extracellular neopullulanase. The neopullulanase gene was also expressed in Saccharomyces cerevisiae placing it under the control of the yeast actin gene (ACT1) promoter. Clones containing the intact neopullulanase gene, including its own bacterial signal sequence, gave rise to the synthesis of active, but intracellular, enzyme by S. cerevisiae transformants. When sequences specifying the signal sequence and leader region of the yeast mating pheromone alpha-factor (MF alpha 1) were fused upstream of the gene encoding the neopullulanase enzyme, the enzyme was secreted by S. cerevisiae. The secreted protein presented the same biochemical properties and the same apparent molecular mass as the Bacillus polymyxa original enzyme. The predicted amino acid sequence of the neopullulanase protein contained sequence motifs conserved among amylolytic enzymes. Northern blot analysis indicated that the transcription of the neopullulanase gene in B. polymyxa was induced by the presence of the substrate, pullulan, in the culture, and was repressed by glucose.
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