Extracellular levansucrase of Bacillus subtilis produced in yeast remains in the cell in its precursor form

Levansucrase, a Bacillus subtilis extracellular enzyme, was not secreted in the culture medium when produced in yeast. The protein accumulated inside the cell in its precursor form which represented 0.3% of total proteins. The absence of any post-translational modifications, such as signal sequence cleavage or addition of N-linked sugars, indicated that this protein did not enter the reticulum secretion pathway. Direct observation of the cells by confocal laser scanning microscopy showed that levansucrase was associated with the cytoplasmic membrane. Subcellular fractionation experiments revealed that levansucrase precursor form is associated with membranes through weak ionic interactions. The purified precursor displayed the same catalytic properties as levansucrase secreted by B. subtilis. Thus yeast could be used as a source of levansucrase precursor allowing its isolation as a pure form on a milligram scale.