Expression of Pichia anomala INV1 gene in Saccharomyces cerevisiae results in two different active forms of hypoglycosylated invertase.
The Pichia anomala invertase gene (INV1) was introduced at different copy numbers into a sucrose-nonfermenting mutant of Saccharomyces cerevisiae and expressed from its own promoter sequences. The level reached in the production of invertase by the transformants (up to 540 units/10(10) cells) was in agreement with the INV1 gene dosage. Two forms of multimeric active and glycosylated invertase displaying different subcellular locations and molecular masses could be detected in the transformants. One was found to be present in the culture medium and in the periplasm, and the other could only be detected inside the cell. Each of the two heterologous forms of invertase was shown to be an oligomer composed of identical subunits. The difference found in the apparent molecular masses of their monomers (81.5 and 78.3 kDa, respectively) seems to be due to the size of their N-linked oligosaccharide chains (on average 2.4 and 1.9 kDa, respectively), since the number of sugar chains (9) and the molecular mass of the protein moiety (60.5 kDa) are identical in both forms. The shorter size of their oligosaccharides must also be the reason for the lower apparent molecular masses of the heterologous invertases when compared with the enzyme purified from P. anomala. The hypoglycosylated invertase accumulated within the cells of the transformants to an unusual level (up to 130 units/10(10) cells). Such accumulation of active enzyme inside the cells, as well as its underglycosylation, could be due to intrinsic properties of the P. anomala invertase that are determined by the particular primary structure of its protein moiety.
Pérez
J A
JA
Departamento de Microbiología y Biología Celular, Facultad de Farmacia, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez, s/n, 38071 La Laguna, Tenerife, Spain.
Rodríguez
J
J
Ruiz
T
T
Rodríguez
L
L
D006026:Glycoside Hydrolases / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D006031:Glycosylation; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D043324:beta-Fructofuranosidase
D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase