Expression of Pichia anomala INV1 gene in Saccharomyces cerevisiae resultsin two different active forms of hypoglycosylated invertase

Citation
Ja. Perez et al., Expression of Pichia anomala INV1 gene in Saccharomyces cerevisiae resultsin two different active forms of hypoglycosylated invertase, ARCH MICROB, 175(3), 2001, pp. 189-197
Citations number
56
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
ARCHIVES OF MICROBIOLOGY
ISSN journal
0302-8933 → ACNP
Volume
175
Issue
3
Year of publication
2001
Pages
189 - 197
Database
ISI
SICI code
0302-8933(200103)175:3<189:EOPAIG>2.0.ZU;2-U
Abstract
The Pichia anomala invertase gene (INV1) was introduced at different copy n umbers into a sucrose-non-fermenting mutant of Saccharomyces cerevisiae and expressed from its own promoter sequences. The level reached in the produc tion of invertase by the transformants (up to 540 units/10(10) cells) was i n agreement with the INV1 gene dosage. Two forms of multimeric active and g lycosylated invertase displaying different subcellular locations and molecu lar masses could be detected in the transformants. One was found to be pres ent 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 oligosacchari de chains (on average 2.4 and 1.9 kDa, respectively), since the number of s ugar 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 a lso be the reason for the lower apparent molecular masses of the heterologo us invertases when compared with the enzyme purified from P. anomala. The h ypoglycosylated invertase accumulated within the cells of the transformants to an unusual level (up to 130 units/10(10) cells). Such accumulation of a ctive 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.