The Influence of Yarrowia lipolytica Glycosylation on the Biochemical Properties and Oligomerization of Heterologous Invertase

Abstract

Invertases are important enzymes used in the food industry. Despite many studies on the invertase-encoding SUC2 gene expression in the industrial yeast Yarrowia lipolytica, no biochemical characteristics of this enzyme expressed as heterologous protein have been provided. Here, two isoforms of extracellular invertase produced by Y. lipolytica were detected using ion-exchange chromatography. Specific activities of 226.45 and 432.66 U/mg for the first and second isoform, respectively, were determined. Basic characteristics of this enzyme were similar to the one isolated from Saccharomyces cerevisiae (optimum pH and temperature, metal ions inhibition, substrate specificity and fructooligosaccharides (FOS) biosynthesis). The apparent differences were higher KM for sucrose (67 mM) and lower molecular mass (66 kDa) resulting from lower N-glycosylation level (9.1% of mass). The N-glycan structures determined by MALDI-TOF and HPLC represented high mannose structures, though with much shorter chains than hypermannosylated glycans from S. cerevisiae. Furthermore, galactose was detected as the modifying sugar in the glycan structures of invertase expressed in Y. lipolytica. N-glycans did not affect invertase activity but were important for its oligomerization. The expressed enzyme aggregated into dimers, tetramers, hexamers, and octamers, as well as structures of higher molecular mass, which might be decamers, which have not been described so far in the literature.