Purification and characterization of invertase from a novel industrial yeast, Schwanniomyces occidentalis.
The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates. Other yeasts have similar disadvantages in addition to producing a variety of proteases. We have investigated the use of Schwanniomyces occidentalis as a host for developing a gene expression system in which these and several disadvantages are minimized. The present paper describes the isolation and characterization of an invertase from cell free supernatants of the yeast Schwanniomyces occidentalis grown on lactose. The enzyme is a beta-D-fructofuranoside-fructohydrolyase, composed of two identical subunits of 76,000 to 78,000 da. with a native molecular mass of 125,000 +/- 25,000 da. of which approximately 17% can be attributed to N-linked carbohydrate. The enzyme has a Vmax of 0.49 +/- 0.025 units, a Km of 21 +/- 1.5 mM, and temperature and pH optima of 55 degrees C and 3.9-4.5, respectively. The amino acid sequences of the amino terminal region and an internal tryptic peptide support an 81% identity with the invertase from Saccharomyces cerevisiae. The enzyme is induced by low glucose and is catabolite repressed.
Klein
R D
RD
Molecular Biology Research, Upjohn Company, Kalamazoo, Michigan 49007.
Deibel
M R
MR
Sarcich
J L
JL
Zurcher-Neely
H A
HA
Reardon
I M
IM
Heinrikson
R L
RL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D004718:Saccharomycetales / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D043324:beta-Fructofuranosidase
D000596:Amino Acids; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase