title: Secreted amylolytic enzymes from Schwanniomyces occidentalis: purification by fast protein liquid chromatography (FPLC) and preliminary characterization.
abstract: Amylolytic enzyme preparations are used extensively for the liquefaction and saccharification of starch in the production of ethanol and SCP (single cell protein). We report the first purification of two amylolytic enzymes from the yeast Schwanniomyces occidentalis using fast protein liquid chromatography (FPLC) in a two step process: size exclusion (Superose 12) followed by anion exchange (Mono Q). The procedure is amenable to direct scale up processes. The enzymes glucoamylase (E.C. 3.2.1.2) and alpha-amylase (E.C. 3.2.1.1) were found in the cell free supernatant of S. occidentalis when grown on a variety of carbon sources. The enzymes are substrate induced and catabolite repressed. Both amylolytic enzymes were purified from three separate culture broths containing either starch, maltose or cellobiose and their physical properties compared. Native molecular masses of glucoamylase and alpha-amylase were determined to be 122,000 +/- 28,000 daltons and 47,000 +/- 11,000 daltons, respectively, while subunit size was approximated at 143,000 +/- 2,000 daltons and 54,500 +/- 1,000 daltons, respectively. Both proteins are N-glycosylated with carbohydrate representing 10-15% of the total mass. The correlation of native mass and denatured subunit structure, while not identical due to slight aberrant behavior on gels and columns as a result of glycosylation, suggest that both proteins exist as monomeric polypeptides. Isoelectric points for both proteins under native conditions could not be determined since alpha-amylase failed to enter native polyacrylamide gels. However, a pI for glucoamylase of 6.2 +/- 0.2 (native) and a pI for alpha-amylase of 6.3 +/- 0.3 (in 6M urea) were determined. Glucoamylase and alpha-amylase specific activities (for the homogeneous proteins) were determined to be 48-67 x 10(3) units/mg and 214-457 x 10(3) units/mg respectively. We could find no apparent differences in either glucoamylase or alpha-amylase proteins obtained from three separate cultures which had been grown on different carbon sources. The purification method we have utilized is easily scaled up to larger protein concentrations, and provides a rapid procedure for analyzing and purifying these amylolytic enzymes.
mesh_terms: D000681:Amylases / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003470:Culture Media / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005087:Glucan 1,4-alpha-Glucosidase / Q000302:isolation & purification; D008970:Molecular Weight; D004718:Saccharomycetales / Q000201:enzymology*; D000516:alpha-Amylases / Q000302:isolation & purification
chemical_list: D003470:Culture Media; D000681:Amylases; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase