Isolation, purification, and characterization of a killer protein from Schwanniomyces occidentalis

Abstract

The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. The protein was stable between pH 2.0 and 5.0 and inactivated at temperatures above 40 degrees C. The killer protein was chromosomally encoded. Mannan, but not beta-glucan or laminarin, prevented sensitive yeast cells from being killed by the killer protein, suggesting that mannan may bind to the killer protein. Identification and characterization of a killer strain of S. occidentalis may help reduce the risk of contamination by undesirable yeast strains during commercial fermentations.

FIG. 1

(A) Native PAGE analysis of the killer protein stained with Coomassie blue. Lane 1, purified killer protein in an 18% acidic native polyacrylamide gel (pH 4.2); lane 2, an inhibition zone on methylene blue agar plate overlaid with a native gel containing the purified killer protein. (B) Tricine SDS-PAGE of the killer protein stained with Coomassie blue. M, protein molecular mass markers (Promega Corporation, Madison, Wis.), including carbonic anhydrase (31 kDa), soybean trypsin inhibitor doublet (20.4/19.7 kDa), horse heart myoglobin (16.9 kDa), lysozyme (14.4 kDa), aprotinin (6.1 kDa), and insulin β chain (3.5 kDa); lane 1, with β-mercaptoethanol; lane 2, without β-mercaptoethanol. Lanes 1 and 2 contain 1.4 μg of protein. (C) Nonreducing Tricine SDS-PAGE of the killer protein stained with Coomassie blue. M, protein marker. Lanes 1 (1.6 μg) and 2 (0.7 μg) are 10% mercaptoethanol-treated killer protein with and without dialysis, respectively.

FIG. 2

Effect of pH on the killer activity (○) or on the stability (●) of the purified killer protein from S. occidentalis. The change of pH values was adjusted with 0.1 M citrate phosphate buffer. The 100% killer activity is 500 aU, under conditions containing 0.4 μg of killer protein. To determine the optimal pH, the killer protein solution was adjusted to various pH values, and the killer activity of samples was then determined with the assay plate that had identical pH values. For pH stability, after incubation in different pH values at 24°C for 8 h, the pH value of samples was adjusted to a final pH of 4.4, and the residual killer activity was determined. Error bars represent the mean ± the standard deviation of the mean for duplicate samples.

FIG. 3

Effect of temperature on the stability of killer protein from S. occidentalis. The 100% killer activity is 500 aU, under conditions containing 0.4 μg of killer protein. ●, 20°C; ▿, 30°C; ■, 40°C; ◊, 50°C. Error bars represent the mean ± the standard deviation of the mean for duplicate samples.

FIG. 4

N-terminal amino acid sequences of a 4.9-kDa subunit of the S. occidentalis killer protein. Fifteen NH₂-terminal amino acid residues were displayed in a one-letter code. Residues 3 to 14 from the 4.9-kDa subunit were aligned with residues 271 to 282 from the K2 toxin. The solid lines indicate identical residue; the dotted line indicates similar residue.

FIG. 5

Agarose gel electrophoresis of dsDNA plasmids extracted from killer yeasts. Lane 1 (125 ng), K. lactis; lane 2 (215 ng), S. occidentalis; lane 3 (215 ng), the plasmids of S. occidentalis with ExoIII digestion; lanes 4 to 6 (15, 15, and 10 ng, respectively), UV irradiation-cured S. occidentalis isolates.