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. 2020 Dec 28;9(1):56.
doi: 10.3390/microorganisms9010056.

Purification and Characterization of WA18, a New Mycocin Produced by Wickerhamomyces anomalus Active in Wine Against Brettanomyces bruxellensis Spoilage Yeasts

Francesca Comitini  1 , Alice Agarbati  1 , Laura Canonico  1 , Edoardo Galli  1 , Maurizio Ciani  1
Affiliations

Purification and Characterization of WA18, a New Mycocin Produced by Wickerhamomyces anomalus Active in Wine Against Brettanomyces bruxellensis Spoilage Yeasts

Francesca Comitini et al. Microorganisms. .

Abstract

Wickerhamomyces anomalus strain 18, isolated from a natural underground cheese ripening pit, secretes a mycocin named WA18 that inhibits wine spoilage yeasts belonging to Brettanomyces bruxellensis species, with a broad-spectrum of activity. WA18 was purified, and the purified protein was digested with specific restriction enzymes (lysine K and arginine R cut sites). The LC-MS and LC-MS/MS analysis after enzymatic digestions revealed a molecular weight of 31 kDa. Bioinformatics processing and database research of digested pure killer protein showed 99% identity with a UDP-glycosyltransferase protein. Competitive inhibition assay of killer activity by cell-wall polysaccharides suggests that branched glucans represent the first receptor site of the toxin on the envelope of the sensitive target. The WA18 partially purified crude extract (PPCE) showed high stability of antimicrobial activity at the physicochemical conditions suitable for the winemaking process. Indeed, in wine WA18 was able to counteract B. bruxellensis and control the production of ethyl phenols. In addition, the strain WA18 was compatible with Saccharomyces cerevisiae in co-culture conditions with a potential application together with commercial starter cultures. These data suggest that WA18 mycocin is a promising biocontrol agent against spoilage yeasts in winemaking, particularly during wine storage.

Keywords: Wickerhamomyces anomalus; bioactive yeasts; biocontrol; killer toxin; killer yeasts; spoilage yeasts.

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Conflict of interest statement

The authors do not have financial and/or personal conflicts of interest regarding the study reported in the present manuscript.

Figures

Figure 1
Figure 1
Correlation between W. anomalus strain 18 growth evaluated by measuring optical density O.D. 600 nm (line) and production of mycocin (bars) expressed as the diameter of the halo produced against sensitive strain B. bruxellensis DiSVA 46. Results were obtained from two experiments carried out separately, and relative standard errors were added.
Figure 2
Figure 2
Fractions eluted using SP Sepharose-cation exchange chromatography. Circled peak corresponds to the active fractions (with killer activity).
Figure 3
Figure 3
SDS Page of purified WA18 mycocin. MK marker; (a) purified fraction; (b) SP Sepharose-cation exchange chromatography active fractions.
Figure 4
Figure 4
Signal peptide analysis graphic of consensus amino acid sequence of digested WA18 purified mycocin.
See this image and copyright information in PMC

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