Production of a fusion lysozyme enzyme by wine yeasts active against gram-positive and gram-negative spoilage bacteria


Project Number
IWBT 01-09

Project title
Production of a fusion lysozyme enzyme by wine yeasts active against gram-positive and gram-negative spoilage bacteria

Project leader
Du Toit, M A

Institution
University of Stellenbosch. Department of Viticulture and Enology. Institute for Wine Biotechnology

Team members
Lambrechts, M G
Felix-Minnaar, J V
Krieling, S J
Potgieter, N

Project description
The aim of this study is to produce a wine yeast with pasteurisation capabilities. This will be done by expressing the hen egg white lysozyme (HEWL) gene as well as a fusion lysozyme gene, active against gram-positive and gram-negative bacteria, respectively, in wine yeast. This should inhibit the growth of spoilage bacteria in bottled wine and enable winemakers to use lower levels of sulphur dioxide and other chemical preservatives.

Lysozyme is a bacteriolytic enzyme isolated initially by Alexander Fleming in 1922 from human mucousal secretions. It has since been reported in bovine and human milk, and the major commercial source is egg white. Lysozyme has been used in the pharmaceutical industry since 1954 and, additionally, is currently used in cheese. Recent work has suggested its applicability in prevention and/or management of malolactic fermentation (MLF).

Lysozyme is a low molecular-weight protein (15 kDa) with muramidase and chitinolytic activity. It exhibits optimal activity in the pH range of wine where it brings about lysis of the peptidoglycan structure of gram-positive bacteria. The cell wall of gram-positive bacteria consists largely of the heteropolymer peptidoglycan, which is composed of alternating units of N-acetylglucosamine and N-acetylmuramic acid linked via ß-1,4-glycosidic bonds.

Lysozyme brings about hydrolysis of the bacterial cell wall, specifically, the ß-1,4-glycosidic bonds. Activity towards gram-negative species is limited because of the protective outer cell membrane in this group. Lysozyme has no effect on yeasts.

Lysozyme exhibits activity toward all species and strains of lactic acid bacteria (LAB) studied. At the suggested usage level of 250-500 mg/L, the enzyme preparation may be added before alcoholic fermentation to prevent malolactic fermentation or during malolactic fermentation, or upon completion to prevent subsequent activity of unwanted lactic acid bacteria. Its activity is not affected by ethanol, although upon reaction with tannin and pigment complexes, activity decreases. Similarly, bentonite dramatically decreases activity. In white juices, this ranged from 37% reduction after bentonite additions of 0.25 g/L to complete inactivation with additions of 1 g/L. In white wines, the impact of bentonite was less, ranging from 10% reduction with 0.25 g/L to 75% reduction at 1.0 g/L. The effect of sulphur dioxide additions in white wine was also investigated. In this case, sulphur dioxide at 80 mg/L had an indirect effect on lysozyme activity compared with a control that did not receive sulphur dioxide. The decrease in lysozyme activity was attributed to increased prefermentation phenolic extraction by sulphur dioxide. Use of gelatine or potassium caseinate did not affect enzyme activity, whereas silica gel brought about a 35% reduction in activity.

As noted previously, lysozyme complexes are partially inactivated by red wine phenolics and bentonite. In red wines, the use of lysozyme may have an indirect sensory impact on palate structure similar to that of proteinaceous fining agents. In white wines, potential protein instability may result from its utilisation. Thus, treated wines should be evaluated and treated with bentonite as appropriate. Lysozyme has recently been approved by the IV for winemaking purposes.

As mentioned above, lysozyme does not act on gram-negative bacteria including foodborne pathogens. Thus any attempts to convert lysozyme to be active in killing gram-negative bacteria would be an important contribution to the control of gram-negative bacteria in wine spoilage. Another application would be to use this modified lysozyme during the making of active dried wine yeast. During this process the carbon source, molasses, has to be heat treated to inactivate the natural bacteria (gram-positive and gram-negative) present in the molasses. Without this treatment, the yeast fermentations would be heavily contaminated by bacteria. This heat treatment elevates the production costs.

The cell envelope of gram-negative bacteria is a complex structure composed of three morphologically distinct layers, a cytoplasmic membrane, a rigid peptidoglycan layer external to the cytoplasmic membrane and the outer membrane at the surface of the cell. Access of lysozyme to the peptidoglycan layer of the cell wall to perform its action is hindered by the outer membrane permeability barrier. Therefore, in order for lysozyme to bind and fuse into the outer membrane, it was postulated that it may be converted into a membrane-penetrating form if equipped with a hydrophobic carrier. Recently, it was shown that by attaching a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro), which can assume an extended ß-strand configuration and thus provide the same length as a palmitoyl residue, to the C-terminus of lysozyme, this fusion lysozyme was able to fuse into the outer membrane. It was also shown that the fusion lysozyme is a potent bactericidal molecule against gram-negative bacteria in both neutral and acidic pH ranges.

Article
Du Toit, M A, Pretorius, I S. 2000. Microbial spoilage and preservation of wine: Using weapons from nature’s own arsenal – a review, South African Journal of Enology and Viticulture, v. 21 Special issue (p. 74-96)

FinalReport.pdf

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