Zymolyase®, produced by a submerged culture of Arthrobacter luteus(1), has strong lytic activity against living yeast cell walls(2),(3) to produce protoplast or spheroplast of various strains of yeast cells. An essential enzyme for the lytic activity of Zymolyase® is beta.-1,3-glucan laminaripentaohydrolase. It hydrolyzes linear glucose polymers at beta.-1,3-linkages and releases specifically laminaripentaose as the main and minimum product unit(4), (5), (10), (11).
There are two preparations of Zymolyase®, Zymolyase®-20T and Zymolyase®-100T, having lytic activity of 20,000 units/g and 100,000 units/g respectively. Zymolyase®-20T is ammonium sulfate precipitate while Zymolyase®-100T is a further purified preparation by affinity chromatography(9). Lytic activity varies depending on yeast strain, growth stage of yeast, or cultural conditions(6-8). Further information related to Zymolyase® can be obtained in the reference section below(12-16).
CW: Cell Wall CM: Cell Membrane M: Mitochondria Mb: Microbody N: Nucleus V: Vacuole
|Purification||Ammonium Sulfate Precipitation||Affinity Chromatography|
|Activity||20,000 units/g||100,000 units/g|
|Essential enzyme||Beta-1,3-glucan laminaripentaohydrolase|
|Other activities contained *1||Beta-1,3-glucanase||ca. 1.5 x 106 units/g||ca. 1.0 x 107 units/g|
|Protease||ca. 1.0 x 104 units/g||ca. 1.7 x 104 units/g|
|Mannanase||ca. 1.0 x 106 units/g||ca. 6.0 x 104 units/g|
|Contaminants||Amylase, Xylanase, Phosphatase||Trace amount||Non detectable|
|Optimum pH & Temperature||pH 7.5, 35°C (for lysis of viable yeast cells)
pH 6.5, 45°C (for hydrolysis of yeast glucan)
|Stability||4°C||No loss of activity was found after storage for 1 year|
|Heat stability||30°C||70% of lytic activity is lost after storage for 3 months||90% of lytic activity is lost after storage for 3 months|
|60°C||Lytic activity is lost on incubation for 5 minutes|
|Specificity (Lytic Spectrum)||Ashbya, Candida, Debaryomyces, Eremothecium, Endomyces, Hansenula, Hanseniaspora, Kloeckera, kluyveromyces, Lipomyces, Metschnikowia, Pichia, Pullularia, Torulopsis, Saccharomyces, Saccharomycopsis, Saccharomycodes, Schwanniomyces, etc.|
|Activators||Cystein, 2-mercaptoethanol, Dithiothreitol etc.|
*1 See reference, Kitamura, K., Kaneko, T. and Yamamoto, Y.: J. Gen. Appl. Microbiol., 18, 57 (1972) as to the definition of each enzyme units.
|Enzyme solution||1 ml (0.05-0.1 mg/ml for Zymolyase®-20T)
1 ml (0.012-0.024 mg/ml for Zymolyase®-100T)
|Brewer's yeast cell suspension||3 ml (2 mg/ml)|
|M/15 Phosphate buffer, pH 7.5||5 ml|
|Distilled water||1 ml|
After incubation for 2 hours at 25°C with gentle shaking, A800 of the mixture is determined. When 60% of A800 decrease, equivalent to 2 units, is observed in the reaction system, the brewer's yeast cells are completely lysed, namely 1 unit of Zymolyase® lyses 3 mg dry weight of brewer's yeast.