(Zymolyase® 20T & Zymolyase® 100T)
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).
Zymolyase® vs Lyticase & Glusulase
- Protoplast/spheroplast preparation
- Yeast cell fusion
- Transformation of yeast cells
- Yeast genetics
Karas BJ (2014) Transferring whole genomes from bacteria to yeast spheroplasts using entire bacterial cells to reduce DNA shearing. Nature Protocols 9, 743–750
Rodríguez A (2012) Using Heterologous Expression Systems to Characterize Potassium and Sodium Transport Activities. Methods in Molecular Biology. Volume 913, Part 4, 371-386
Pizzolitto R (2012) Analysis of fumonisin B1 removal by microorganisms in co-occurrence with aflatoxin B1 and the nature of the binding process. International Journal of Food Microbiology. March In Press
Chatterjee A (2011) Saccharomyces cerevisiae THI4p is a suicide thiamine thiazole synthase. Nature. Oct 26;478(7370):542-6.
Park JN (2011) Functional analysis of a Hansenula polymorpha MNN2-2 homologue encoding a putative UDP-N-acetylglucosamine transporter localized in the endoplasmic reticulum. The Journal of Microbiology. Vol. 49, No. 6, pp. 1012-1017
Stringer DK and Piper RC (2011) A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination. The Journal of Cell Biology, vol. 192 no. 2 229-242
Oh J & Nislow C (2011) Signature-tagged Mutagenesis to Characterize Genes Through Competitive Selection of Bar-coded Genome Libraries. Methods in Molecular Biology, 2011, Volume 765, Part 2, 225-252
Walker SC et. al (2011) The Dual
Use of RNA Aptamer Sequences for Affinity Purification and Localization Studies of RNAs and RNA–Protein Complexes. Methods in Molecular Biology, Volume 714, Part 5, 423-444
||Zymolyase® -20T:(NH4)2SO4 precipitation
Zymolyase® -100T:Affinity Chromatography
||Zymolyase® -20T:20,000 units/gram
Zymolyase® -100T:100,000 units/gram
||beta.-1, 3-glucan laminaripentaohydrolase
|Other activities contained:
||Zymolyase® - 20T
||Zymolyase® - 100T
||ca. 1.5 x 106 units/g
||ca. 1.0 x 107 units/g
||ca. 1.0 x 104 units/g
||ca. 1.7 x 104 units/g
||ca. 1.0 x 106 units/g
||ca. 6.0 x 104 units/gf
||Trace amounts of amylase, xylanase, phosphatase.
No DNase, RNase detected
|Optimum pH & temperature:
||pH 7.5, 35oC (for lysis of viable yeast cells)
pH 6.5, 45oC (for hydrolysis of yeast glucan)
||The lytic activity is lost on incubation at 60oC for 5 minutes.
|Specificity (lytic spectrum)(5):
||Ashbya, Candida, Debaryomyces, Eremothecium, Endomyces, Hansenula, Hanseniaspora, Kloeckera, Kluyveromyces, Lipomyces, Metschikowia, Pichia, Pullularia, Torulopsis, Saccharomyces, Saccharomycopsis, Saccharomycodes, Schwanniomyces, etc.
||SH compound such as cystein, 2-mercaptoethanol of dithiothreitol
||No loss of activity was found after storage for 1 year at 4oC
Electron Microscope Images of Yeast Cell:
CW: Cell Wall CM: Cell Membrane M: Mitochondria Mb: Microbody N: Nucleus V: Vacuole
Data courtesy of Professor Masako Osumi, Nippon Women's University
Properties of Zymolyase®:
Lytic Spectrum of Zymolyase®
1) Susceptible strains in low concentration (0.2 units/ml)
Ashbya, Endomyces, Kloeckera, Kluyveromyces, Pullularia, Saccharomyces
2) Susceptible strains in high concentration (2.0 units/ml)
Candida, Debaryomyces, Eremothecium, Hansenula, Hanseniaspora, Lipomyces, Metschikowia, Saccharomycopsis, Saccharomycodes, Schizosaccahromyces, Selenozyma, Trigonopsis,Wickerhamia
3) Susceptibility depending on strains
Bretanomyces, Cryptococcus, Nadsonia, Pichia, Rodosporidium, Schwanniomyces, Stephnoascus, Torulopsis
4) No susceptible strains
Bullera, Pityrosporum, Rhosotorula, Sporidiobolus, Sporobolomyces, Stetigmatomyces, Trichosporon
Assay for Enzyme Activity:
One unit of lytic activity is defined as that amount which indicates 30% of decrease in absorbance at 800 nm (A800) of the reaction mixture under the following condition. Reaction
||1ml - 0.05-0.1 mg/ml for Zymolyase® - 20T
1ml - 0.012-0.024 mg/ml for Zymolyase® - 100T
||3ml - Brewer's yeast cell suspension (2 mg dry weight/ml)
||5ml - M/15 Phosphate buffer, pH 7.5
After incubation for 2 hours at 25oC with gentle shaking, A800 of the mixture is determined. As a reference, 1 ml of distilled water is used instead of enzyme solution.
Percentage decrease in A800 = (A800 of reference - A800 of reaction mixture) x 100 / initial A800 of reference. 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® - 100T lyses 3 mg dry weight of brewer's yeast.
Precautions on Use:
1) Avoid using nitrocellulose filters and use of material other than nitrocellulose, when sterilizing. Zymolyase® may be adsorbed on nitrocellulose membranes.
2) Zymolyase®, especially Zymolyase® -100T, may not be completely dissolved in buffers. Use Zymolyase® as suspension.
3) When sterilized, Zymolyase® is used in a concentration higher than 0.05%, prepare 2% Zymolyase® solution in buffers containing 5% glucose, filter the suspension
and dilute the solution with the appropriate buffer.
1) Kaneko, T., Kitamura, K and Yamamoto,Y.: J. Gen. Appl.Microbiol. 15, 317 (1969)
2) Kitamura, K., Kaneko, T. and Yamamoto,Y.: Arch. Biochem. Biophys., 145, 402 (1971)
3) Kitamura, K., Kaneko, T. and Yamamoto,Y.: J. Hen. Appl.Microbiol., 18, 57 (1972)
4) Kitamura, K. and Yamamoto,Y.: Arch. Biochem. Biophys., 153, 403 (1972)
5) Kaneko, T., Kitamura, K. and Yamamoto,Y.: Agric. Biol. Chem., 37, 2295 (1973)
6) Kitamura, K., Kaneko, T. and Yamamoto,Y.: J. Gen Appl.Microbiol., 20, 323 (1974)
7) Kitamura, K. and Yamamoto,.: Agric. Biol. Chem., 45, 1761 (1981)
8) Katamura, K. and Tanabe, K.: Agric, Biol. Chem., 46, 553 (1982)
9) Katamura, K.: J. Ferment. Technol., 60, 257 (1982)
10) Kitamura, K.: Agric. Biol. Chem., 46, 963 (1982)
11) Kitamura, K.: Agric. Biol. Chem., 46, 2093 (1982)
12) Calza R. E., Schroeder A. L.: J. Ben.Microbiol., 129, 413 (1983)
13) Iizuka Masaru, Torii Yasuhiko,Yamamoto Takehiko: Agric. biol. Chem., 47 (12), 2267 (1983)
14) Shibata Nobuyuki, Kobayashi Hidemitsu, tojo Menehiro, Suzuki Shigeo: Arch. Biochem. Biophys., 251 (2), 697 (1986)
15) Iijima Y.,Yanagi S. O.: Agric. biol. CHem., 50 (7), 1855 (1986)
16) Herrero Enrique, Sanz Pascual. Sentandreu Rafael: J. Gen.Microbiol., 133 (10), 2895 (1987)