Overview
Zymolyase®, purified from culture fluid of Arthrobacter luteus, has strong
lytic activity against living yeast cell walls to produce protoplast or
spheroplast of various strains of yeast cells. Essential enzyme lytic activity
of Zymolyase® is beta.-1, 3-glucan laminaripentaohydrolase, which hydolyzes glucose
polymers linked by beta.-1, 3-bonds and produces laminaripentaose. Zymolyase® is
reported to be a complex enzyme of Zymolyase® A, beta.-1, 3-glucan
laminaripentaohydrolase and Zymolyase® B, alkaline protease, which may change the
structure of the yeast cell wall to facilitate penetration of Zymolyase® A.
Zymolyase® A alone was unable to lyse yeast cell walls. There are two
preparations of Zymolyase®, Zymolyase® - 20T and 100T, having lytic activity of
20,000 units/gram and 100,000 units/gram respectively. Zymolyase® - 20T is
ammonium sulphate precipitate while Zymolyase® 100T is a further purified
preparation by affinity chromatography. Lytic activity varies depending on
strains, fermentation conditions and growth phases of yeast substrate.
Product Specifications
|
Form: |
Lyophilized powder |
|
Purification: |
Zymolyase® -20T: (NH4)2SO4
precipitation
Zymolyase® -100T: Affinity
Chromatography
|
|
Activity: |
Zymolyase® -20T: 20,000
units/gram
Zymolyase® -100T: 100,000
units/gram
|
|
Essential enzyme: |
beta.-1, 3-glucan laminaripentaohydrolase
|
|
Other activities contained: |
|
|
Zymolyase® - 20T |
Zymolyase® - 100T |
|
B-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/gf |
|
|
Contaminants: |
Trace amounts of amylase, xylanase, phosphatase.
No DNase, RNase detected
|
|
Optimum pH & temperature: |
pH 7.5, 35 deg. C (for lysis of viable yeast cells)
pH 6.5, 45 deg. C (for hydrolysis of yeast glucan)
|
|
Stable pH: |
5~10
|
|
Heat stability: |
The lytic activity is lost on incubation at 60 deg. C 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.
|
|
Activators: |
SH compound such as cystein, 2-mercaptoethanol of dithiothreitol
|
|
Stability: |
No loss of activity was found after storage for 1 year at 4 deg. C
|
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
Unit Definition
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
Reaction mixture
|
Enzyme Solution: |
0.05 - 0.1 mg/ml for Zymolyase® - 20T |
1ml |
|
|
0.012 - 0.024 mg/ml for Zymolyase® - 100T |
|
|
Substrate: |
Brewer's yeast cell suspension (2 mg dry weight/ml) |
3 ml |
|
Buffer: |
M/15 Phosphate buffer, pH 7.5 |
1 ml |
|
Distilled Water: |
|
1 ml |
Procedure
After incubation for 2 hours at 25 deg. C with gentle shaking, A800 of the mixture
is determined. As a reference, 1 ml of distilled water is used instead of enzyme
solution.
Calculation
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.
Applications
- Protoplast/spheroplast preparation
- Yeast cell fusion
- Transformation of yeast cells
- Yeast genetics
Product List
References
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)