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 ?1, 3-glucan laminaripentaohydrolase, which hydolyzes
glucose polymers linked by ?1, 3-bonds and produces laminaripentaose. Zymolyase is reported
to be a complex enzyme of Zymolyase A, ?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: |
?1, 3-glucan laminaripentaohydrolase
|
| Other activities contained: |
| |
Zymolyase?- 20T |
Zymolyase?- 100T |
| ?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°C (for lysis of viable yeast cells)
pH 6.5, 45°C (for hydrolysis of yeast glucan)
|
| Stable pH: |
5~10
|
| Heat stability: |
The lytic activity is lost on incubation at 60°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°C
|
Properties of Zymolyase
Lytic Spectrum by 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°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)
Ordering Information
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