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Protocol for Z1000 Zymolyase 20T
(Lyticase, Yeast Lytic Enzyme)









Synonyms: Lyticase*, Yeast Lytic Enzyme

Essential Enzyme: b-1,3-glucan laminaripentaohydrolase

Other activities contained:

b-1,3-glucanase: ~1.5x10e6u/g

Protease:
~1.5x10e4u/g

Mannanase:
~1.5x10e6u/g

Amylase, xylanase, phosphatase:
Minute amounts

DNase, Rnase
: None detected

(See reference No. 3 for the definition of each enzyme unit. Activity varies slightly between lots.)


Optimum pH:


For cell wall lysis:
7.5, 35°C

For yeast glucan hydrolysis:
6.5, 45°C

Stable pH:
5-10

Heat Stability:
The lytic activity is lost on incubation at 60°C for 5 minutes.
 

Properties of Zymolyase:

Specificity (lytic spectrum)(5):
Ashbya, Candida, Debaryomyces, Eremothecium, Endomyces, Hansenula, Hanseniaspora, Kloekera, Kluyveromyces, Lipomyces, Metschikowia, Pichia, Pullularia, Torulopsis, Saccharomyces, Saccharomycopsis, Saccharomycodes, Schwanniomyces, etc

Susceptible strains in low concentration (0.2 units/ml):

Ashbya, Endomyces, Kloekera, Kluyveromyces, Pullularia, Saccharomyces

Susceptible strains in high concentration (2.0 units/ml):

Candida, Debaryomyces, Eremothecium, Hansenula, Hanseniaspora, Lipomyces, Metschnikowia, Saccharomycopsis, Saccharomycodes, Schizosaccharomyces, Selenozyma, Trigonopsis, Wickerhamia

Susceptibility depending upon strains:

Bretanomyces, Cryptococcus, Nadsonia, Picia, Rodosporidium, Schwanniomyces, Stephnoascus, Torulopsis

No susceptible strains:

Bullera, Pityosporum, Rhosotorula, Sporidiobolus, Sporobolomyces, Stetigmatomyces, Trichosporon

 

Activators: SH compound such as cysteine, 2-mercaptoethanol or dithiothreitol


* Lyticase is similar but testing has shown that Zymolyase exhibits greater lytic activity.


Experimental Precautions:

1. Avoid nitrocellulose filters due to non-specific adsorption onto the membrane.

2. If using sterilized Zymolyase in concentrations higher than 0.05%, prepare a 2% solution in buffers containing 5% glucose. Filter the suspension and then dilute with an appropriate buffer.


Recommended Reconstitution Buffers:

Buffer A:      0.06M phosphate buffer, pH 7.5

 

Buffer B:   1M sorbitol
10mM magnesium chloride
2mM dithiothreitol
50mM potassium phosphate, pH 7.8
100ug/ml phenylmethylsulfonyl fluoride


 

Assay for Enzymatic Activity:

Unit Definition: One unit of lytic activity is defined as that amount which indicates 30% decrease in absorbance at 800 mm (A800) of the reaction mixture under the following condition.

Reaction Mixture:

Enzyme (0.05-01mg/ml solution): 1ml
Substrate [Brewer's yeast cell
suspension (2mg dry weight/ml)]:   
 
3ml
Buffer (M/15 Phosphate buffer, pH 7.5):    5ml
Distilled water: 1ml
Total Volume: 10ml


 

Procedure

Incubation for 2 hours at 25ºC with gentle shaking
Determine A800 of the mixture. As a reference, 1ml of distilled water is used instead of enzyme solution.


Calculation

% 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 3mg dry weight of brewer's yeast.

Transformation of Saccharomyces cerevisiae by Protoplast Method:

Materials:

  1. 1M Sorbitol, 25mM EDTA, pH 8, 50mM DTT. The sorbitol/EDTA is prepared separately and sterilized.
Filter Sterilized 1M DTT stock solution (Molecular Biological Grade) is prepared and frozen in 500ul aliquots. The DTT is added to 9.5ml of sorbitol/EDTA just prior to use.

 2. 1M Sorbitol: Sterilized by autoclaving

 3. For Zymolyase: 1M Sorbitol, 1mM EDTA, 10mM sodium citrate buffer, pH 5.8. Dissolve Zymolyase in dH2O at 60u/ml
                                  
 4. 1M Sorbitol, 10mM Tris, pH 7.5, 10mM CaCl2

  5. 20% PEG, 10mM Tris, pH 7.5, 10mM CaCl2 (prepared fresh).

  6. Leu-selective agar plates: 1% glucose, 0.67% yeast nitrogen base, 1M sorbitol.

  7. Leu-selective top agar tubes: 10ml of 1% glucose, 0.67% yeast nitrogen base, 1M sorbitol in a large foam stoppered test tube. Melt and store at 47°C when using.
dH2O: Sterile, Molecular Biological Grade

  8. 5% SDS in dH2O.


Protocol:

  1. Streak cryopreserved yeast on a YPD agar plate (1% yeast extract, 2% peptone, 2% dextrose, 2% agar) and incubate for 24-48 hours at 30°C.

  2. In a 500ml flask, inoculate 100ml YPD broth from an isolated colony. Incubate overnight at 30°C with shaking (250-300rpm).

  3. Measure the OD520 of the yeast culture the following morning using sterile YPD to zero the spectrophotometer. If necessary, dilute the yeast in YPD broth in order to attain a readable OD between 0.1-1. If the yeast OD is between 0.2-0.3, harvest the cells as described below. Alternatively, dilute the yeast to an OD of 0.2 and continue culturing. When the culture OD reaches 0.3, harvest the cells by centrifuging at RT for 10 minutes at 1500xg. Pour off the supernatant and save the yeast pellet.

  4. Thoroughly resuspend the yeast pellet in 10ml of sterile dH2O. Transfer the cells to a sterile 15ml screw capped tube.

  5. Centrifuge the yeast at 1500xg for 5 minutes at RT. Decant the supernatant and save the yeast pellet.

  6. Resuspend the yeast in 10ml freshly prepared 1M sorbitol. Pellet the yeast by centrifuging at 1500xg for 5 minutes.
 
  7. Resuspend the yeast in 10ml freshly prepared 1M sorbitol. Pellet the yeast by centrifuging at 1500xg for 5 minutes.

  8. Resuspend the cells in 10ml of sorbitol/citrate buffer (1M sorbitol, 1mM EDTA, 10mM sodium citrate buffer, pH 5.8). To assess the time required and efficiency of protoplast formation, transfer 1.6ml of the cells to a small tube and add 2.5ul of Zymolyase. Proceed to step 9. Save the remaining cells.

  9. Following the addition of cell wall lytic enzyme, incubate the cells at 30°C. Remove 200ul aliquots of treated cells at 0, 5, 10, 20, 30, 40, 50, and 60 minutes. Add the aliquots to 2ml 1M sorbitol. Add 800ul 5% SDS. Mix. Measure cell lysis by culture clearing at OD800. The spectrophotometer should be blanked against 1M sorbitol.

  10. The efficiency and time required for protoplast formation can be determined by plotting OD800 of the treated cells vs time. To calculate the efficiency, divide the OD800 at each time point by the starting OD800. Then multiply by 100. A protoplast efficiency of 70% is needed for transformation.

  11. To protoplast the remaining yeast, add 13ul Zymolyase to the respective tubes and incubate for the determined time to reach 70% protoplast efficiency. Handle the protoplast carefully because they are fragile and will easily rupture.

  12. Centrifuge the protoplasts at 750xg for 10 minutes at RT. Decant and save the pellet.

  13. Gently resuspend the protoplasts in 1ml of 1M sorbitol. Centrifuge at 750xg for 10 minutes at RT.

  14. Resuspend the protoplasts in 5ml of 1M sorbital, 10mM Tris, pH 7.5, 10mM CaCl2. Centrifuge at 750xg for 10 minutes at RT. Decant supernatant; save the cell pellet. Resuspend the cells in 300ul 1M sorbitol, 10mM Tris, pH 7.5, 10mM CaCl2.

  15. For each transformation, aliquot 100ul of the protoplasts into sterile 15ml tubes. Add 10ug of YEp351/A/M control plasmid and incubate at RT for 10 minutes.

  16. Add 1ml of fresh 20% PEG, 10mM Tris, pH 7.5, 10mM CaCl2 to the protoplasts. Mix gently. Incubate at RT for 10 minutes.

  17. Centrifuge the protoplasts at 750xg for 10 minutes at RT. Carefully decant the supernatant. Invert the tube and drain excess solution.

  18. Gently resuspend the protoplasts in 1ml of 1M sorbitol.

  19. Add 100ul of protoplasts to 10ml of molten top agar and pour onto the selective agar plates. Allow the top agar to solidify.

  20. Invert plates and incubate at 30°C. Transformed cells should appear in 4-6 days.