What is zymolyase used for?

What is zymolyase used for?

Zymolyase (also known as lyticase) is an enzyme mixture used to degrade the cell wall of yeast and form spheroplasts. Essential activities of zymolyase include β-1,3-glucan laminaripentao-hydrolase activity and β-1,3-glucanase activity. A common source of zymolyase is the Actinobacteria Arthrobacter luteus.

How do you make zymolyase?

Mix thoroughly 50mg of Zymolyase powder with 2275 mg of sorbitol (molecular biology grade). Aliquot 46.5 mg of this mixture in individual eppendorf tubes (make sure you tare each time) and store at 4°C. Before use: resuspend in 500 ul water to obtain a solution of 2mg/ml Zymolyase, 0.5M Sorbitol.

How do you make Lyticase solution?

Preparation Instructions

1. Pipette 1.33 mL of 1 M Potassium phosphate monobasic solution (Catalog Number P8709) into a suitable beaker.
2. Add 5.37 mL of 1 M Potassium phosphate dibasic solution (Catalog Number P8584).
3. Dilute to 100 mL with ultrapure water.
4. Adjust to pH 7.5 with 1 M KOH.

What is yeast cell wall?

The yeast cell wall is a dynamic organelle that determines the cell shape and integrity of the organism during growth and cell division. It must provide the cell with mechanical strength in order to withstand changes in osmotic pressure imposed by the environment (Gibson et al. 2007; Dague et al. 2010).

What are Spheroplast cells?

A spheroplast (or sphaeroplast in British usage) is a microbial cell from which the cell wall has been almost completely removed, as by the action of penicillin or lysozyme. According to some definitions, the term is used to describe Gram-negative bacteria.

How do you lyse a yeast cell?

For efficient cell lysis use 2ml microcentrifuge tubes or other tubes with near flat bottom (in 1.5 ml tubes with sharp conic bottom the lysis is much less efficient). To avoid heating the samples, disrupt cell during 30-60 sec with 20-30 sec pauses on ice.

How do you dilute Lyticase?

Procedure

1. Dilute stock of lyticase to 50 U/mL in TE.
2. Aliquot lyticase in 50uL quantities.
3. Pick colonies (I use a pipette tip) and add to lyticase aliquots, pipette up and down or agitate to break up colony.
4. Incubate at 37°C for 30 min.
5. Incubate at 95°C for 10 min.

Is yeast cell wall made of chitin?

The fibrous component of yeast cell walls usually consists of β-glucan and/or chitin. N-glycosylated proteins form an amorphous, cross-linking matrix as well as fibres on the outer surfaces of the walls.

How does yeast reproduce?

Most yeasts reproduce asexually by budding: a small bump protrudes from a parent cell, enlarges, matures, and detaches. A few yeasts reproduce by fission, the parent cell dividing into two equal cells. Torula is a genus of wild yeasts that are imperfect, never forming sexual spores.

How do you make a Spheroplast?

Spheroplasts are prepared by resuspending the pelleted cells in 50mL of 20mM potassium phosphate buffer (pH 7.4), containing 20% glycerol, 1mM PMSF, and 10mM β-mercaptoethanol.

What is the difference between a Spheroplast and a protoplast?

Both protoplasts and spheroplasts refer to altered forms of plant, bacterial or fungal cells from which the cell wall has been partially or completely removed. Protoplasts are bounded by a single membrane while spheroplasts have two – an inner membrane and an outer membrane.

How do you break yeast?

Breaking yeasts is easy using glass beads (Ballotini, SIGMA). It can be done manually in a vortex or using a Fast-prep machine. If done manually you should use glass tubes. Keep temperature as low as you can by maintaining samples on ice between breaking pulses.

What is zymolyase 20T made of?

Zymolyase-20T is an enzyme preparation from a submerged culture of Arthrobacter luteus which effectively lyses cell walls of viable yeast cells. This preparation is a lyophilized powder prepared by salting out from the culture fluid with ammonium sulfate and contains 20,000 units enzyme/g. …

How is the zymolyase from Zymo Research prepared?

The Zymolyase from Zymo Research is prepared from Arthrobacter luteus, lyophilized, and packaged with a resuspension buffer. The buffer… …Zymoprep-96 is a simple and efficient yeast plasmid Miniprep kit that is based on the classic E. coli alkaline lysis method with our Zymolyase added in the first solution.

What is zymolyase 100T used for in yeast?

Applications Zymolyase 100T is used to lyse living yeast cell walls to produce protoplast or spheroplast of various strains of yeast cells. It may also be used for glucan hydrolysis. Product Description Zymolyase-100T is an enzyme preparation from a submerged culture of Arthrobacter…

What is the difference between zymolyase-100t and zymolite lb?

Product Description Zymolyase-100T is an enzyme preparation from a submerged culture of Arthrobacter… …LB™ is a proprietary improvement on the Zymolyase® based spheroplast preparation and extraction of soluble proteins from yeast cells.