Enzymatic Assay of Lyticase

Description

This procedure may be used for the determination of Lyticase activity using Baker’s yeast as the substrate. The turbidimetric rate determination [Absorbance at 800 nm (A800), Light path = 1 cm] is based on the following reaction:


Unit Definition: One unit of lyticase will produce a ΔA800 of 0.001 per minute at pH 7.5 at 25 °C, using a suspension of yeast as the substrate in a 3 ml reaction mixture.

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

Potassium phosphate, monobasic (Catalog No. P5379)
Yeast from Saccharomyces cerevisiae (Catalog No. YSC1)

Preparation Instructions

Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.

Phosphate Buffer (67 mM Potassium Phosphate Buffer, pH 7.5, at 25 °C) – Prepare a 9.1 mg/ml solution of potassium phosphate, monobasic (Catalog No. P5379) in ultrapure water. Adjust the pH to 7.5 at 25 °C with 1 M KOH.

Substrate [0.4% (w/v) suspension of baker’s yeast (yeast from Saccharomyces cerevisiae)] –

  • Using a mortar and pestle grind a suitable amount of yeast (Catalog No. YSC1) to obtain greater than 800 mg of powder. The ground powder should be between 25–30 mesh size.
  • Using the powder, prepare a 4 mg/ml yeast solution in ultrapure water.
  • Allow the solution to stir at 300 rpm for ~2 hours at room temperature.
    Note: Stirring at higher speeds could cause premature lysing of the yeast cells.
  • After 2 hours, cease stirring to allow the larger sediments to settle. Carefully decant the upper two thirds of the supernatant into a new beaker. Removing the larger sediments will minimize the amount of noise seen in the spectrophotometer readings when performing the assay.
  • The substrate concentration must be verified prior to running the assay. Measure the A800 of the yeast mixture versus ultrapure water. The absorbance of the yeast mixture versus ultrapure water must be between 0.6–1.0. If necessary, adjust the absorbance using appropriate amount of yeast powder or ultrapure water.
  • Mix the Substrate at 300 rpm at room temperature throughout the assay. It is stable for up to 8 hours.

Enzyme Solution – Immediately before use, prepare a solution containing 500 units/ml of Lyticase in cold ultrapure water.
Note: For some crude lyticase products it may take more than 15 minutes to go into solution. Short sonication periods of 1–15 seconds can aid in dissolving the enzyme without affecting the enzymatic activity.

Procedure

In a 3.00 ml reaction mixture, the final concentrations are 34 mM Potassium Phosphate, ~0.12% (w/v) baker’s yeast, and 25–35 units of Lyticase.

1. Pipette the following reagents into suitable containers:

Reagent Test 1
(ml)
Test 2
(ml)
Test 3
(ml)
Blank
(ml)
Ultrapure Water 0.55 0.54 0.53 0.60
Phosphate Buffer 1.50 1.50 1.50 1.50
Substrate 0.90 0.90 0.90 0.90


2. Mix the tests and the blank by inversion and equilibrate to 25 °C for 5 minutes. Then add the Enzyme Solution:

Reagent Test 1
(ml)
Test 2
(ml)
Test 3
(ml)
Blank
(ml)
Enzyme Solution 0.050 0.060 0.070

Note: Do not deviate from the enzyme volumes listed.


3. Immediately mix the blank and all tests by inversion and record the decrease in absorbance at A800 for ~10 minutes. It is important to mix all cuvettes immediately and sequentially to capture the settling effects of the substrate.


4. Using a 2 minute time period, obtain the ΔA800/min using the maximum linear rate for each Test. Each Test rate needs to be corrected using the corresponding time frame for the Blank. The corrected ΔA800/min must fall between 0.025 and 0.035 in order for the assay to be valid. If the rate falls outside this range, make the appropriate adjustments to the enzyme concentration and repeat.

Results

Calculations

1.

Units/ml enzyme = (ΔA800/min Test – ΔA800/min Blank) (df)
(0.001) (Ve)

where:
df = dilution factor of enzyme
0.001 = Change in absorbance at 800 nm as per the unit definition
Ve = Volume (ml) of Enzyme Solution used in reaction


2.

Units/mg solid = units/ml enzyme
mg solid/ml enzyme

Materials

     

03/14-1

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