Enzymatic Assay of Cellulase

1. Objective

To standardize an enzymatic assay procedure of cellulase.

2. Scope

This procedure applies to all cellulase and hemicellulase products.

3. Definitions

3.1 Method: Spectrophotometric Stop Rate Determination

3.2 Conditions: T = 37°C, pH = 5.0, Abs340nm, Light Path = 1 cm

3.3 ATP = Adenosine 5’-Triphosphate

3.4 ADP = Adenosine 5’-Diphosphate

3.5 β-NAD = β-Nicotinamide Adenine Dinucleotide, Oxidized Form

3.6 β-NADH = β-Nicotinamide Adenine Dinucleotide, Reduced Form

3.7 G-6PDH = Glucose 6-Phosphate Dehydrogenase

3.8 G-PG = 6-Phospho-D-Gluconate

3.9 Cellulose + H2O Cellulase> D-Glucose

3.10 D-Glucose + ATP Hexokinase> D-Glucose 6-Phosphate + ADP

3.11 D-Glucose 6-Phosphate + β-NAD G-6PDH> 6-PG + β-NADH

3.12 Unit definition: One unit will liberate 1.0 μmole of glucose from cellulose in one hour at pH 5.0 at 37ºC (2 hour incubation time).

3.13 Final assay concentration: In a 5.00 ml reaction mix, the final concentrations are 40 mM sodium acetate, 4% (w/v) Sigmacell and 2-6 units of cellulase.

4. Discussion

N/A

5. Responsibilities

It is the responsibility of trained Analytical Services, New Product Support and Research and Development personnel to follow this procedure as written.

6. Safety

Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 Reagents

Product Number Description
S8625 Sodium Acetate, Trihydrate
H3162 1 N Hydrochloric Acid
S3504 Sigmacell Microcrystalline Type 20
G3293 Glucose (HK) Assay Reagent

 

7.2 Materials/Equipment

Disposable, borosilicate glass, culture tubes
Adjustable volume air-displacement pipettes and tips
Balance
Vortex
Shaker bath
37ºC oven
Spectrophotometer
Spectrophotometer cuvettes
Centrifuge

 

7.3 Related Operating Procedures
N/A

7.4 Solution Preparation

7.4.1   50 mM Sodium Acetate Buffer, pH 5.0 at 37ºC

7.4.1.1    Prepare 200 ml in deionized water using 1.36 g of Sodium Acetate, Trihydrate, Sigma Prod. No. S8625. Adjust to pH 5.0 at 37ºC with 1N HCl, Sigma Prod. No. H3162.

7.4.2   5% (w/v) Sigmacell Solution (Sigmacell)

7.4.2.1    Prepare 100 ml in 50 mM Sodium Acetate Buffer using 5 g of Sigmacell Microcrystalline Type 20, Sigma Prod. No. S3504. Mix and heat gently to make a uniform suspension.

7.4.3   Glucose (HK) Determination Vial

7.4.3.1    Use Glucose (HK) Assay Reagent, Sigma Prod. No. G3293. Dissolve contents per label instructions.

7.4.4   Cellulase Enzyme Solution (Cellulase)

7.4.4.1    Immediately before use, prepare a solution containing 2-6 units/ml of Cellulase in cold deionized water.

7.5 TEST METHOD

7.5.1   Pipette (in milliliters) the following into disposable, borosilicate glass, culture tubes:

Reagent Test Blank
Sigmacell 4.00 4.00

 

7.5.1.1    Equilibrate to 37°C.

7.5.1.2    Then add:

Reagent Test Blank
Cellulase 1.00 ----
Deionized water ---- 1.00

 

7.5.1.3    Immediately mix both the Test and Blank by swirling. Using a shaker bath incubate both the Test and Blank at 37ºC for exactly 120 minutes with moderate shaking.

7.5.1.4    Immediately transfer both the Test and Blank into an ice bath. Allow each to stand until the suspensions have settled. Centrifuge at approximately 4000 rpm for approximately 2 minutes to clarify. Use the supernatant in step 7.5.2.2.

7.5.2    Pipette (in milliliters) the following into suitable cuvettes:

Reagent Test Blank
Glucose (HK) Determination Vial 3.00 3.00

 

7.5.2.1    Equilibrate to 25ºC. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer. Record the initial A340nm for both the Test and Blank.

7.5.2.2    Then add:

Reagent Test Blank
Test Supernatant (step 7.5.1.4) 0.10 ----
Blank Supernatant (step 7.5.1.4) ---- 0.10

 

7.5.2.3    Immediately mix by inversion and record the increase in A340nm until complete (for approximately 5 minutes). Obtain the final A340nm for both the Test and Blank.

7.6 CALCULATIONS

7.6.1    ΔA340nm Test = A340nm Test Final – A340nm Test Initial

7.6.2    ΔA340nm Blank = A340nm Blank Final – A340nm Blank Initial

7.6.3 Units/ml enzyme = (ΔA340nm Test – ΔA340nm Blank) (3.1) (5) (DF)
(6.22) (2) (1) (0.1)

 

7.6.3.1    3.1 = Final volume (in milliliters) of step 7.5.2

7.6.3.2    5 = Total volume (in milliliters) of reaction mix of step 7.5.1

7.6.3.3    DF = Dilution Factor

7.6.3.4    6.22 = Millimolar extinction coefficient of β-NADH at 340nm

7.6.3.5    2 = Conversion factor from 2 hours to 1 hour as per the Unit Definition

7.6.3.6    1 = Volume (in milliliters) of cellulase used in step 7.5.1

7.6.3.7    0.1 = Volume (in milliliters) from step 7.5.1 used in step 7.5.2

7.6.3.8 Units/mg solid = Units/mL enzyme
mg solid/ml enzyme

8. References & Attachments

8.1 Attachment: Report Form

8.2 Reference: Worthington, C.E. (1988) Worthington Enzyme Manual, pp76-79, Worthington Biochemical Corporation, Freehold, NJ

9. Approval

Review, approvals and signatures for this document will be generated electronically using DocCompliance (QUMAS). Please print a “For Use” copy if hardcopy with signature verification is required.

Materials

     
Related Links