Cellulase (CL) Activity Assay Kit (DNS, Micro Method)

Cat. No.: C1507302

GRADE & PURITY BioReagent

Overview

Cellulase (β-1,4-glucan-4-glucanohydrolase, abbreviated as CES) is a general term for a group of enzymes that degrade cellulose into glucose. It is not a single enzyme but a synergistic multi-component enzyme system, specifically a complex enzyme. It primarily consists of exo-β-glucanase, endo-β-glucanase, and β-glucosidase, and often exhibits high xylanase activity. It acts as a biocatalyst in cellulose decomposition, being a protein capable of breaking down cellulose into oligosaccharides or monosaccharides.

Cellulase is widely distributed in organisms throughout nature. Bacteria, fungi, and animals can all produce cellulase. Cellulase used for industrial production typically comes from fungi, notably from the genera Trichoderma, Aspergillus, and Penicillium. Microbial cellulase is highly significant for converting insoluble cellulose into glucose and for disrupting cell walls in fruit and vegetable juices to increase juice yield. Cellulase has extensive applications in both the food and environmental industries. During alcohol fermentation, adding cellulase can increase raw material utilization and improve liquor quality.

Detection Principle

Using carboxymethyl cellulose sodium salt (CMC) as the substrate, cellulase hydrolyzes it to produce reducing sugars (glucose). Under alkaline and heating conditions, the reducing sugars are oxidized to sugar acids, while 3,5-dinitrosalicylic acid (DNS) is reduced to a brown-red amino compound. Within a certain range, the amount of reducing sugar is proportional to the intensity of the brown-red color. The absorbance of this brown-red product is measured at 540 nm using a microplate reader, allowing the determination of the reducing sugar content and subsequently the cellulase activity. This product is for research use only and not intended for clinical diagnosis or other purposes.

C1507302	Component	100T	Storage
C1507302A	Glu Standard (1 mg/mL)	1.8 mL	2-8℃
C1507302B	CES Assay Buffer	20 mL	RT
C1507302C	CMC Solution	30 mL	RT
C1507302D	DNS Reagent	30 mL	RT. Store in the dark.

Materials Required but Not Provided

1. Deionized water or distilled water

2. Electronic balance, shaker, gauze, centrifuge, centrifuge tubes or test tubes

3. Conical flasks, volumetric flasks, water bath, incubator, microplate reader, 96-well plate

Experimental Procedure

1. Standard Curve Preparation

Take 6 test tubes and set them up according to the table below. Precisely pipette the Glu Standard (1 mg/mL) and mix with CES Assay Buffer to obtain glucose solutions of different concentrations.

Reagent (μL)	Std.0	Std.1	Std.2	Std.3	Std.4	Std.5
Glu Standard (1 mg/mL)	0	50	75	100	125	150
CES Assay buffer	500	450	425	400	375	350
Glucose Std (μg/mL)	0	100	150	200	250	300

Add 0.3 mL of DNS Reagent to each tube, mix well, and place in a boiling water bath for exactly 5 minutes. Cool under running water. Transfer 280 μL from each tube sequentially to a 96-well plate. Using "Std.0" to zero the instrument, measure the absorbance of each tube at 540 nm with the microplate reader.

2. Sample Preparation

Weigh 10 g (or 10 mL) of the sample into a conical flask containing glass beads. Add a certain volume of distilled water for dilution, let stand for 20 minutes, shake at 200 rpm for 30 minutes, then filter through four layers of gauze. Centrifuge the filtrate at 3000 rpm for 10 minutes. Transfer the supernatant to a 50 mL volumetric flask, bring to volume with water – this is the cellulase extraction solution, used for CES activity detection. If the sample enzyme activity is high, dilute to an appropriate concentration before re-testing.

3. Assay Setup

Pre-warm the CMC Solution in a 60°C water bath. Take 2 centrifuge tubes and add reagents as follows:

Reagent (mL)	Control Tube	Test Tube
Enzyme Extract	--	0.1
CES Assay buffer	0.1	0.1
CMC Solution	0.3	0.3

Incubate at 60°C water bath for 20 min.

DNS Reagent	0.3	0.3
Enzyme Extract	0.1	--

Mix immediately, place in a boiling water bath for color development for 5 min, remove immediately, and cool under running water.

4. Measurement

Transfer 280 μL from each tube sequentially to a 96-well plate. Using "Std.0" to zero the instrument, measure the absorbance of each tube at 540 nm with the microplate reader.

5. Calculation

Plot the standard curve using the series of glucose standard concentrations (μg/mL) (tubes 1–5) as the x-axis and the corresponding absorbance values as the y-axis. Calculate the glucose concentrations corresponding to the absorbances of the enzyme extraction solution (Control and Test tubes) based on the standard curve. Then calculate the cellulase activity using the formula below.

Cellulase Activity Unit Definition: One unit of cellulase activity (U) is defined as the amount of enzyme that catalyzes the hydrolysis of carboxymethyl cellulose sodium to produce 1 μg of glucose per minute per mL of enzyme extraction solution under the conditions of 60°C. Calculate the cellulase activity in the sample based on this definition.

U = k × (C₁-C₀) / t OR U = k × (C₁-C₀) × V₀/ (m × t)

Parameter Description:

• U: Enzyme activity of the sample, in μg/(mL·min) or μg/(g·min)

• k: Sample dilution factor

• C_₁: Glucose concentration in the sample test tube, in μg/mL

• C_₀: Glucose concentration in the sample control tube, in μg/mL

• t: Reaction time between enzyme and substrate, in min (=20)

• V_₀: Total volume of enzyme extraction solution for solid samples, in mL

• m: Mass of the weighed sample, in g (=10)

Precautions

1. Avoid repeated freeze-thaw cycles of the Glu Standard to prevent inactivation or decreased efficiency.

2. If the enzyme extraction solution cannot be assayed immediately, store at -20°C; it remains stable for 3 days.

3. If the sample enzyme activity is too high, dilute the enzyme extraction solution with distilled water and re-assay, multiplying the result by the dilution factor.

4. The test sample must not contain cellulase inhibitors, and repeated freeze-thaw cycles should be avoided.

5. The optimum pH for cellulase is generally 4.5–6.5. Gluconolactone effectively inhibits cellulase. Heavy metal ions like copper and mercury also inhibit cellulase, but cysteine can eliminate their inhibition and even further activate cellulase. Plant tissues contain natural cellulase inhibitors, which are phenolic compounds protecting plants from fungal decay. If plant tissues have high oxidase activity, it can oxidize phenolic compounds into quinones, which inhibit cellulase.

6. This kit is also suitable for assaying cellulase activity in other samples, but relevant reference data should be available.

7. If the CES Assay Buffer becomes turbid or develops flocculent matter, discard it.

8. For your safety and health, wear a lab coat and disposable gloves during operation.

9. Use reagents promptly after opening to prevent potential effects on subsequent experimental results.

Appendix

Following the kit instructions at room temperature, measure the absorbances corresponding to glucose standard concentrations of 100, 150, 200, 250, 300 μg/mL at 540 nm using a microplate reader, and plot the standard curve as shown below.

Using a microplate reader at 540 nm, the absorbance of the "Std.0" tube is about 0.12 and appears yellow. Concentrations of 100 μg/mL and above appear brown-red and gradually intensify. Results show that when the glucose concentration exceeds 350 μg/mL, the OD value begins to deviate. Glucose concentrations within 100–300 μg/mL are more suitable. Therefore, if the measured result is significantly high, dilute and re-measure. Deviations may occur due to differences in instruments and operational techniques.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.