Cellulase (CL) / carboxymethyl cellulase activity assay kit instructions

Cellulase (CL) / carboxymethyl cellulase activity assay kit instructions

Spectrophotometry 50 tubes / 24 samples

Before the formal measurement, it is necessary to take 2-3 samples with large expected differences to make predictions:

CL (EC 3.2.1.4) is a kind of enzyme preparation which can be widely used in medicine, food, cotton spinning, environmental protection and renewable resource utilization, etc. in bacteria, fungi and animals.

Measuring principle:

The content of reducing sugars produced by CL-catalyzed degradation of sodium carboxymethylcellulose was determined by anthrone colorimetry.

Need to bring your own instruments and supplies:

Visible spectrophotometer, water bath, adjustable pipette, 1 mL glass cuvette, mortar, ice, concentrated sulfuric acid and distilled water.

Composition and preparation of reagents:

Extract: liquid 50mL × 1 bottle, stored at 4 ° C; reagent one: liquid 6mL × 1 bottle, stored at 4 ° C; reagent two: liquid 40mL × 1 bottle, stored at 4 ° C;

Reagent 3: powder × 1 bottle, stored at 4 ° C; 5 mL of distilled water and 45 mL of concentrated sulfuric acid before use, fully dissolved for use.

Preparation of sample determination:

1. Bacteria or cultured cells: first collect bacteria or cells into a centrifuge tube, discard the supernatant after centrifugation; according to the number of bacteria or cells

(10 ⁴ ): The volume of the extract (mL) is 500~1000:1 (recommended 5 million bacteria or cells added to 1mL extract), ultrasonically disrupted bacteria or cells (ice bath, power 20% or 200W, ultrasound 3s) , 10s interval, repeated 30 times); 8000g centrifuged at 4 ° C for 10min, take the supernatant, set on ice to be tested.

2. Tissue: According to the tissue quality (g): the volume of the extract (mL) is 1:5~10 (recommended to weigh about 0.1g tissue, add 1mL extract), and homogenize in ice bath. Centrifuge at 8000g for 10min at 4°C, remove the supernatant, and place on ice for testing.

3. Serum (plasma) samples: direct detection.

Determination steps:

1. Preheat the spectrophotometer for more than 30 minutes, adjust the wavelength to 620 nm, and dilute the distilled water.

2. Loading the sample (add the following reagents in the EP tube):

After shaking at 37 ° C for 1 h, the water bath at 90 ° C for 15 min (tighten to prevent water loss), after cooling

Centrifuge at 8000g for 5min at 25°C, take the supernatant, and obtain the saccharification solution.

Mix, 90 °C water bath for 10 min (tighten to prevent water loss), cool, distilled water at 620 nm to zero, determine absorbance

A, Calculate ΔA=A Tube-A control tube. A control tube is provided for each assay tube.