β-Glucosidase (β-GC) Activity Assay Kit - Microplate Method

Product Information

Product Introduction

β-Glucosidase (β-GC, EC 3.2.1.21) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the hydrolysis of β-glycosidic bonds and has multiple physiological functions.

During cellulose saccharification, β-GC further hydrolyzes cellobiose and cello-oligosaccharides to produce glucose. β-GC also hydrolyzes terpene aroma precursors, converting glycosidically bound forms into free forms and producing aroma. In plants, β-GC can hydrolyze prunasin and release HCN, helping prevent insect feeding.

β-GC decomposes p-nitrophenyl-β-D-glucopyranoside to produce p-nitrophenol, which has a maximum absorption peak at 400 nm. β-GC activity is calculated by measuring the rate of increase in absorbance.

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Transportation and Storage

Instructions for Use

1. Preparation of Crude Enzyme Extract

1.1 Bacteria or Cultured Cells

1. Collect the bacteria or cells into a centrifuge tube.
2. Centrifuge and discard the supernatant.
3. Add Extraction Solution according to the ratio of bacteria or cells (10⁴cells) to Extraction Solution volume (mL) at 500-1000:1. The recommended ratio is 5 million bacteria or cells with 1 mL Extraction Solution.
4. Ultrasonically disrupt the bacteria or cells in an ice bath using 20% power or 200 W, ultrasound for 3 s, interval for 10 s, repeated 30 times.
5. Centrifuge at 15000 g and 4°C for 10 min.
6. Collect the supernatant and keep it on ice for testing.

1.2 Tissue

1. Add Extraction Solution according to the ratio of tissue mass (g) to Extraction Solution volume (mL) at 1:5-10. It is recommended to weigh approximately 0.1 g tissue and add 1 mL Extraction Solution.
2. Homogenize in an ice bath.
3. Centrifuge at 15000 g and 4°C for 10 min.
4. Collect the supernatant and keep it on ice for testing.

1.3 Liquid Samples

Culture medium, serum, plasma, and other liquid samples can be tested directly.

2. Reagent Preparation

Before use, add 12 mL distilled water to each bottle of Reagent I and dissolve thoroughly. Store unused prepared Reagent I at -20°C.

3. Assay Procedure

1. Preheat the microplate reader for at least 30 min.
2. Set the wavelength to 400 nm and zero with distilled water.
3. Add reagents according to the table below.

1. Mix thoroughly and place in a 37°C water bath for exactly 30 min.
2. Immediately place in a 95°C water bath for 5 min. Cap tightly to prevent water loss.
3. Cool under running water and mix thoroughly to ensure the concentration remains unchanged.
4. Centrifuge at 8000 g and 4°C for 5 min.
5. Collect the supernatant and add the following reagents to a 96-well plate.

1. Mix thoroughly and let stand at room temperature for 2 min.
2. Measure the absorbance at 400 nm as A.
3. Calculate ΔA = A_assay- A_control.

Each assay tube requires one control tube.

β-GC Activity Calculation

The regression equation measured under standard conditions is y = 0.0039x - 0.0027, where x is the standard concentration (nmol/mL) and y is the absorbance value.

4.1 Calculation by Liquid Volume

Unit definition: the production of 1 nmol p-nitrophenol per mL sample per minute is defined as one unit of enzyme activity.

β-GC activity (nmol/min/mL) = [(ΔA + 0.0027) ÷ 0.0039 × V_{total reaction}] ÷ V_sample÷ T = 85.47 × (ΔA + 0.0027)

4.2 Calculation by Sample Protein Concentration

Unit definition: the production of 1 nmol p-nitrophenol per mg tissue protein per minute is defined as one unit of enzyme activity.

β-GC activity (nmol/min/mg prot) = [(ΔA + 0.0027) ÷ 0.0039 × V_{total reaction}] ÷ (V_sample× Cpr) ÷ T = 85.47 × (ΔA + 0.0027) ÷ Cpr

4.3 Calculation by Sample Fresh Weight

Unit definition: the production of 1 nmol p-nitrophenol per g tissue per minute is defined as one unit of enzyme activity.

β-GC activity (nmol/min/g, fresh weight) = [(ΔA + 0.0027) ÷ 0.0039 × V_{total reaction}] ÷ (W × V_sample÷ V_{sample total}) ÷ T = 85.47 × (ΔA + 0.0027) ÷ W

4.4 Calculation by Bacterial or Cell Density

Unit definition: the production of 1 nmol p-nitrophenol per 10⁴bacteria or cells per minute is defined as one unit of enzyme activity.

β-GC activity (nmol/min/10⁴cells) = [(ΔA + 0.0027) ÷ 0.0039 × V_{reaction total}] ÷ (500 × V_sample÷ V_{sample total}) ÷ T = 0.171 × (ΔA + 0.0027)

Formula Parameters

Precautions

1. This 100T kit can test 48 samples.