α-Amylase(α-AL) Activity Assay Kit

The α-Amylase (α-AL) Activity Assay Kit is designed for the quantitative detection of α-amylase enzyme activity in plant or biological tissue samples. α-Amylase (EC 3.2.1.1) randomly hydrolyzes α-1,4-glycosidic bonds in starch, yielding reducing sugars such as glucose, maltose, maltotriose, and dextrin, while reducing starch viscosity. This enzyme is commonly referred to as a liquefying enzyme due to this property.

This kit uses a 3,5-dinitrosalicylic acid (DNS) colorimetric method to measure reducing sugars generated from enzymatic hydrolysis. The red-brown reaction product exhibits strong absorbance at 540 nm, providing a reliable measure of α-amylase activity.

Key Features

• ✅ Specific for α-Amylase: Heat treatment at 70°C selectively inactivates β-amylase, enabling precise measurement of α-amylase activity.
• ✅ Quantitative and Reproducible: Uses a glucose standard curve for accurate enzyme activity calculation.
• ✅ Convenient Format: Supplied with ready-to-use and reconstitutable reagents, including standard glucose powder.
• ✅ Flexible Normalization: Activity can be expressed per gram of fresh sample or per milligram of protein.
• ✅ Reliable and Cited: Referenced in peer-reviewed publications for plant physiology and post-harvest studies.

Kit Components

Required Materials (User Supplied)

• Spectrophotometer (set to 540 nm)
• Water bath (40°C, 70°C, and 90°C)
• Desk centrifuge
• Adjustable pipettes
• Mortar or homogenizer
• 1 mL cuvettes
• Distilled water

Assay Procedure Overview

1. Sample Extraction
   • Homogenize 0.1 g sample in 0.8 mL distilled water
   • Extract for 15 min at room temp
   • Centrifuge at 6000 ×g for 10 min
   • Dilute supernatant to 10 mL – this is the stock enzyme solution
2. Glucose Standard Curve
   • Prepare a series of dilutions: 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 mg/mL
3. Reaction Setup
   • Add 250 μL enzyme solution to test and control tubes (control sample is boiled)
   • Incubate samples at 70°C for 15 min (inactivates β-amylase)
   • Add Reagent II (only to test tube), then incubate at 40°C for 5 min
   • Add Reagent I (all tubes), followed by a 90°C bath for 10 min
   • Read absorbance at 540 nm
4. Calculation
   • Construct a standard curve: ΔA(S) vs glucose concentration
   • Determine glucose concentration (x) from ΔA(T)
   • Calculate α-amylase activity:
     • U/min/g fresh weight = (2 × x) ÷ W
     • U/min/mg protein = (0.2 × x) ÷ Cpr

Definitions:

W = sample weight (g)

Cpr = protein concentration (mg/mL)

Vs = sample volume in reaction (0.25 mL)

T = reaction time (5 min)

Performance Notes

• Dilute the sample if absorbance > 1.0
• Concentrate if absorbance is too low
• Assay optimized for plant, fungal, or microbial samples

Recent Literature Using This Kit

• Yu et al., Postharvest Biology and Technology, 2019
• Chen et al., Journal of Experimental Botany, 2019
• Qin et al., International Journal of Biological Macromolecules, 2018