C4H is also called trans cinnamic acid-4-monooxygenase. It is an enzyme that catalyzes cinnamic acid to
form coffee bean and coumaric acid. C4H mainly exists in higher plants, yeasts and fungi. It is a key
enzyme in the process of lignin synthesis.
C4H catalyzes the cinnamic acid and NADPH to form 4-coumarite and NADP. The decrease rate of
NADPH at 340nm can reflect the activity of C4H.
Required but Not Provided:
Spectrophotometer, desk centrifuge, water-bath, transferpettor, 1 mL quartz cuvette, mortar /homogenizer,
ethanol, ice and distilled water.
Protocol
I. Preparation:
1. Tissue:
Add 1 mL of extract solution to 0.1 g of tissue. Homogenate on ice. Centrifuge at 12000 g 4℃ for 15
minutes. Take the supernatant on ice for test.
2. Cells or bacterial
Collect bacteria or cells into the centrifuge tube. Discard the supernatant after centrifugation. It is
suggested to take about 5 million bacteria/cell and add 1 mL extract reagent. Bacteria/cell is split by
ultrasonication (power 20%, ultrasonic 3s, interval 10s, repeat for 30 times). Centrifuge at 12000 g 4℃ for
15 minutes. Take the supernatant on ice for test.
II. Determination
