Shikimic acid pathway is an important metabolic pathway in plants and microorganisms. Shikimate
dehydrogenase (EC 1.1.1.25) is the key enzyme to catalyze the fourth step of shikimate synthesis and
metabolism.
Shikimate dehydrogenase catalyzes the production of NADPH from shikimate and NADP. The SD activity
can be expressed by measuring the increasing rate of absorbance value at 340 nm.
Required but Not Provided:
Spectrophotometer/microplate reader, desk centrifuge, water-bath, transferpettor, micro quartz cuvette/96
well flat-bottom UV plate, mortar/homogenizer and distilled water.
Protocol
I. Preparation:
1. Tissue: according to the ratio of mass (g): extract solution volume (mL): 1:5-10 to add the extract. It is
suggested that add 1 mL of extract solution to 0.1 g of tissue. Homogenate on ice. Centrifuge at 8000 g 4℃
for 10 min. Take the supernatant on ice for test.
2. Cells: first collect bacteria or cells into the centrifuge tube. Discard the supernatant after centrifugation.
According to the number of the cells (104): the volume of the extract solution (mL) is 500~1000:1. It is
suggested that add 1 mL of extract solution to 5 million of cells. Breaking cells by ultrasonic wave in ice
bath (power 300W, ultrasonic 3s, interval 7s, total time 3 min). Centrifuge at 8000 g 4℃ for 10 min. Take
the supernatant on ice for test.
3. Liquid: direct detection.
II. Determination
