Procedure

1.Prepare the following reaction mixture in a test tube (32φ ×200mm) and equilibrate at 20℃ for about 5 minutes.

2.Add 1.0 ml of the enzyme solution* and mix.

3.After exactly 20 seconds at 20℃, add 1.0 ml of 2.0N H₂SO ₄ solution (D) to stop the reaction.

4.Extract the produced purogallin from the above stopped reaction mixture in five times with 15ml portions of ether and fill up the combined ether extracts to 100ml with fresh ether.

5.Measure the optical density at 420nm against water (OD test).
At the same time, prepare the blank by first mixing the reaction with 1.0ml of 2.0N H₂SO₄ solution (D) after 20 sec-incubation at 20℃, followed by the addition of the enzyme solution and extracting with ether by the same procedure as the test (OD blank).

*Dissolve the enzyme preparation in ice-cold 0.1M phosphate buffer, pH6.0 (C), dilute to 3.0-6.0 purpurogallin U/ml with the same buffer and store on ice.

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  Fig.1. Stability (Powder form)

  (kept under dry conditions)

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  Fig.2. pH-Activity

  37℃, in 0.1M buffer solution; pH 3.3-6, Acetate; pH 6-7.5, K-Phosphate; pH 7.5-9, Tris-HCl

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  Fig.3. pH-Stability

  25℃, 20hr-treatment with 0.1M buffer solution; pH 3-6, Acetate; pH 6-8, K-Phosphate; pH 9-11, Glycine-NaOH

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  Fig.4. Thermal stability< /p>

  10 min-treatment with 0.05M K-Phosphate buffer, pH6.0 Enzyme concentration : 2 U/ml