P-Hydroxybenzoate Hydroxylase is a biocatalyst and a key enzyme in new biocatalyst technology. Enzyme engineering focuses on enhancing enzyme reaction kinetics, substrate selectivity, and activity under harsh conditions such as low or high pH. By introducing stimulus responsiveness to these enzyme modifications, dynamic control of activity is also possible.
PREPARATION and SPECIFICATION
| Appearance: | Yellowish amorphous powder, lyophilized |
| Activity: | ≥20U/mg enzyme powder (containing approximately 40% of stabilizers) |
| Contaminant: | NADPH oxidase ≤0.1% |
| Stabilizers: | Sugars, FAD |
PROPERTIES
| Stability | Stable at −20℃ for at least one year | (Fig.1) |
| Molecular weight | 55,000～60,000 |
|
| Michaelis constants | 2.0×10⁻⁵M (p-Hydroxybenzoate), 4.0×10⁻⁵M (NADPH) |
|
| Structure | One mol of FAD per mol of enzyme |
|
| Inhibitors | Ag⁺, Hg²⁺, PCMB, SDS |
|
| Optimum pH | 7.7－7.9 | (Fig.3) |
| Optimum temperature | 35℃ | (Fig.4) |
| pH Stability | pH 5.0－7.5 (25℃, 72hr) | (Fig.5) |
| Thermal stability | below 40℃ (pH 6.0, 15min) | (Fig.6) |
| Substrate specificity | (Table 1) |
| Effect of various chemicals | (Table 2) |
APPLICATIONS
This enzyme is useful for enzymatic determination of choline esterase in combination with protocatechuate 3, 4-dioxygenase.
ASSAY
Principle
The formation of NADPH is measured at 340 nm by spectrophotometry.
Unit definition
One unit causes the oxidation of one micromole of NADPH per minute under the conditions described below.
Method
Reagents
A. Tris-malate buffer, pH 8.2 50 mM: Dissolve 3.03 g of Tris (MW＝121.14) in approx. 300 mL of H₂O, and, after adjusting the pH to 8.2 at 25 ℃ with 1.0 M maleic acid, make up to 500 mL with H₂O.
B. p-hydroxybenzoate solution 5.0 mM: 80 mg of sodium p-hydroxybenzoate / 100 mL of buffer solution A (should be freshly prepared).
C. FAD solution 0.2 mM: 19 mg of FAD·Na₂ / 100 mL of buffer solution A (should be freshly prepared)
D. NADPH solution 3.0 mM: 272 mg NADPH·Na₄·4H₂O / 100 mL of buffer solution A (should be freshly prepared)
E. Enzyme diluent 50mM Potassium phosphate buffer, pH 6.0, containing 0.2% BSA
Procedure
1.Prepare the following working solution (for 10 tests) in a brownish bottle and store on ice.
21.0 mL Buffer solution (A)
3.0 mL Substrate solution (B)
3.0 mL FAD solution (C)
3.0 mL NADPH solution (D)
Concentration in assay mixture
| Tris-malate buffer | 49 mM |
|---|
| p-Hydroxybenzoate | 0.49 mM |
|---|
| FAD | 20 μM |
|---|
| NADPH | 0.30 mM |
|---|
2.Pipette 3.0 mL of working solution into a cuvette (d ＝ 1.0 cm) and equilibrate at 37 ℃ for approximately 5 minutes.
3.Add 0.05 mL of the enzyme solution* and mix by gentle inversion.
4.Record the decrease in optical density at 340nm against water for 3 to 4 minutes with a spectrophotometer thermostated at 37 ℃ and calculate the ΔOD per minute from the portion of the curve from 1.5 to 3 minutes (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) using the same method as the test except that the enzyme diluent (E) is added instead of enzyme solution.
*Dissolve the enzyme preparation in ice-cold enzyme diluent (E) (1.0 mg/mL or more) and dilute to 0.2−0.6 U/mL with the same buffer, immediately before the assay.
Calculation
Activity can be calculated by using the following formula:
Vt : Total volume (3.05 mL)
Vs : Sample volume (0.05 mL)
6.22 : Millimolar extinction coefficient of NADPH (cm²/micromole)
1.0 : Light path length (cm)
df : Dilution factor
C : Enzyme concentration (c mg/mL)
Table 1. Substrate Specificity of p-Hydroxybenzoate hydroxylase
|
| Substrate (0.5mM) | Relative activity (%) | Substrate (0.5mM) | Relative activity (%) |
|---|
| p-Hydroxybenzoic acid | 100 | Protocatechuic acid | 3.3 |
| Methyl-p-hydroxybenzoic acid | < 0.05 | β-Resorcylic acid | 4.5 |
| Ethyl-p-hydroxybenzoic acid | < 0.05 | Gentisic acid | < 0.05 |
| n-Propyl-p-hydroxybenzoic acid | < 0.05 | p-Chlorobenzoic acid | < 0.05 |
| m-Hydroxybenzoic acid | < 0.05 | p-Aminobenzoic acid | 0.12 |
| o-Hydroxybenzoic acid |
|
Table 2. Effect of Various Chemicals on p-Hydroxybenzoate hydroxylase (Residual activity after 1 hr-treatment at 30℃)
| Chemical | Concn.(mM) | Residual activity(%) | Chemical | Concn.(mM) | Residual activity(%) |
|---|
| None | — | 100
| MIA | 1.0 | 91 |
Metal salt
| 1.0
|
| PCMB | 1.0 | 3.7 |
| CoCl₂ |
| 106 | NaN₃ | 1.0 | 97 |
| ZnCl₂ |
| 94 | NaF | 1.0 | 96 |
| CuSO₄ |
| 103 | o-Phenanthroline | 1.0 | 95 |
| AgNO₃ |
| 0 | α,α'-Dipyridyl | 1.0 | 90 |
| MgSO₄ |
| 107 | EDTA | 5.0 | 96 |
| BaCl₂ |
| 107 | Borate | 50 | 104 |
| FeCl₃ |
| 106 | Tween 20 | 0.1% | 91 |
| MnCl₂ |
| 90 | Brij 35 | 0.1% | 101 |
| NiCl₂ |
| 104 | Span 20 | 0.1% | 94 |
| CaCl₂ |
| 97 | Triton X-100 | 0.1% | 97 |
| SnCl₂ |
| 102 | Na-cholate | 0.1% | 88 |
| HgCl₂ |
| 1.1 | SDS | 0.05% | 34 |
| CrCl₃ | | 93 |
| CdCl₂ |
| 104 |
| FeSO₄ |
| 90 |
MIA, Monoiodoacetate; PCMB, p-Chloromercuribenzoate; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate.
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