NEUTRAL PROTEINASE from Bacillus sp.
Microbial metalloproteinase (EC3. 4. 24. 4)
Appearance: | White amorphous powder or granule | |
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Activity: | 1000U/mg-solid or more |
|
Contaminants: | Other proteases ≤2 X 10⁻⁻²% | |
Stability: | Stable at 5°C for at least one year | |
Stabilizer : | Ca⁺⁺ |
Molecular weight: | approx. 25,000 (SDS-PAGE) |
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Inhibitors: | EDTA |
Optimum pH : | pH 7.0-8.0(Fig.1) |
Optimum temperature: | 80℃(Fig.2) |
pH Stability: | pH 6.0-9.0 (40°C,30min)(Fig.3) |
Thermal stability: | below 70℃ (pH 7.5, 10min) (Fig.4) |
Effect of various chemicals: | (Table 1) |
APPLICATIONS
This enzyme is useful for synthesis of oligopeptides, for proteolysis of insoluble protein and for structural investigation of the protein.
NEP-801
ASSAY
Principle:
The appearance of the peptides is measured as tyrosine equivalent at 275nm by spectrophotometry.
Unit definition:
One unit causes an increase in the optical density at 275nm corresponding to one microgram of tyrosine per minute under the conditions described below.
Method:
A.Casein solution: | 2.0% (Suspend 20.0g of Hammarsten milk casein (Merck) in 400ml of 0.5% NaOH solution and dissolve. Adjust the pH to 7.2 with 1N HCI, add 500ml of 20mM borate buffer containing 4mM CaSO₄(pH 7.2) and fill up to 1000ml with H₂O.) |
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B. TCA solution: | 20% Trichloroacetic acid (TCA) |
C. Enzyme diluent: | 10mM Borate buffer containing 2mM CaSO₄(pH 8.0) |
Procedure
Concentration in assay mixture | |
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Borate buffer | 10 mM |
Casein | 1.0% |
CaSO₄ | 2mM |
1. Pipette 1.0ml of substrate solution (A) and 0.9ml of enzyme diluent (C) into a test tube, and equilibrate at 35°C for about 5 minutes.
2. Add 0.1ml of the enzyme solution* and mix.
3. After exactly 10 minutes at 35°C, add 2.0ml of TCA solution (B) to stop the reaction.
4. Incubate for an additional 30 minutes at 35°C.
5. Filter the mixture with a filter paper ( Toyo ROshi No.131) and measure the optical density of the filtrate at 275nm (OD test).
At the same time, prepare the blank by first mixing the substrate solution with 2.0ml of TCA solution (B) after 10min-incubation at 35°C, followed by the addition of the enzyme solution, and carry out the same procedure (procedure4-5) at test (OD blank).
* Dissolve the enzyme preparation in the enzyme diluent (C) 60-100U/ml, immediately before assay.
Calculation
Activity can be calculated by using the following formula :
OD (OD test−OD blank ) × Vt × df
Volume activity (U/ml) = =OD × 541×df
0.0074× 1.0 x t ×Vs
Weight activity (U/mg) = (U/ml) × 1/C
- Vt
- : Total volume (4.0ml)
- Vs
- : Sample volume (0.1ml)
- 0.0074
- : Extinction coefficient of tyrosine (㎠/microgram)
- t
- : Reaction time (10 minutes)
- df
- : Dilution factor
- C
- : Enzyme concentration in dissolution (c mg/ml)
REFERENCES
- T.Nakanishi, Y.Matsumura, N.Minamiura; Agric. Biol. Chem., 38, 37-44 (1974).
- T.Nakanishi and T.Yamamoto; Agric. Biol. Chem., 38, 2391-2397 (1974).
- B.Hagihara, H.Matsubara, M.Nakai and K.Okunuki; J.Biol.Chem., 45, 185 (1958).
Chemical | Concn.(mM) | Residual activity |
Chemical | Concn.(mM) | Residual activity |
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None | − | 100% | NaF | 2.0 | 97% |
Metal salt | 2.0 | NaN₃ | 20.0 | 95 | |
MgCl₂ | 92 | EDTA | 5.0 | 0 | |
CaCl₂ | 100 | o-Phenanthroline | 2.0 | 0 | |
Ba(OAc)₂ | 97 | α,α'-Dipyridyl | 2.0 | 96 | |
FeCl₃ | 3 | Borate | 50.0 | 95 | |
CoCl₂ | 100 | IAA | 2.0 | 20 | |
MnCl₂ | 100 | NEM | 2.0 | 94 | |
ZnSO₄ | 100 | Hydroxylamine | 2.0 | 94 | |
Cd(OAc)₂ | 20 | Triton X-100 | 0.10% | 100 | |
NiCl₂ | 43 | Brij 35 | 0.10% | 5 | |
CuSO₄ | 7 | Tween 20 | 0.10% | 7 | |
Pb(OAc)₂ | 72 | Span 20 | 0.10% | 100 | |
AgNO₃ | 95 | Na-cholate | 0.10% | 100 | |
HgCl₂ | 3 | SDS | 0.05% | 100 | |
PCMB | 1.0 | 80 | DAC | 0.05% | 100 |
MIA | 0.1 | 8 |
Ac, CH₃CO;PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetete; EDTA, Ehtyenediaminetetraacetete; IAA,
lodoacetemide; NEM, N-Ethylmalemide; SDS, Sodium dodecyl sulfate; DAC, Dimethy-benzyl-alkyl-ammonium chloride.
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