NEUTRAL PROTEINASE from Bacillus sp.

Microbial metalloproteinase (EC3. 4. 24. 4)

PREPARATION and SPECIFICATION
Appearance White amorphous powder or granule
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⁺⁺
PROPERTIES ¹⁾
Molecular weight approx. 25,000 (SDS-PAGE)
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:

Reagents
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.)
B. TCA solution 20% Trichloroacetic acid (TCA)
C. Enzyme diluent 10mM Borate buffer containing 2mM CaSO₄(pH 8.0)

Procedure

Concentration in assay mixture
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

  1. T.Nakanishi, Y.Matsumura, N.Minamiura; Agric. Biol. Chem., 38, 37-44 (1974).
  2. T.Nakanishi and T.Yamamoto; Agric. Biol. Chem., 38, 2391-2397 (1974).
  3. B.Hagihara, H.Matsubara, M.Nakai and K.Okunuki; J.Biol.Chem., 45, 185 (1958).
Table 1. Effect of Various Chemicals on Neutral Proteinase
[The enzyme dissolved in 1.0mM HBO-NaBO₇ ; pH 8.0 (6 KU/ml) was incubated at 25°C for 1hr.]
Chemical Concn.(mM) Residual
activity
Chemical Concn.(mM) Residual
activity
None 100% NaF 2.0 97%
Metal salt 2.0   NaN 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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