i worked before in these enzymes but i need to find an easier protocol
Dear Maher,
Please read the following text:
Amylase ( -amylase EC. 3.2.1.1): The Amylase activity was calculated by using
Jayaraman, (1981) method. Treated and control seedlings were collected and cut into small pieces. Accurately 1.0 gm of seedling sample was homogenized in pre chilled mortar with pestle, in 5 ml of 0.1 M sodium acetate buffer (pH4.8) supplemented with (10 mM NaCl). The extract was centrifuged at 4 0C in cooling centrifuge at 15000xg for 10 minutes and supernatant was used as sources of crude enzyme. Enzyme reaction mixture contained 2.0 ml of 0.1M sodium
acetate buffer (pH 4.7), 0.5ml of 1% starch and 0.5ml enzyme extract in total 3 ml
volume. The reaction was initiated by adding 0.5 ml of enzyme extract and
reaction mixture was incubated for 10 minutes at 37 O C temperatures in water bath. After 10 minutes of incubation 2ml of Dinitrocalysilic acid (DNS) was added and reactions were heated in water bath at 100 oC for 10 minutes. Same procedure was followed for control but enzyme reaction was terminated at zero minutes by adding DNS reagent. After heating reaction mixture was diluted with distilled water to 10 ml and OD was taken at 510 nm. One unit of amylase activity was defined as the amount required for liberating 1 mg of maltose in 1 min at 37 oC. The enzyme activity was expressed as units g-1 fresh tissue. Protease: The Protease activity was
calculated by using Issac and Gokhale (1982) method. Accurately 1.0 gm of
seedling sample was homogenized in pre chilled mortar with pestle, in 5ml of 0.1 M
phosphate buffer (pH 7.4). The extract was centrifuged at 4 oC in cooling centrifuge at 15000xg for 10 minutes and supernatant was used as sources of crude enzyme. Enzyme reaction mixture contained 2.0 ml of 0.1M (pH 7.5) phosphate buffer, 0.5ml casein and 0.5ml plant extract in total 3 ml of volume. The reaction was initiated by adding 0.5 ml of enzyme extract and incubated for 10 minutes at 40 oC temperatures in water bath. The enzyme activity was stopped by adding 3ml of 5% H2SO4 solution. The proteins precipitated in reaction mixture after 60 minutes of resting were separated by centrifugation at 10,000 xg for 10 minutes. Exactly 2ml of supernatant was mixed 3ml of 2% Na2CO3 and 1ml of folin phenol reagent. The blue colour developed was read at 660nm. Similar procedure was followed
for control but enzyme reaction was terminated at zero minutes by adding 3ml of
5% H2SO4 solution. The protease activity was measured by estimating the release of tyrosine calculated from the standard curve prepared with tyrosine. One unit of protease activity was defined as the amount required for liberating 1 mg of tyrosine in 1 min at 40 oC. The protease activity was expressed as units g-1 fresh tissue
For more details, see attached file.
Hoping this will be helpful,
Rafik
The below mentioned link will be helpful for performing the protease assay.
http://www.jove.com/video/899/sigma-s-non-specific-protease-activity-assay-casein-as-a-substrate
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