Page 66 - Enzymes in Tropical Soils

54 Enzymes In Tropical Soils

As used by Salam et al. (1998d), to analyze soil phosphatase activities, a 1
o
gram 105 C oven-dry equivalent of soil sample is used. After stopping the soil
microbial activities with toluene, a 4 ml of a modified universal buffer (MUB), of pH
6.5 for acid phosphatase measurement and of pH 11 for alkaline phosphatase
measurement, and a 1 ml of 0.025M p-nitrophenyl phosphate (p-NPP) dissolved in
MUB solution with the corresponding pH were added to the soil sample. The
o
mixture was then incubated for 1 hour at 30 C. The soil enzymatic reaction was
terminated by the addition of a 4 ml 0.5 M NaOH solution, followed by the
addition of a 1 ml 0.5 M CaCl 2 solution to extract p-nitrophenol adsorbed by the soil
colloids. The concentration of p-nitrophenol in the extract is determined by a
spectorophotometer. Para-nitrophenyl is used in the assays of several soil enzymes
including: -glucosidase, chitinase, cellobiohydrolase, acid phosphatase, and
alkaline phosphatase (Verchot and Borelli, 2005).
The activity of -Glucosidase is also determined by the above method by
employing p-nitrophenyl--D-Glucopyraniside (p-NPG) as a substrate instead of p-
NPP and a MUB solution of pH 6.
In the acid phosphatase assay,the enzyme hydrolyses the phosphate ester
bonds under an optimum pH of 4.8 (Richers-Haunerland, 2013). The artificial of
para-nitrophenylphosphate is hydrolyzed by acid phosphatase releasing p-
nitrophenol and inorganic phosphate (Fig. 4.12). Both para-nitrophenylphosphate
and p-nitrophenol are colorless at acidic pH values. The addition of 0.05 M NaOH
after 30 minute incubation stops the reaction due to an increase in the pH of the
reaction medium. Para-nitrophenol is yellow at alkaline pH and its concentration
can be measured at 405 nm.

Fig. 4.12. The breakdown of para-nitrophenolphosphate by acid phosphatase
(Taken from Richer-Haunerland, 2013).

Abdul Kadir Salam - 2014