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2.3. Analytical methods
The reaction medium containing 250 ll of 2mM pnitrophenyl-
b-xylopyranoside (pNPX) (50 mM phosphate
buffer (pH 5)) and suitably diluted enzyme was incubated
in a total volume of 0.5 ml at 45 C for 30 min. The reaction
was stopped by the addition of 1 ml of 2 M Na2CO3
solution, and the amount of p-nitrophenyl released was
determined by measuring the absorbance at 405 nm and
compared with a standard reference. One unit of b-xylosidase
is defined as the amount of enzyme required to release
of nmol p-nitrophenyl per hour under the conditions
described above (Biely et al., 1980). The initial trials
showed that the enzyme was stable under the assay conditions
and the hydrolysis rates that were measured behave
linear during assay.
The kinetic constants (Km and Vmax) of the purified
enzyme for the hydrolysis of pNPX were estimated by
the method of Lineweaver and Burk (1934). Protein was
determined by the dye-binding method (Bradford, 1976)
using serum albumin as the standard and specific activity
was defined as U mg1 of protein. Growth rate was determined
by direct cell count in a Neubauer chamber (Basaran
et al., 2001).