Biomedical Engineering Reference
In-Depth Information
The culture samples, as whole flasks in duplicate, were collected each 12 h. Mycelial
mass was harvested by paper filtration using a pre-dried and pre-weighted Whatman filter
paper No. 1, washed with distilled water and dried to constant weight at 105ºC. The growth of
the organism was determined as dry weight. Paper-filtered media were used to perform
analytical determinations (pH, enzyme concentration, total sugars and COD).
Method for determination of total sugars was described in a previous work (Guerra &
Pastrana, 2003). COD analyses were carried out in both the MPW and BW media before the
fermentations and in the paper-filtered media at the end of each fermentation using the closed
reflux colorimetric method as described previously (Greenberg et al., 1980). All
determinations were carried out in triplicate.
Assay of Amylase Activity
Total amylase activity (
TAA
) in the submerged and solid state cultures was determined as
described by Murado et al., (1993). Then, 80
μ
L of suitably diluted paper-filtered medium (in
case of submerged cultures) or crude enzyme extract (in case of solid state cultures) were
mixed with 400
μ
L of 0.15 M citrate-phosphate buffer, pH 5.0 (1 volume) and 4% soluble
starch (1.5 volumes) previously maintained at 40ºC/15 min. The reaction mixture was
incubated at 40ºC for 10 min. The reaction was stopped by addition of 480
μ
L of
dinitrosalicylic acid, and the released glucose was determined by 3,5-dinitrosalicylic acid
reaction (Bernfeld, 1951). One unit of amylase activity (enzymatic units (EU)/mL) was
defined as the amount of enzyme that releases 1 mg/mL of reducing sugars (glucose
equivalents) under the assay conditions. In case of SSF in sugar cane bagasse, the amylase
activity units were expressed as EU/g of dry support (gds).
Table 1. Experimental domain and codification of the variables used in the factorial
design to test the effect of temperature (T) and pH on amylase activity
Natural values
Coded values
T
(ºC)
pH
- 1.267
26
3.6
-1
30
4.0
0
45
5.5
+ 1
60
7.0
+ 1.267
64
7.4
Effect of Temperature and pH on Amylase Activity
A second-order orthogonal design (Box et al., 1989) based on five levels and two
variables was used to study the combined influence of pH and temperature on amylase
activity. The design consisted of 13 experiments with four (2
2
) factorial points, four axial
points to form a central composite design with
α
= 1.267 and five center points for
replication.
Samples of cell-free medium containing the enzymes were buffered at different pHs with
the appropriate buffer (potassium hydrogen phthalate-HCl buffer for pH 3.6, 4.0 and 5.5;
