Microbial Enzymes and Biotransformations Microbial Enzymes and ...

Microorganisms for NDOs biosynthesis 109
Fig. 1. Semi-native PAGE of culture samples taken at 24, 48, 72, 96, and 120 h from
a culture of Aspergillus BT18 rich in fructosyltransferase activity. Right: gel stained
with Coomassie brilliant blue. Left: activity stain with 1, 2, 3 triphenyltetrazolium
chloride. M: molecular weight marker. The band with fructosyltransferase activity is
marked by an arrow.
in Subheading 3.1. One-mL samples of the culture broth were taken every 24
h and the enzyme present in the supernatant was analysed by semi-native PAGE
and fructosyltransferase activity staining (Fig. 1) according with the method
described by Heyer and Wendenburg (7).
1. Prepare a 6% semi-native PAGE gel (8) containing 0.1% SDS.
2. Prepare samples of 10 µL of culture supernatant in 20 µL loading buffer containing
the same amount of SDS but omitting β-mercaptoethanol and the heating step.
3. After the electrophoresis, rinse the gel extensively with washing buffer for 15 min
to remove SDS.
4. Incubate the gel in substrate solution for 30 min at room temperature.
5. Wash the gel repeatedly with water to remove sucrose.
6. To visualize glucose release, pour onto the gel 10 mL of staining solution at 100°C.
A red insoluble formazan will form in the areas where a fructosyltransferase is
present.
7. Once the red area is visible, discard the substrate solution, and stop the staining by
adding 10 mL of stop solution (see Note 4).
3.4. Partial Purification of the Enzyme and Enzymatic Synthesis
of Fructooligosaccharides
The results obtained in the semi-native PAGE confirm those previously
described (9–11) suggesting a high molecular weight (>100 kDa) for the
enzyme with fructosyltransferase activity. Thus, in order to concentrate and partially
purify the enzyme, an ultrafiltration step (cut-off 100 kDa) was performed
after the scale-up of the fermentation of the selected strain.