biologia - Studia

M.. DRĂGAN-BULARDA,. R. CARPA, M. MIŞCA, V.-CĂLEAN
the bubbling vessel. The water-bath was adjusted at 30 0 C and the inlet air-flow was
set at 1.5 L air/L medium/minute. The fermentation process took 14 hours.
Analyses performed. Measuring the increase of biomass. It took place at
the beginning of the fermentation and then every 30 minutes. The increase of the
cell mass was determined by measuring the absorbance at 600 nm.
Monitoring the α-amylase production. Every 30 minutes, a small quantity of
culture medium (4 ml) was taken under aseptical conditions. This sample was
centrifugated for 5 minutes at 8000 rpm in order to separate the bacterial cells and the
supernatant was used for enzymatic tests. The quantity of dry cell mass was determined
(after drying in the drying chamber at 105 0 C, for 48 hours). The Metais and Bieth
method (Popa, 1990) was used for determining the α-amylase activity. The principle of
this method consists in using a starch solution that will be treated with an enzymatic
solution. The substrate remained unhydrolised was dosed spectrophotometrically at
565 nm, after the reaction with iodine. There was used a reaction mixture of enzymatic
solution + enzymatic substrate (starch solution 0.4% as buffer Na 2 HPO 4 0.2 M – citric
acid 0.1 M, pH7). The incubation took place at several temperatures (20-70 0 C), for 5
minutes. The enzymatic reaction was stopped with 5 ml of 6% acetic acid solution.
Iodine solution N/1500 was added, and the resulting violet complexe passed trough a
colorimetric analyse at 565 nm, towards distilled water. In order to determine the initial
starch concentration in the reaction medium, the enzymatic reaction was stopped at
zero time. The amylase activity is indicated in U/ml/min and is calculated as follows:
Enzymatic activity = OD control – OD sample x 1.2 x 5 x 1/5 per OD control (where
OD is optical density; 1.2 – quantity of starch, expressed in mg, added to the reaction).
The above relation was multiplied by 5 in order to determine the enzymatic
units in 1 ml of protein preparation, respectively by 1/5 in order to determine the
activity during 1 minute.
Obtaining the raw extract. After 14 hours, the content was centrifugated
for 15 minutes at 800 rpm. The supernatant was collected and filtered, getting the
raw extract (in total 220 ml).
Obtaining the partial purified preparation. To the raw extract there was
added cold acetone up to 80% (V/V) saturation. We worked on ice bath. The
suspension was centrifuged for 30 minutes at 800 rpm. The supernatant was
removed. The precipitation was redisolved in buffer phosphate 0.1 mol/L.
Calculation of K m and V max . In order to determine the reaction rate, there
were used 5 starch solutions of known concentrations. To each of these, there was
added 1 ml of enzymatic preparation. After homogenisation, there was taken 1 ml
of mixture, to which 0.2 ml iodine solution N/100 was added and the adsorbance at
565 nm was measured. The rest of the sample was indroduced in a water bath at
30 0 C. After 1 minute there were added 0.4 ml iodine solution N/100 and the
adsorbance at 565 nm was measured. For calculating the starch concentration in the
sample, there was used a standard curve with 6 concentrations of starch (0.333 – 2
mg/ml). The reaction rate was calculated as follows: V= (C iniŃial -C final )/60.
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