IA on effect of duration on efficiency of immobilized enzyme amylase (yeast extract) in alginate beads, in starch hydrolysis.

1. Effect of duration on efficiency of immobilized enzyme amylase (yeast extract) in alginate beads, in starch hydrolysis
Sodium alginate reacts with calcium chloride to form calcium alginate
These beads can be used to immobilise enzymes so it can be recycle/reuse
Encapsulation of drugs/enzymes can be done using alginate beads.
3% (w/v) sodium alginate and 2% (w/v) calcium chloride investigated.
Sodium alginate + calcium chloride
forming alginate beads.
Procedure:
Weigh 3g of sodium alginate (SA) in 100ml water - 3% SA.
Weigh 2g calcium chloride (CC) in 100ml water – 2% CC.
1ml clear yeast extract after centrifuging was added to 3ml of 3% SA.
Pipette 1ml 3% SA/amylase and drop it into 2%CC, making 12 beads in total.
12 alginate beads added to starch/I2 mix in next slide.
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3% (w/v) SA used
Yeast extract spin down. Clear sol taken
Yeast solution. 1g in 20ml water Beads measured with colorimeter
3% SA beads with yeast enzyme + 2% CC
Beads added to starch/I2 mix

2. Starch react with I2 to form a blue black product.
Blue black colorization can be monitored using visible spec.
Enzyme amylase from yeast will breakdown starch to maltose.
As starch is broken down, I2 wont be able to bind with starch.
Resulting in colorless product.
Rate of starch hydrolysis – measured by change in blue black over time (3mins).
Starch + I2 + enzyme → maltose + simple sugars
Reaction mechanism
Starch + I2 (Blue black) I2 (yellow)
Comparison bet beads with enzyme
and without enzyme
Initial color after adding beads Color fades with enzyme After 30 mins
All blue black sol taken out, and its abs was measured using colorimeter
Effect of duration on efficiency of immobilized enzyme amylase (yeast extract) in alginate beads, in starch hydrolysis
Procedure:
1% (w/v) starch – 1g in 100ml water
1% (w/v) iodine – 1g in 100ml water
5% (w/v) yeast sol – 5g in 20ml water
1ml starch + 20ul I2 was added into a cuvette. (Blue black)
12 alginate beads with enzymes immobilized was added into cuvette.
Ini and final after abs taken over 3 mins. (Day 1)
Rate of hydrolysis – decrease in abs over time.
Beads were kept in fridge and reused for Day 2 and Day 3.

3. Go to expt – press calibrate Insert cuvette (blue black) and press collect
Insert a blank containing water
Press stop, and click on rainbow icon. Select abs vs time. λ max at 584nm will
be automatically chosen
Abs vs time
Effect of duration on efficiency of immobilized enzyme amylase (yeast extract) in alginate beads, in starch hydrolysis

4. 0
20
40
60
80
100
120
1 2 3
Efficiency
Duration/Day
Duration/Day vs Efficiency
3% SA added to 2% CC seems
to be the best
Beads able to breakdown starch after day 3, with 44% efficiency
Day Ini abs Final abs Rate/
absmin-1
Efficiency
%
1 1.672 1.056 0.205 100
2 1.840 1.500 0.113 55
3 1.763 1.491 0.091 44
-ve
control
2.054 2.073 -- --
-ve control - Beads without any enzyme
Procedure:
1% (w/v) starch – 1g in 100ml water
1% (w/v) iodine – 1g in 100ml water
5% (w/v) yeast sol – 5g in 20ml water
1ml starch + 20ul I2 was added into a cuvette. (Blue black)
12 alginate beads with enzymes immobilized was added into cuvette.
Ini and final after abs taken over 3 mins. (Day 1)
Rate of hydrolysis – decrease in abs over time.
Beads were kept in fridge and reused for Day 2 and Day 3.
Color fades with enzyme Beads taken out and abs was measured
Effect of duration on efficiency of immobilized enzyme amylase (yeast extract) in alginate beads, in starch hydrolysis
0
20
40
60
80
100
120
0 0.5 1 1.5 2 2.5 3 3.5
Efficiency
Duration/Day
Duration vs Efficiency
Unsure if it is still able to retain its enzyme activity after 1 month