Experiment – 9

Utkarsh Anand - 2021BB10322

Meenakshi Mina – 2021BB10012

Learning Objective: Immobilized beads in CSTR

Principle:

Immobilising of enzymes by gel entrapment involves the entrapment of the biocatalyst

within a polymeric network. After mixing with the aqueous polymeric solution, the
biocatalyst is forced through a fine orifice (syringe) into a salt solution that insolubilizes
the mixture through ion exchange. The shape and size of beads can be controlled by
choosing the orifice diameter and the distance of the nozzle from the liquid surface. Gel
entrapment usually does not result in any adverse modification of the enzyme
conformation and can provide high yield of immobilisation. The use of immobilized
beads in CSTRs is based on the principle that the catalyst or support material is fixed in
a particular location, allowing the reactants to pass through it, while the products are
formed and removed from the system. This approach o ers several advantages over
traditional batch reactors, including better control over reaction conditions, higher
reaction rates, and improved product purity.

Reagents and Materials required:

1. Invertase-entrapped calcium alginate beads

2. 0.1M sucrose solution

3. Alkaline DNS

4. 50mM sodium acetate bu er, pH 4.8

5. Spectrophotometer

6. Water Bath

7. Glass Wool

8. CSTR set up

Procedure:

1.Add prepared beads (25ml)

2.Adjust the flow rate to 2mL/min.

3.Connect input and output lines and pass substrate through the packed bed.
4.Collect 50 µL sample every 0, 10, 20, 30, 40, 50, 60 mins.

5. Perform kinetic characterisation of the calcium alginate beads –

a. Add 200µL alkaline DNS to 50µL sample obtained from the reactor.

b. Incubate the mixture at 90 degree C for 5 min.

c. Add 200µL 50mM acetate, pH 4.8 to the mixture.

d. Blank (without enzyme) + sucrose + DNS+ acetate

Now, Dilute the samples appropriately with distilled water. Record all the absorbance
readings taken at 540nm and calculate the enzyme activity using a standard curve.

Observation Table:

Calculation Table:
Theoretical conversion factor:

Using the above equation, we get 3.31768 = -29.457ln(1-X) + 100X

Therefore X (theoretical) = 0.0495,

Experimental Conversion factor:

=(Initial subs conc. – final subs conc.)/(initial subs conc.)

= (100-60.843)/100 = 0.392
Precautions:

 Handle prepared beads (25ml) carefully to avoid damage during addition to the
reactor.

 Ensure proper connection of input and output lines to prevent leaks during substrate
passage.

 Use calibrated equipment to adjust the flow rate precisely to 2mL/min.

 Maintain the incubation temperature at 90°C for 5 minutes precisely to ensure

consistent results.

 Use distilled water for appropriate dilution of samples to ensure accuracy in

absorbance readings.