Enzyme immobilisation and application

Preparation of beads: 0.4g of sodium alginate in 10 mls of distilled water and mix to a smooth paste 2g of brewers yeast to 10 mls of distilled water and mix to a smooth paste (The

yeast contains the enzyme sucrase, just in case you forget) Mix the alginate and yeast solutions together In a different beaker, mix 1.5g of calcium chloride to 100 mls of distilled water. Draw the mixture of alginate and yeast solution into a syringe (Draw all of the

mixture because then we know how much free yeast to use for a control) From a height of 10 cm, add a stream of alginate and yeast drops into the CaCl2

(From 10 cm we are sure to get beads due to atmospheric pressure)(The CaCl2 will harden the beads by forming a gel of Calcium Alginate)(Leaving the beads for 15 minutes in the CaCl2 will harden the beads)

Next, filter the beads in distilled water (to remove any yeast cells from the outside of the beads)

The Control 2g of brewers yeast to 10 mls of distilled water

Preparation of Substrate: 1g of sucrose into 100 mls of distilled water

Procedure:

Set up two separating funnels ( A and B ) Pour the immobilised yeast into A and the free yeast into B Pour the 50 mls of sucrose into each To prevent the tap of A clogging, put a straw through it Using glucose strips, test the mixture of each every minute

by opening the tap, collecting a 1 ml sample, and checking for any colour change

Results:The free yeast works quicker than the immobilised yeast but the product is cloudy (unlike the immobilised yeast product)

Advantages of Immobilized Enzymes (Taken from Web)1. Easy recovery of enzymes for reuse.2. Easy harvesting of products (no enzyme contamination – no cloudiness).3. Greater enzyme stability.

You need also to be able to draw the bioreactors and explain fully the industrial uses

Miscellaneous Questions

To what kingdom do yeasts belong? Fungi

What is an immobilized enzyme? In our case it is an enzyme attached to an inert material called sodium alginate and

hardened in calcium chloride to form a gel of calcium alginate

Why is all glassware sterilized before use? To exclude microbes which may affect our experiment

Why should distilled water be used? To exclude microbes and the process of making alginate beads is extremely

delicate

What is the purpose of the sodium alginate? It is an inert material which will mix with the yeast, but when put into calcium

Chloride, it will harden to form beads of calcium alginate

What is the purpose of the calcium chloride solution? To harden the yeast/alginate mixture

Why are the beads left for 15 minutes? To harden sufficiently for the experiment

Why are the beads washed with distilled water? to remove any yeast cells from the outside of the beads

Why do we use brewers yeast? We know that it contains sucrase and it is an easy test with glucose strips to test

when the sucrose solution which is added as the substrate changes to glucose

What was the difference in turbitity (cloudy appearance) between the solution with immobilized yeast and the one with free yeast?

Free yeast was cloudy and Immobilised yeast was not

It was noted at first that the immobilised yeast was slow at first to show reaction. Why? The substrate has to travel through the gel to get to the enzyme. This slows the

reaction initially

State two benefits of immobilized systems to industry? Reused and Don’t have to take enzyme away from the product

Name two examples of industrial processes where immobilized systems are used successfully?

Production of fructose (sweetener) from glucose Convert penicillin to different forms Produce sweet-tasting sugars from whey

What is a bioreactor? A container such as a vat used in alcohol fermentation