Cell and Enzyme Immobilisation

Enzymes
1. Enzymes are protein catalysts.

2. After synthesis within a cell, enzymes can function independently of the cell provided certain 3. 4.
conditions are maintained. Enzyme technology involves the production, isolation and purification of enzymes. Commercial enzymes are obtained generally from (a) Plants (b) Animals (c) Microbes Most enzymes are obtained from microbes (fungi and bacteria). Mutant strains have been selected which maximise enzyme production. Genetic engineering has come to the fore in the tailoring of producer organisms for synthesis. Over 2000 enzymes have been isolated to date, yet only a small number are produced on a large scale. Relatively crude preparations used in the food processing industries and in the manufacture of detergents make up the bulk of the output. Many commercially produced enzymes are hydrolases such as amylases, cellulases, pectinases, and peptidases. Nevertheless, there is a growing demand for purified enzymes such as glucose oxidase for the biochemical analysis of blood glucose concentration and asparaginase for the treatment of a form of leukaemia.

Enzyme a amylases

Examples of microorganisms involved

Aspergillus oryzae

Application
Breakdown of starch in beer Improving of flour Prep. of glucose syrup Thickening of canned sauces Beer production Sweetener for soft drinks Cake fillings Lactose removal from whey Sweetener for milk drinks Flavour development in cheese Clearing of wines and fruit juices Meat tenderiser Soft ice cream manufacture Confectionery production

b- glucanase Glucose isomerase Lactase Lipase Pectinase Protease Pullulanase Sucrase

Bacillus subtilis Bacillus coagulans

Kluyveromyces sp.

Candida sp. Aspergillus sp Bacillus subtilis Klebsiella aerogens Saccharomyces sp.

Procedure for Enzyme Production 1. 2. 3. Selected strains of microorganisms are taken for the culture. They are inoculated into large sterilised vessels containing previously sterilised and cooled nutrient medium. Sterile air is pumped through and the bioreactor is cooled with circulating water. The o o optimum temperature is between 18 C and 37 C.

Extraction of Enzymes 1. The nutrient liquor is separated into: (i) liquid with the dissolved contents and

(ii) the insoluble cells and other substances. If the enzyme is intracellular the cells are separated from the contents of the growth medium by centrifuge or filtration. The cells are then disrupted by ultrasound or liquidising and the contents separated from the membranes/walls again by filtration. If the enzyme is extracellular separation from the liquid is by centrifuge or filtration. 2. The enzyme solution is now concentrated by removing most of the water by one of the following methods: low temperature vacuum evaporation reverse osmosis. 3. Preservatives are then added and the enzyme is then packaged for distribution to industrial users. 4. If pure enzymes are needed then other procedures are involved before packaging, e.g. fractional precipitation. Enzyme Immobilisation The widespread use of enzymes brought with it a problem how to recover the enzyme for reuse. Many enzymes are expensive to produce and extract. The answer came with recent development of cell / enzyme immobilisation. Immobilisation is the conversion of: Enzyme (water soluble) Enzyme (water insoluble) The enzyme still however maintains its catalytic activity.

There are four ways of bringing about enzyme immobilisation.

1. Adsorption onto insoluble surfaces e.g. charcoal, clay (Fig. 1).

(Fig. 1) 2. Covalent attachment to collagen (Fig. 2). Not the easiest method.

(Fig. 2)

3. Entrap the enzymes in gels (Fig. 3). The enzymes are trapped in the fibres of the gels. The

disadvantage of this method is that substrate or product may not be small enough to enter or leave the gel.

(Fig. 3)

4. Direct cross linking (Fig.4). Glutaraldehyde is used to join the enzyme molecules to each
other to form a polymerised enzyme. This is then precipitated out of solution and is thus immobilised without the need for a solid support.

(Fig.4) Advantages of Immobilized Enzymes

1. 2. 3. 4.

Easy recovery of enzymes for reuse. Easy harvesting of products (no enzyme contamination). Greater enzyme stability. Extends the life of proteolytic enzymes by preventing them digesting each other.

There is an increased demand for cell-free enzymes because: When whole cells are used some of the substrate is used in the cell metabolism The optimum condition for individual enzymes is often different to that of a whole cell. No wasteful side reactions.

In some cases, whole cells are advantageous when the enzyme is expensive or difficult to extract or involves a chain of interconnected reactions. Uses of Industrial Enzymes

1. The production of fructose from corn for use in canned drinks

Many millions of tonnes of fructose are produced every year for use in soft drinks because it is sweeter than glucose or sucrose. amylase, amyloglucosidase Corn Starch Glucose

glucose isomerase Glucose Fructose

Glucose isomerase is not an easy enzyme to produce therefore it is economical to be able to reuse this enzyme again

1. 2. 3. 4. 5. 6. 7. 8.

The crude enzyme is mixed with gelatine and heated to 40 C. The mixture is sprayed onto a cold immiscible solvent e.g. butyl acetate. The gelatine with the enzyme solidifies and falls to the bottom of the column. The particles are dehydrated in alcohol. The particles are cross-linked with glutaraldehyde. The particles are then washed and put into a large column. Glucose is then poured into the top of the column; it is converted to fructose, which is harvested at the bottom free from contamination of the enzyme. The enzyme remains active for 100 days. Its activity is monitored regularly by testing for the presence of glucose.

2. To make lactose-free milk A large percentage of the human population are lactose intolerant. They are missing an enzyme, lactase, which converts lactose to glucose and galactose. When lactose is consumed in the diet it remains undigested and passes into the colon where bacteria may convert it into a variety of different chemicals e.g. ethanoic acid, butanoic acid, carbon dioxide, and methane. These cause a variety of symptoms cramps, distension, acid diarrhoea. Lactose is removed from milk to make it acceptable to those who are lactose intolerant. Immobilised lactase is used. Heat-treated skimmed milk is passed through a column containing immobilised lactase. The lactose-

free milk emerges at the bottom and is then packaged for consumers. 3. Clarification of fruit juices Fruit juices contain pectins, which are a complex group of carbohydrates that cement adjacent cells together. They occur in the middle lamella and in the presence of calcium ions form gels. This poses a viscosity problem for fruit juice manufacturers. Also, pectins can form colloids with proteins giving an unwelcome haze in the juice, for example in apple juice. Immobilised pectinase is used to remove pectin from the juices. 4. Meat tenderisation In olden days freshly slaughtered meat was hung for several days or weeks to allow the meat to tenderise. Proteolytic enzymes released from lysosomes gradually broke down the muscle structure. Today, immobilised enzymes are used to shorten the hanging time. Many fungi, bacteria, and plants e.g. pineapple, contain proteolytic enzymes, which can be used for this purpose. These are non-toxic and are capable of tenderising the meat. However the difficulty with the use of these immobilised enzymes is that, if the enzyme is injected into the meat after slaughter, it causes uneven tenderisation. The most effective way of introducing these enzymes to the muscle tissue is to inject the enzyme into the animals jugular vein before slaughter (usually up to half an hour prior to slaughter).

5. Production of vinegar Ethanoic acid is produced by the following reaction: C2H5OH + O2 CH3COOH + H2O.

In the fermenter, the bacteria are immobilised on wood shavings or some other inert material while the ethanol is sprinkled over them. Air is forced upwards through the shavings and vinegar is drawn off at the bottom.

6. Diagnostic reagents Enzymes are immobilised onto reagent dip strips for diagnostic processes e.g. in glucose concentration determination in blood samples. The enzymes glucose oxidase and peroxidase are immobilised. References Chenn, P. Microorganisms and Biotechnology (1997) John Murray, London. ISBN O719575095

OToole, G. & S. Understanding Biology for Advanced Level (1999) Nelson Thornes, Gloucestershire. ISBN 0003222764

Simpkins, J. & Williams, J.I. Advanced Biology (1997) Collins Education Ltd. ISBN 0748744673