Production of Enzymes Learning objective: To be able to explain why it is more efficient to use isolated enzymes than whole cells. To be able to explain how contamination of end products is eradicated. To be able to describe how this process occurs.

Uses of enzymes commercial uses- e.g. biological washing powders medical uses- e.g. biosensors, therapeutic enzymes, drugs industrial uses - e.g. bioconversion, fruit juice extraction, sweeteners

Enzymes are being used more and more for industrial bioconversion i. e Enzymes are being used more and more for industrial bioconversion i.e. making a chemical product using purified enzymes rather by pure chemical methods (e.g. citric acid production) or using whole cells (e.g. yeast in brewing).

Considerations when selecting a strain: Does it do what is required Is it safe Is it cost effective Enzymes may be intracellular or extracellular. What is the advantage of extracellular production? Already outside cell Limited number secreted so easier to isolate More robust so less likely to be broken down by heat of chemicals

Why are intracellular enzymes more difficult to isolate than extracellular ones? Because they are inside the cell, first the cell has to be broken open then the enzyme separated from the mixture of all the cellular contents Why is it more efficient to use isolated enzymes than whole cells. Isolated enzymes are usually more efficient in biotechnology than whole cells because enzyme concentration is higher and no unwanted enzymes are present

Enzyme production Surface and submerged techniques: Surface = enzyme produced on the surface of a solid medium Submerged = the mould or bacterium producing the enzyme is grown throughout a liquid medium Advantages and disadvantages? Submerged – more yield as growth throughout but aeration necessary

The maximum enzyme production is usually in stationary phase of microbe growth, so a batch or fed-batch process are usually used. The medium must be chosen to stimulate the microbe into synthesising the correct enzyme. For example to stimulate a microbe to synthesise amylase enzymes, a medium with starch but no sugars is used.

What type of medium would you use to stimulate a microbe to synthesise a protease? A medium with proteins but no amino acids is used.

Microbes are encouraged into the log phase initially with a medium with a lot of protein This encourages rapid increase in the number of cells, but not much protease is produced. Cells are then introduced into the fermentation vessel and allowed to grow for a further 1-8 days. The medium now has very little protein in it. Why?

The microbe must produce a lot of protease because as the enzyme leaves the cell it doesn’t immediately come into contact with protein that it can break down – more protease produced to maximise the amount of amino acids from the small amount of protein.

Down stream processing The remaining mixture contains enzymes, waste materials, nutrients and cells The enzyme is extracted by downstream processing

The nature of the downstream processing depends on two considerations: Whether enzyme is intracellular or extracellular How pure the final product needs to be. Industrial enzymes can be quite crude, but medicinal enzymes must be extremely pure. The purer the enzyme, the more complex the downstream processing, and the more expensive it is.

Break open cells by grinding or ultra-sonics Extracellular enzyme Intracellular enzyme Break open cells by grinding or ultra-sonics Cell biomass (useful waste product) Filter Enzyme in solution Crude enzyme in solution eg protease in chemical industry Concentrate by evaporation at low temperature and pressure or by osmosis Powdered crude enzyme eg pectinase Precipitate Pure enzyme for medicine eg glucose oxidase Chromatography

Immobilised enzymes When purified enzymes are used to make large quantites of another product, downstream processing can be difficult and expensive. Immobilising enzymes is cheaper. Enzyme molecules are attached to a support matrix rather than free in solution. They still function properly but can be kept separate from the reactants and the products. Immobilised enzymes are usually used in continuous flow-through re-actors, which have a low volume.

Isolated enzymes can be immobilised so that they do not contaminate the end product and can be used again and again

Methods of Immobilisation Entrapment The enzyme is entrapped within an inert matrix, such as alginate, silica or collagen, and cannot be washed out. The substrate and product molecules can diffuse in and out of the matrix, but this diffusion may limit the rate of the reaction. This is the most gentle method of entrapment, and does little damage to the enzymes.

Adsorption The enzyme molecules are attached by weak physical forces to a support matrix, such as glass beads or carbon particles. This does not chemically modify the enzyme molecules, but the adsorption process may cause the enzymes to loose their shape and therefore their activity. The molecules may also become detached during the bioconversion reaction.

Cross-linking Enzyme molecules are chemically cross-linked by covalent bonds using glutaraldehyde: E=CH–CH2–CH2–CH2– CH=E. This only works for some enzymes, but is very successful. Used in Biosensors