© SSER Ltd.

Enzyme technology is concerned with the application of enzymesas tools of industry, agriculture and medicine

Enzymes are biological catalysts that fulfil their roleby binding specific substrates at their active sites

This specificity is one property of enzymes thatmakes them useful for industrial applications

The value of using enzymes over inorganic catalysts in the technological field is their efficiency, selectivity and specificity

Enzymes are able to operate at room temperature, atmospheric pressure and within normal pH ranges (around 7)

– all of which create energy savings for industry

Enzymes possess specifically shaped active sites for reacting with one specific substrate thereby generating pure products

free from unwanted by-products

Enzymes are biodegradable and, unlike many inorganiccatalysts, cause less damage to the environment

The microorganisms (such as yeast) are really used as a source of enzymes during

the manufacture of these products of biotechnology

Many industrial processes now make use of pure sources of enzymes, i.e. the enzymes have been ISOLATED from the microorganisms before use

Microorganisms have beenused for thousands of yearsfor making products such as

wine, beer, vinegar, soy sauce,bread and cheese

Microbial enzymes are ISOLATED from a variety of sourcesand these include bacteria, fungi and yeast cells

Microorganisms produce enzymes that function inside their cells(intracellular enzymes) and they may also produce enzymes that are

secreted and function outside the cells (extracellular enzymes)

Electron micrograph of bacteria (Bacillus)

Intracellular enzymes function within cellsExtracellular enzymes are secreted from cells

and function in the external environmentExtracellular enzymes are more commonly used in biotechnological processes

Extracellular Enzymes Intracellular Enzymes

Isolation is easier Isolation is more difficult

Enzymes are secreted into the medium in which the microorganisms are growing – the cells do not require breaking apart

Microorgansims need to be harvested from the culture medium and broken apart to obtain the enzymes

The required enzyme is generally secreted as a single product

A mixture of enzymes, other chemicals and cell debris are present when the cells are broken apart and these have to be separated from the desired enzyme

Extracellular enzymes are more stable in an isolated form

Intracellular enzymes are less stable when isolated from the cells

Downstream processing (extraction and purification of the enzyme product) is relatively easy and low cost

Downstream processing is generally more difficult and more expensive

The large scale production of enzymes involves culturing microorganismsin chambers called FERMENTERS or BIOREACTORS

Microorganisms are suitable for use in the large scale production of enzymes in fermenters because:

• They have rapid growth rates and are able to produce larger numbers of enzyme molecules per body mass than many other organisms

• Microorganisms can be genetically engineered to improve the strain and enhance yields

• Microorganisms are found in a wide variety of different habitats such that their enzymes are able to function across a range of temperatures and pH

• Microorganisms have simple growth requirements and these can be precisely controlled within the fermenter

• Microorganisms can utilise waste products such as agricultural waste as substrates

MODIFICATION – possibleapplication of genetic

engineering to improvethe microbial strain

LABORATORY SCALE PILOT – to determine the optimumconditions for growth of the

microorganism

PILOT PLANT – small scalefermenter to clarify optimum

operating conditions

SCREENING – choosing anappropriate microorganism

for the desired enzyme

INDUSTRIAL SCALEFERMENTATION

The microorganisms thatproduce the desired enzymeare grown on a large scalein fermenters containing asuitable growth medium

Temperature probepH probe

Stainless steel vat withsmooth inner surfaces which allow for easy steam cleaningand prevents:• microbes becoming trapped• corrosion of the surfaces

Sterilised nutrient solution inoculated with a pureculture of the microorganism

Sterilised air

Sparger for forcingair into the medium

Vent forwastegases

Sampling tube

Water jacket forcirculating coolingwater to removethe heat generatedby the metabolicactivity of themicroorganisms

Foam breaker

Motor

The microorganisms thatproduce the desired enzymeare grown on a large scalein fermenters containing asuitable growth medium

Temperature probepH probe

Stainless steel vat withsmooth inner surfaces which allow for easy steam cleaningand prevents:• microbes becoming trapped• corrosion of the surfaces

Sterilised nutrient solution inoculated with a pureculture of the microorganism

Sterilised air

Sparger for forcingair into the medium

Vent forwastegases

Sampling tube

Water jacket forcirculating coolingwater to removethe heat generatedby the metabolicactivity of themicroorganisms

Foam breaker

Motor

Impellers (stirrers) for mixingthe microorganisms andnutrients and distributingthe heat

The nutrients added to the fermenter must allow for optimum growthof the microorganisms being used – different microorganisms

may have specific nutritional requirements All microorganisms require a source of carbon as it is a component of

all their organic molecules, e.g. carbohydrate, protein, fat, nucleic acid.Some microorganisms can rely on an inorganic source of carbon suchas CO2 whereas others require specific organic sources such as sugars

All microorganisms require a nitrogen source for the manufacture ofproteins necessary for growth. The nitrogen source may be inorganic,

e.g. ammonium and nitrate salts but for most microorganisms thenitrogen requirement is met by organic sources such as amino acids

Sulphur is a nutrient requirement for all microorganisms and may bemet by inorganic sulphates or organic sulphur-containing amino acids

Microorganisms require a supply of vitamins in relatively smallquantities to satisfy all of their synthetic and growth needs

Aerobic microorganisms require a supply of molecular oxygen for aerobicrespiration. O2 must be excluded from fermenters when anaerobic organisms

are being used

Microorganisms need to be grown in aseptic conditions to prevent contamination of the culture medium with other unwanted

microorganisms and to maintain yield

If unwanted microorganisms were to grow in the same culture medium asthe desired organisms, competition for nutrients would occur and the yieldof product (e.g. enzyme) would be reduced – resulting in economic losses

Aseptic conditions are ensured by:

• Cleaning, steam sterilising and polishing the fermenter before use

• Heat sterilising the culture medium before use

• Sterilising all materials to be added to the fermenter such as the air supply and anti-foaming agents

• Sterilising the air leaving the fermenter

There are two main types of fermentation processes:

BATCH FERMENTATION – in which the fermentation process is set up, nutrients are added at the start and no further modifications are made. Fermentation is stopped when sufficient product has formed, then the productis removed and purified and the fermenter is cleaned and sterilised ready forthe next batch – this method is generally used for enzyme production

CONTINUOUS FERMENTATION – involves long-term fermentation overa number of weeks. Nutrients are added continuously to the fermenter andproducts are removed at regular intervals without shutting down the system

A third process known as FED-BATCH FERMENTATION is really acompromise between the two main processes and is increasingly being usedfor a number of industrial processes

Fed-batch fermentation involves adding nutrients and other materials inlow concentrations throughout the operation. In many cases, better yieldsare obtained from this method as the growth period is monitored,controlled and extended

Following fermentation, the desired product is extracted and purified ina procedure known as DOWNSTREAM PROCESSING

The steps involved in downstream processing depend upon whether thedesired product (e.g. enzyme) is located within the microbial cells

or in the culture solutionIf the desired product is dissolved in the culture solution, it must be

concentrated and then purified

If the desired product is located within the microbial cells, then these mustfirst be broken open to release the product before extraction and purification

Irrespective of whether the product is intracellular or extracellular, the firststep in the procedure is to separate the microbial cells from the culture solution;

this is usually achieved by filtration followed by centrifugation

Cells are separatedfrom the solution

Microbial cellsare broken open

to release intracellularenzymes

Extracted enzymesare purified

Purified enzyme is packaged,stored and marketed

(Formulation)

Cell debris is removedby centrifugation

The resulting DILUTE solutionis distilled to concentrate the

enzyme solution

Enzyme solution is purified and dried

Purified enzyme is packaged,stored and marketed

(Formulation)

Solution containingmicrobial cells is

centrifuged to obtaina cell-free system

Pectin is an insoluble substance found in the cell walls of plants

In the drinks industry, juice extracted from fruitsappears cloudy due to the presence of pectin

PRODUCTION OF PECTINASE

Pectinase is an enzyme that is used in the industry to break down the pectin

The effect of pectinase is to clarify the fruit juice and to make it flow more freely

Pectinase is obtained from the fungus Aspergillus niger

Aspergillus niger produces pectinase as an extracellular enzyme

PRODUCTION OF PECTINASE

Aspergillus niger is grown ina fermenter with a source ofnitrogen, with sucrose as the

carbon source and the substratepectin to stimulate pectinase

production by the fungus

Filtration or centrifugation to obtaina cell-free system containing

pectinase in solution

Evaporate to concentrate the enzyme

Precipitate the pectinaseout of the solution and

filter the solid

Dry and purify the crudepectinase

Pure, powdered pectinase

Enzymes are used in industrial processes and as analytical reagents in medicine

Immobilisation of enzymes is an important technique used in industry as it enables economical operation of a process

and protection of enzymes during their use

Because of their sensitivity and specificity, enzymes are used as analytical reagents in systems such as the detection

of glucose in human blood and urine

Thermostability and an ability to withstand extremes of pH are

essential properties for enzymes usedin many industrial processes

The optimum temperature for some of these enzymes is above 100oC

One problem associated with thermophilic bacteria is that they often donot produce large quantities of the required enzyme

This problem has been overcome using gene technology: the gene forthermostable enzyme production has been transferred into higher yielding

bacteria such as Bacillus subtilis, creating strains that demonstrate good enzyme production and thermostability

The advantages of using thermostable enzymes for industrial processes include:

• Chemical reactions can occur at faster rates at higher temperatures and the rate of product formation is significantly increased

• The fermentation process requires less monitoring and control providing economic benefits to the industry

                                              

Certain species of bacteria, called Thermophilic Bacteria, possess enzymes

that can operate at temperatures that would denature most known enzymes

Thermophilic bacteria inhabit hot springs

Thermophilic bacteria inhabit volcanic vents

The costs associated with the use of enzymes for industrial purposes can also be reduced by immobilising the enzymes

Enzymes for industrial processes are more valuable when they are able to act in an insolubilised state rather than in

solution

Enzymes are immobilised by binding them to, or trapping them in a solid support

Various methods for immobilising enzymes are available

Enzymes are held on to a solidsupport (matrix) by weak forcessuch as hydrogen bonding

Enzymes are trapped withinthe structure of a solid polymer(usually in the form of beads)– the enzyme is trapped ratherthan bound

Methods for Immobilising Enzymes

Enzymes are covalently bondedto a matrix such as celluloseor collagen

Another more expensive method involvesenzymes which are both covalently bonded to,and cross-linked within, a matrix.Cross-linking and covalent bonding may causesome enzymes to lose their catalytic activityespecially if the active site is involved in formingthe linkages

Compared with free enzymes in solution, immobilised enzymeshave a number of advantages for use in industrial processes

The stability of many enzymes is increased when they are in an immobilised state; they are less susceptible to changes in

environmental conditions such as temperature and pH fluctuations

Immobilised enzymes can be recovered and re-used,reducing overall costs

The products of the reaction are not contaminated with enzyme eliminating the need to undertake costly separation of

the enzyme from the product

Immobilising enzymes allows for continuous production of a substance with greater automation

Enzyme Immobilisation and Thermostable Enzymes inThe Production of High Fructose Syrup

This industrial process involves the conversion of cheap corn starch into a high fructose syrup for use as a sweetener in confectionary and drinks

Starch Paste Starch paste is incubated with thethermostable enzyme alpha amylase

at 90oC for a couple of hours

Dextrins(short chains

of glucosemolecules)

Alpha amylase catalyses the hydrolysis of the starchinto short glucose chains called dextrins

The temperature is raised to 140oC to denature theamylase and then lowered to around 55oC before

adding the fungal enzyme amyloglucosidase

Glucose

Amyloglucosidase catalyses the hydrolysis ofdextrins into glucose molecules

Fructose syrup emergesfrom the end of the column

free from contaminationwith enzyme

The final stage involvesthe conversion of glucose

syrup into the much sweeterfructose syrup using the

enzyme glucose isomerase

Glucose isomerase is immobilisedin rigid granules and packed into

a column

Glucose syrup is poured intothe top of the column and ishydrolysed as it contacts the

immobilised enzyme

The sensitivity and specificity of enzymes makes them usefultools in medicine for the detection and measurement of chemicals

in fluids such as blood and urine

Because of their specificity, enzymes will bind to only one substrate – they can therefore be used for the identification

of a specific substance in a biological sample

Because of their sensitivity, enzymes are able to detect thepresence of specific molecules even when they are

present at very low concentrations

The enzyme glucose oxidase is used in an immobilised formfor the detection of glucose in biological fluids

This method relies upon the specificity of the enzyme glucoseoxidase, allowing glucose to be detected in the presence of

other sugars. N.B. Benedict's test is not specific for glucoseas it gives a positive reaction with ALL reducing sugars)

At the tip of the clinistix is a cellulose fibre pad on to whichglucose oxidase, peroxidase and a chromagen dye are immobilised

When the clinistix is dipped into a urine sample(containing glucose), the glucose oxidase catalysesthe conversion of glucose to hydrogen peroxide:

This test uses a plastic strip (clinistix) for thedetection of glucose in the urine of diabetics

Glucose + O2

gluconic acid + hydrogen peroxide (H2O2)

In the presence of the enzyme peroxidase, the chromagen dye is oxidised by the hydrogen peroxide

to produce a colour change on the fibre pad

DH2 ( chromagen dye) + H2O2 2H2O + D

The amount of coloured compound (D) produced is a directmeasure of the amount of glucose in the sample

The colour of the pad on the clinistix is compared witha colour chart to determine the amount of glucose

present in the sample

Increasing amounts of glucoseNo

glucose

Biosensors are electronic monitoring

devices that makeuse of an enzyme’sspecificity and the

technique of enzyme immobilisation

Biosensors are electronic monitoring devices that make use of an

enzyme’s specificity and the technique of enzyme immobilisation

Transducer

Amplifier Read-out

Immobilised enzymes bindwith specific

moleculeseven when they

are presentin very low

concentrations

The enzyme reaction brings about a change

that is converted into an electrical

signal by a transducer

The electrical signal is amplified

and gives aread-out on a small display

screen

A biosensor has been developed for detecting

glucose in the blood of diabetics

Glucose oxidaseoxidises any glucose

present in the blood torelease electrons – these

are detected by the transducer and convertedinto an electrical current

Transducer

Amplifier

The current generated isproportional to the amount

of glucose present in thesample and this is displayed

as a digital read-out

Glucose molecules

in the blood

Glucoseoxidase

END SHOW