• Enzymes are proteins specialized to catalyze biological reactions.

• Most remarkable biomolecules due to their extraordinary specificity and catalytic power

• Far greater than those of man-made catalysts

Enzymes are obtained from• Plant source – Papain, ficin (latex)• Animal source – Rennet• Microbial source – amylase, proteases,

cellulase, xylanase etc.,

Application Enzymes UsesFood processing Amylase, protease To produce sugars and to digest the

proteins in flour

Baby foods Trypsin To predigest baby foods

Brewing industry Amylase, glucanases, proteases, Acetolactatedecarboxylase (ALDC)

To degrade proteins and polysaccharides, improve the wort and fermentation process, increase the flavour.

Fruit juices Cellulases, pectinases Clarify fruit juices

Dairy industry Rennin, lipase, lactase Production of cheese and other diary products

Meat tenderizers papain To soften meat for cooking.

Starch industry Amylase, glucoisomerases Convert starch into glucose and simple sugars

Paper industry Amylase, cellulase, xylanase, ligninases Degrade starch, aid in sizing, decolorizing, soften the paper

Biofuel industry Cellulase, xylanase, ligniniase, lipase Production of ethanol and biodiesel

Detergent industry Amylase, protease, lipase, cellulase Remove starch, protein and lipid stains and as fabric conditioner

Photographic industry ficin Dissolve gelatin off scrap film, allowing recovery of its silver content.

Stages involved in commercial production of enzymes:

1.Isolation of microbes.

2.Screening of microbes.

3.Fermentation.

4.Increase the yield of the enzymes.

The yield has to be increased in order to minimize theproduction cost. This can be done by:

(i) developing a suitable medium for fermentation

(ii) refining the fermentation process and

(iii)improving the strain for higher production.

• The potential productivity of the organisms is controlled by its genes and hence their genome must be altered for the maximum production of enzymes.

• The techniques involved are

* Mutations

* Recombination – Protoplast fusion

* Recombinant DNA technology

• One of the most successful approaches for strain improvement.

• A mutation is any change in the base sequence of DNA - deletion, insertion, inversion, substitution.

• The types include - Spontaneous mutation- Induced mutation- Site directed mutation

1.Spontaneous mutation:Occur spontaneously at the rate of 10-10 and 10-15 per generation and per

gene.Occur at low frequency and hence not used much in industrial strain

improvement.

2. Induced mutation:The rate of mutation can be increased by various factors and agents called

mutagens. ionizing radiations (e.g. X-rays, gamma rays) non-ionizing radiations (e.g. ultraviolet radiations)various chemicals (e.g. mustard gas, benzene, ethidium bromide,

Nitrosoguanidine-NTG)

3. Site directed mutations(SDM) (site-specific mutagenesis ):

• Change in the base sequence of DNA • changing the codon in the gene coding for that

amino acid. • Can be done by protein engineering method• Desired improvements might be

*increased thermostability*altered substrate range*reduction in negative feedback inhibition*altered pH range etc.,

• Karana and Medicherla (2006)- lipase from Aspergillus japonicus MTCC 1975- mutation using UV, HNO2, NTG showed 127%, 177%, 276% higher lipase yield than parent strain respectively

• Sandana Mala et al., 2001- lipase from A. niger - Nitrous acid induced mutation – showed 2.53 times higher activity.

• Kim et al., 1998 did a comparative study on strain improvement of Aspergillus oryzae for protease production by both mutation and protoplast fusion.

• UV radiation – 14 times higher yield.• Ethyl methanesulphonate – 39 times higher yield.• Protoplast fusion – using PEG and CaCl2 – 82 times

higher yield.

• The more advanced method• to increase the yields and consistencies of enzymes.• Genetic material derived from one species may be

incorporated into another where it is expressed• Increases the production of heterologous proteins

by: - increasing the gene expression using strong promoters - deletion of unwanted genes from the genome- manipulation of metabolic pathways.

Steps involved:

• Preparation of desired DNA

• Insertion of desired DNA into vector DNA

• Introduction of recombinant DNAs into host cells

• Identification of recombinants

• Expression of cloned genes

• Sidhu et al., 1998 tried both mutagenesis and cloning in E.coli for increased production by amylase in Bacillus sp. MK716.- Mutation-ethyl methane sulphonate – 40 times higher.- Mutated gene-cloned in E.coli pBR322 - 107 times

higher yield than parent strain.• Calado et al., 2004 – cutinase enzyme – Arthrobacter

simplex - 205 fold higher.

Enzymes are now used in a wide range of industrial processes. The study of industrial enzymes and their uses is called enzyme technology. The advantages and disadvantages of using enzymes are directly related to their properties:

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

Enzyme TechnologyEnzyme Technology

Isolating the Enzyme • Pure enzymes are needed for commercial use;

therefore microbes must be grown in aseptic conditions, free from contaminants - such as unwanted chemicals - and other microbes.

• It is necessary to prevent contamination with other bacteria since: - there may be competition for nutrients; - the required enzyme may not be produced as readily; - the end-product may be contaminated and unsafe.

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

Micro-organisms 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)

The required enzyme that is finally produced must also be isolated from the microbial cells

• Extracellular enzymes are present in the culture outside the microbial cells, since they have been secreted. They are often soluble in water, so they can readily be extracted from the culture medium and purified.

• These enzymes are cheaper to produce and tend to be more stable – they are therefore the preferred choice, when available!

• To obtain an intracellular enzyme, the microbe cells are harvested (by filtration or centrifugation) from the culture and are then broken up.

• The mixture is next centrifuged to remove large cell fragments and the enzymes are precipitated from solution by a salt or alcohol.

• The required enzyme must then be purified by techniques such as electrophoresis or column chromatography.

The micro-organisms(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 micro-organisms before use

Micro-organisms have beenused for thousands of yearsfor making products such as

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

Products of Enzyme TechnologyProducts of Enzyme Technology

Non-recombinant Sources

GRAS

Bacillusprotein is secreted into fermentation mediumeasier purification

Aspergillus

Yeast

Recombinant SourcesMost industrial enzymes are produced recombinantly

Why?A. Higher expressionB. Higher purity (% protein:other junk)C. cheapD. can engineer proteinE. can express enzymes which are found in pathogenic organisms

Protein engineering

Make oxidation resistant

make enzymes tolerant of processes used in industry

less substrate specificity

more thermostable

more stable in presence of detergent

The large scale production of enzymes involves culturing micro-organismsin chambers called FERMENTERS or BIOREACTORS

Micro-organisms 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

• Micro-organisms can be genetically engineered to improve the strain and enhance yields

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

• Micro-organisms have simple growth requirements and these can be precisely controlled within the fermenter

• Micro-organisms can utilise waste products such as agricultural waste as substrates

Large Scale Production of EnzymesLarge Scale Production of Enzymes

MODIFICATION – possibleapplication of genetic

engineering to improvethe microbial strain

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

Micro-organism

PILOT PLANT – small scalefermenter to clarify optimum

operating conditions

SCREENING – choosing anappropriate micro-organism

for the desired enzyme

INDUSTRIAL SCALEFERMENTATION

The Biotechnological Process of Enzyme ProductionThe Biotechnological Process of Enzyme Production

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

Commercial Enzyme Production - An ExampleCommercial Enzyme Production - An Example

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

Enzymes in BiotechnologyEnzymes in Biotechnology

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

Immobilised EnzymesImmobilised Enzymes

Four main methods available for immobilising enzymes1. Adsorption in glass or alginate beads –

enzyme is attached to the outside of an inert material

2. Cross-linkage to another chemical e.g. cellulose or glyceraldehydes.

3. Entrapment in a silica gel – enzyme is held in a mesh or capsule of an inert material.

4. Membrane confinement

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 bondedto, and cross-linked within, a matrix

Cross-linking and covalent bonding maycause some enzymes to lose their catalyticactivity especially if the active site is involvedin forming the 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

Advantages of Immobilising EnzymesAdvantages of Immobilising Enzymes

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

Enzymes as Analytical AgentsEnzymes as Analytical Agents

This method relies upon the specificity of the enzyme glucose oxidase, 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 which glucoseoxidase, 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 + DThe amount of coloured compound (D) produced is a direct

measure of the amount of glucose in the sample

Glucose Measurement using 'Clinistix'Glucose Measurement using 'Clinistix'

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 glucoseNoglucose

Glucose Measurement using 'Clinistix'Glucose Measurement using 'Clinistix'

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

BiosensorsBiosensors

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

BiosensorsBiosensors

The industrial use of enzymes (using the whole microbe)

1. Brewing: • Yeast (S. cerevisiae) reacts with the sugars in

fruit or malted barley to produce ethanol and carbon dioxide

• The process of fermentation takes several days or weeks and results in a product with a maximum alcohol content of about 12% - above which the yeast is itself killed

2. Vinegar production:

• To make vinegar, wine is slowly pored over oak chips in a tall tower, open to the air.

• Bacteria (Acetobacter) on the wood oxidise the ethanol in the wine and turn it into ethanoic acid or vinegar, giving out a great deal of heat as well.

• If the vinegar is made from fermented raisins and stored in oak vats (similar to the solera system used for making sherry) then the sweet, highly-prized Balsamic vinegar is made – mainly around Modena in Italy.

3. Yoghurt production• Milk goes sour within a few hours in the hot

conditions common in the Middle East. • If stored in a leather bag and mixed with a

suitable starter culture, however, it rapidly turns into yoghurt, which will keep for several days.