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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

Uses

Food 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
Dairy industry
Meat tenderizers

Cellulases, pectinases
Rennin, lipase, lactase
papain

Clarify fruit juices


Production of cheese and other diary
products
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 the


production 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
Enzyme technology is concerned with the application of enzymes
as tools of industry, agriculture and medicine
Enzymes are biological catalysts that fulfil their role
by binding specific substrates at their active sites
This specificity is one property of enzymes that
makes 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 inorganic
catalysts, cause less damage to the environment

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 sources


and these include bacteria, fungi and yeast cells

Electron micrograph of bacteria (Bacillus)

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)

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.

Products of Enzyme Technology


Micro-organisms have been
used for thousands of years
for making products such as
wine, beer, vinegar, soy sauce,
bread and cheese

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

Non-recombinant Sources
GRAS
Bacillus
protein is secreted into fermentation medium
easier purification
Aspergillus
Yeast

Recombinant Sources
Most industrial enzymes are produced recombinantly
Why?
A. Higher expression
B. Higher purity (% protein:other junk)
C. cheap
D. can engineer protein
E. 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

Large Scale Production of Enzymes


The large scale production of enzymes involves culturing micro-organisms
in 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

The Biotechnological Process of Enzyme Production


SCREENING choosing an
appropriate micro-organism
for the desired enzyme

MODIFICATION possible
application of genetic
engineering to improve
the microbial strain

LABORATORY SCALE PILOT


to determine the optimum
conditions for growth of the
Micro-organism

PILOT PLANT small scale


fermenter to clarify optimum
operating conditions

INDUSTRIAL SCALE
FERMENTATION

Commercial Enzyme Production - An Example


PRODUCTION OF PECTINASE
Pectin is an insoluble substance found in the cell walls of plants
In the drinks industry, juice extracted from fruits
appears cloudy due to the presence of pectin
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

Filtration or centrifugation to obtain


a cell-free system containing
pectinase in solution

Evaporate to concentrate
the enzyme
Aspergillus niger is grown in
a fermenter with a source of
nitrogen, with sucrose as the
carbon source and the substrate
pectin to stimulate pectinase
production by the fungus

Pure, powdered
pectinase

Precipitate the pectinase


out of the solution and
filter the solid
Dry and purify the crude
pectinase

Enzymes in Biotechnology
Enzymes are used in industrial processes and as analytical
reagents in medicine
Thermostability and an ability to
withstand extremes of pH are
essential properties for enzymes used
in many industrial processes
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

Immobilised Enzymes
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

Four main methods available for


immobilising enzymes
1. 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

Methods for Immobilising Enzymes

Enzymes are held on to a solid


support (matrix) by weak forces
such as hydrogen bonding

Enzymes are trapped within


the structure of a solid polymer
(usually in the form of beads)
the enzyme is trapped rather
than bound

Enzymes are covalently bonded


to a matrix such as cellulose
or collagen

Another more expensive method involves


enzymes which are both covalently bonded
to, and cross-linked within, a matrix
Cross-linking and covalent bonding may
cause some enzymes to lose their catalytic
activity especially if the active site is involved
in forming the linkages

Advantages of Immobilising Enzymes


Compared with free enzymes in solution, immobilised enzymes
have 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 in


The 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 the


thermostable enzyme alpha amylase
at 90oC for a couple of hours
Alpha amylase catalyses the hydrolysis of the starch
into short glucose chains called dextrins

Dextrins
(short chains
of glucose
molecules)

The temperature is raised to 140oC to denature the


amylase and then lowered to around 55oC before
adding the fungal enzyme amyloglucosidase

Amyloglucosidase catalyses the hydrolysis of


dextrins into glucose molecules

Glucose

The final stage involves


the conversion of glucose
syrup into the much sweeter
fructose syrup using the
enzyme glucose isomerase

Glucose isomerase is immobilised


in rigid granules and packed into
a column
Glucose syrup is poured into
the top of the column and is
hydrolysed as it contacts the
immobilised enzyme
Fructose syrup emerges
from the end of the column
free from contamination
with enzyme

Enzymes as Analytical Agents


The sensitivity and specificity of enzymes makes them useful
tools 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 the
presence of specific molecules even when they are
present at very low concentrations
The enzyme glucose oxidase is used in an immobilised form
for the detection of glucose in biological fluids

Glucose Measurement using 'Clinistix'


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 glucose
as it gives a positive reaction with ALL reducing sugars
This test uses a plastic strip (clinistix) for the
detection of glucose in the urine of diabetics
When the clinistix is dipped into a urine sample
(containing glucose), the glucose oxidase catalyses
the conversion of glucose to hydrogen peroxide:
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 direct


measure of the amount of glucose in the sample
At the tip of the clinistix is a cellulose fibre pad on to which glucose
oxidase, peroxidase and a chromagen dye are immobilised

Glucose Measurement using 'Clinistix'


The colour of the pad on the clinistix is compared with
a colour chart to determine the amount of glucose
present in the sample

No
glucose

Increasing amounts of glucose

Biosensors
Biosensors are electronic monitoring devices that make use of
an
enzymes specificity and the technique of enzyme
immobilisation

Amplifier

Read-out

Transducer

Immobilised
enzymes bind
with specific
molecules
even when they
are present
in 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 a
read-out on a
small display
screen

Biosensors
A biosensor has been developed for detecting
glucose in the blood of diabetics

Amplifier
Transducer

Glucose
molecules
in the blood

Glucose
oxidase

Glucose oxidase
oxidises any glucose
present in the blood to
release electrons these
are detected by the
transducer and converted
into an electrical current

The current generated is


proportional to the amount
of glucose present in the
sample and this is displayed
as a digital read-out

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, highlyprized 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.

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