Enzyme Technology

Enzymology and Enzyme Technology
BIOT. 552
Haramaya University
Dr. Zekeria Yusuf 1

Topics to be covered
1. What are enzymes, nomenclature, classification,

properties etc
2. Applications of enzymes
3. Enzyme kinetics
4. Regulations of enzyme activity
5. Enzyme productions: microbial, plant and animal cells,
genetic engineering, and protein engineering.
6. Enzyme immobilizations
7. Enzymes as biosensors
8. The role of nanoparticles in enzyme technologies
Dr. Zekeria Yusuf 2
Dr. Zekeria Yusuf 3
Dr. Zekeria Yusuf 4
Enzymes
• named by adding the suffix “-ase” to the name of their substrate or
to a word or phrase describing their activity.
• Other enzymes were named by their discoverers for a broad
function, before the specific reaction catalyzed was known. For
example, an enzyme known to act in the digestion of foods was
named pepsin, from the Greek pepsis, “digestion,” and lysozyme
was named for its ability to lyse bacterial cell walls.
• Still others were named for their source: trypsin, named in part
from the Greek tryein, “to wear down,” was obtained by rubbing
pancreatic tissue with glycerin.
• The distinguishing feature of an enzyme-catalyzed reaction is that
it takes place within the confines of a pocket on the enzyme called
the active site.
• The molecule that is bound in the active site and acted upon by the
enzyme is called the substrate.
Dr. Zekeria Yusuf 5
Dr. Zekeria Yusuf 6
Dr. Zekeria Yusuf 7
Dr. Zekeria Yusuf 8
Dr. Zekeria Yusuf 9
Dr. Zekeria Yusuf 10
Enzyme technology…

Sources of enzymes
• Microbes are still the most common source of industrial enzymes.

• Microorganisms produce enzymes inside their cells (intracellular enzymes)
and may also secrete enzymes for action outside the cell (extracellular
enzymes).
• The microorganisms selected are usually cultured in large fermentation

chambers (known as fermenters) under controlled conditions to maximise
enzyme production.
• The microorganisms may have specific genes introduced into their DNA
through genetic engineering, so that they produce enzymes naturally made
by other organisms - this is explained in further detail under the genetic
engineering section of this unit.
Sources of enzymes
• Micro-organisms have been used for thousands

of years for making products such as wine,
beer, vinegar, soy sauce, bread and cheese.
• Many industrial processes now make use of
pure sources of enzymes that have been
isolated from micro-organisms: 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).
Growing microbes in a fermenter
• Given a suitable nutrient medium and the right conditions (temperature,

pH, oxygen levels (many microbes are obligate anaerobes, i.e. are killed
by oxygen), it is easy to grow microbes on a laboratory scale in Petri
dishes, test tubes and flasks. However, producing substances such as
penicillin from microbes on an industrial scale causes serious problems
because massive numbers of organisms have to be grown for
commercial use.
Requirements for the production of microbes in fermenters:
• Oxygen is needed for aerobic respiration of (some) micro-organisms –
others are strict anaerobes and oxygen must be excluded
• a source of Carbohydrate is needed as an energy source for respiration
to release energy needed for growth.
• a source of Nitrogen is needed need nitrogen for protein synthesis –
Ammonia (NH3) and urea ((NH2)2CO) are both widely used as (cheap)
sources of useable nitrogen
• The microorganisms are grown in very large
vessels called fermenters – as shown in this
simplified diagram:
• The large stainless steel cavity is filled with a
sterile nutrient solution, which is then
inoculated with a pure culture of the
carefully selected fungus or bacterium.
• Paddles rotate the mixture so that the
suspension is mixed well. As the nutrients
are used up, more can be added. Probes
monitor the mixture and changes in pH,
oxygen concentration and temperature are
all computer controlled. A water jacket
surrounding the fermenter contains fast
flowing cold water to cool the fermenter
since fermentation is a heat generating
process. Most of the air, including carbon
dioxide and other gases produced by cell
metabolism, leave the fermenter by an
exhaust pipe.
Typical flowchart for enzyme production using fermentation
process

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. Microorganisms
such as bacteria and fungi are saprobionts i.e. they feed
saprophytically, secreting enzymes onto their food – making
them a good source of extracellular enzymes.
For example, the fungus Aspergillus niger produces an enzyme
called pectinase, which breaks down pectin, a substance found in
the cell walls of plant cells. The fruit juice industry uses pectin
widely, since when fruit is crushed to extract the juice, pectin
prevents some being released and also makes the juice cloudy.
Comparison of intra- and extra-cellular enzymes:

• 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. Less common in
Nature (though genetic engineering can be used to modify cells to promote
this), 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 (all of
them!) are precipitated from solution by a salt or alcohol. The required
enzyme must then be purified by techniques such as electrophoresis or
column chromatography.
• This last process is complicated and expensive, so these enzymes are only
used when no other alternative is available. By their very nature, they tend to
be more sensitive to their operating conditions, which makes their commercial
use less easy. On the other hand, they are much more common in Nature!

General properties of enzymes

The fungus named as Aspergillus tubingensis which breaks down the plastic
materials using its enzymes.
• Cellulases, beta-glucanases, alphaamylases,
proteases, maltogenic amylases are used for
liquefaction, clarification and to supplement
malt enzymes in brewery industry.
• Amyloglucosidase: for conversion of starch to
sugar.

Enzymes in baking industries/bakery
Glucose oxidase Stability of dough

Lactose: used to remove lactose sugar from milk and other dairy
products

Enzymes in food industry
• Enzymes break down specific components within fruit
& vegetables such as pectin, starch, proteins and
cellulose which results in increased yields, shortening
of processing time and improving sensory
characteristics.
Some examples:
• Pectinases and Cellulases are used to break down cell
walls in fruit and vegetables, resulting in improved
extraction and increase in yield. They can also be
used to decrease the viscosity of purees or nectars,
and to provide ‘cloud stability’ and texture in juices.
Medical uses of enzymes
Diagnostic:
• Reagent strips have been designed to perform rapid and semi-quantitative
analysis for glucose. They are easy to use and require no addition
laboratory equipment or reagents. A Clinistix contains molecules of two
enzymes fixed onto the end of a plastic strip. When this is dipped into a
sample, the first, glucose oxidase, converts any glucose molecules, by
reaction with atmospheric oxygen, into gluconic acid and hydrogen
peroxide.
• The second enzyme, peroxidase, then enables the hydrogen peroxide to
react with an indicator to give a purple colour. A colour chart on the strip
will match the shade of purple to the glucose concentration.
• The idea of fixing an enzyme to a plastic support is the basic principle of
biosensors - mobile, cheap and accurate sensors which can monitor a
number of biochemicals in blood, urine and also in food and soil. Over
the next few years, the use of biosensors is likely to increase
dramatically.
Enzyme Catalysis
• A simple enzymatic reaction might be written:
• where E, S, & P represent the enzyme, substrate, and product;

• ES and EP are transient complexes of the enzyme with the substrate & with the
product.
• The activation energy is the energy barrier needed to overcome during chemical
reactions.
• Catalysts enhance reaction rates by lowering activation energies.
• Enzymes are considered to be biological catalysts and do not affect reaction
equilibria.
• Any enzyme that catalyzes the reaction SP also catalyzes the reaction PS.

Enzyme kinetics

Enzymes Are Subject to Reversible or Irreversible Inhibition
• Enzyme inhibitors are molecular agents that interfere with

catalysis, slowing or halting
• enzymatic reactions applicable in pharmaceuticals (drug
development). Forex, aspirin (acetylsalicylate) inhibits the
enzyme that catalyzes the first step in the synthesis of
prostaglandins, compounds involved in many processes,
including some that produce pain.
• There are two broad classes of enzyme inhibitors: reversible and
irreversible. The double reciprocal
• plot offers an easy way of determining whether an enzyme
inhibitor is competitive, uncompetitive, or mixed.

• Allosteric means "other site" or "other
structure".
• The interaction of an inhibitor at an allosteric
site changes the structure of the enzyme so
that the active site is also changed.

Two Processes Involving the Allosteric Enzyme

2. Feedback Inhibition
• An enzyme regulation process in which formation of a
product inhibits an earlier reaction in the sequence.
• It controls the allosteric enzymes.
• This occurs when an end-product of a pathway
accumulates as the metabolic demand for it declines.
• This end-product in turn binds to the regulatory
enzyme at the start of the pathway and decreases its
activity -the greater the end-product levels the greater
the inhibition of enzyme activity.
3. Proenzymes (Zymogen)
• The inactive form of enzyme which can be activated
by removing a small part on their polypeptide chain.
• Mostly are the digestive enzymes and blood clotting
enzymes.
• Why is it that digestive enzymes are in inactive state
before it becomes active?
• This is necessary to prevent digestion of pancreatic
and gastric tissues.

4. Protein Modification
• Another mechanism that can on
and off the enzyme.
• This a process in which a chemical
group is covalently added to
removed from the protein.
• Phosphorylation, whereby a
phosphate is transferred from an
activated donor (usually ATP) to
an amino acid on the regulatory
enzyme, is the most common
example of this type of
regulation.

Enzyme inhibitors are chemicals that can bind to enzyme
and either eliminate or drastically reduce their catalytic
ability.

Classification of enzyme inhibition
1. Reversibility: deals whether the inhibition will

eventually dissociate from the enzyme
releasing it in the active form.
2. Competition:
- Refers whether the inhibitor is a structural
analog or look – alike of the natural substrate.
- If so, the inhibitor and substrate will compete
for the enzyme’s active site.

1. Irreversible Inhibitors
• usually covalently modify an enzyme, and inhibition can
therefore not be reversed.
• Often contains highly reactive functional group such as
aldehydes, alkenes, haloalkenes, etc.,

2. Reversible Inhibitors
• bind to enzymes with noncovalent interactions such as

hydrogen bonds, hydrophobic interactions and ionic bonds.
• generally do not undergo chemical reactions when bound to
the enzyme and can be easily removed.
a. Competitive Inhibitor:
• They are molecules that resemble the structure and charge
distribution of the natural substrate for a particular enzyme.
• The inhibition is competitive because the inhibitor and
substrate compete for the binding to the enzyme’s active
site.

b. Uncompetitive Inhibition:
• the inhibitor binds only to the substrate-
enzyme complex, it should not be confused
with non-competitive inhibitors.

c. Mixed Inhibition:
• The inhibitor can bind to the enzyme at the
same time as the enzyme's substrate.
• However, the binding of the inhibitor affects the
binding of the substrate, and vice versa.
• Although it is possible for mixed-type inhibitors
to bind in the active site, this type of inhibition
generally results from an allosteric effect where
the inhibitor binds to a different site on an
enzyme.
d. Non – competitive Inhibitor:
• is a form of mixed inhibition where the binding of the inhibitor
to the enzyme reduces its activity but does not affect the
binding of substrate.
• Binds to R groups of amino acids or perhaps to the metal ion
cofactor.
• Modifies the shape of the active site.

Specificity of the Enzyme – Substrate Complex
• There are four classes of Enzyme Specificity
1. Absolute Specificity:
- An enzyme that catalyzes the reaction of only one substrate.
2. Group Specificity:
• - an enzyme that catalyzes processes involving similar molecule
containing the same functional group. e.g., hexokinase
3. Linkage Specificity:
- an enzyme that catalyze the formation of breakage of only certain
binds in molecule. e.g., proteases – hydrolyzes peptide bonds
5. Stereochemical Specificity:
- an enzyme that can distinguished an enantiomer from the other.

Enzyme Production
• Enzymes are the biocatalysts synthesized by living cells. They are
complex protein molecules that bring about chemical reactions
concerned with life.
• It is fortunate that enzymes continue to function (bring out catalysis)
when they are separated from the cells.
• Enzyme technology broadly involves production, isolation, purification
and use of enzymes (in soluble or immobilized form) for the ultimate
benefit of humankind.
• In addition, recombinant DNA technology and protein engineering
involved in the production of more efficient and useful enzymes are also
a part of enzyme technology.
• The commercial production and use of enzymes is a major part of
biotechnology industry. The specialties like microbiology; chemistry and
process engineering, besides biochemistry have largely contributed for
the growth of enzyme technology.
History of enzyme production
• Microbial enzymes have been utilized for many centuries without
knowing them fully. The first enzyme produced industrially was
taka-diastase (a fungal amylase) in 1896, in United States. It was
used as a pharmaceutical agent to cure digestive disorders.
• A German scientist (Otto Rohm) demonstrated in 1905 that
extracts from animal organs (pancreases from pig and cow) could
be used as the source of enzymes-proteases, for leather
softening..
• The utilization of enzymes (chiefly proteases) for laundry
purposes started in 1915. However, it was not continued due to
allergic reactions of impurities in enzymes.
• A real breakthrough for large scale industrial production of
enzymes from microorganisms occurred after 1950s.
Enzymes from animal and plant sources
• In the early days, animal and plant sources largely

contributed to enzymes.
• Even now, for certain enzymes they are the major
sources.
• Animal organs and tissues are very good sources
for enzymes such as lipases, esterases and
proteases. The enzyme lysozyme is mostly
obtained from hen eggs.
• Some plants are excellent sources for certain
enzymes-papain (papaya), bromelain (pineapple).
Limitations
• The quantities are limited and there is a wide
variation in their distribution.
• The most important limitations are the difficulties in
isolating, purifying the enzymes, and the cost factor.
• As regards extraction of industrial enzymes from
abovine sources, there is a
• heavy risk of contamination with bovine spongiform
encephalopathy (BSE is prion disease caused by
ingestion of abnormal proteins)
• For these reasons, microbial production of enzymes
is preferred.
Example
• Aspergillus niger— A unique organism for production
of bulk enzymes
• Among the microorganisms, A. niger (a fungus)
occupies a special position for the manufacture of a
large number of enzymes in good quantities.
• There are well over 40 commercial enzymes that are
conveniently produced by A. niger.
• These include a-amylase, cellulase, protease, lipase,
pectinase, phytase, catalase and insulinase.
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 lessDr.damage
Zekeria Yusufto the environment 183
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 Dr. Zekeria Yusuf 184
The Biotechnological Process of Enzyme Production
SCREENING – choosing an INDUSTRIAL SCALE
appropriate micro-organism FERMENTATION
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
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 Precipitate the pectinase
nitrogen, with sucrose as the out of the solution and
carbon source and the substrate filter the solid
pectin to stimulate pectinase
production by the fungus
Pure, powdered Dry and purify the crude

pectinase pectinase
Enzyme production

Enzyme production…
• The first enzyme produced industrially was the fungal amylase
Takadiastase which was employed as a pharmaceutical agent for
digestive disorders.
• By 1969, 80% of all laundry detergents contained enzymes,
chiefly Proteases.
• Due to the occurrence of allergies among the production workers
and consumers, the sale of such enzyme utilizing detergents
decreased drastically.
• Special techniques like micro-encapsulation of these enzymes were
developed which could provide dustless protease preparation. It
was thus made risk free for production workers and consumers.
• Microbial rennin is also one of the most significant enzymes. It has
been used instead of Calf’s rennin in cheese production.

Location of Enzyme production
• Enzymes which are produced within the cell or

at the cytoplasmic membrane are called as
Endocellular enzymes.
• Enzymes which are liberated in the
fermentation medium which can attack large
polymeric substances are termed as Exocellular
enzymes. Eg: Amylases & Proteases.

Improved Prospects of Enzyme Application
• Microbial Genetics – High yields can be obtained by Genetic
manipulation.
• Example – Hansenula polymorpha has been genetically modified so
that 35% of it’s total protein consists of the enzyme alcohol oxidase.
• Optimization of fermentation conditions (Use of low cost nutrients,
optimal utilization of components in nutrient solution, temperature and
pH)
• New cell breaking methods like Homogenizer, Bead mill, Sonication etc
• Modern purification processes like Counter current distribution, Ion-
exchange chromatography, Molecular-sieve chromatography, Affinity
chromatography and precipitation by using alcohol, acetone.
• Immobilization of enzymes
• Continuous enzyme production in special reactors.

Submerged Culture
• Fermentation equipment used is the same as in the manufacture

of antibiotics.
• It’s a cylindrical tank of stainless steel and it is equipped with an
agitator, an aerating device, a cooling system and various
ancillary equipment (Foam control, pH monitoring device,
temperature, oxygen tension etc)
• Good growth is not enough to obtain a higher enzyme yield.
• Presence of inhibitors or inducers should also be checked in the
medium.
• Example – Presence of Lactose induces the production of β-
galactosidase.
• As the inducers are expensive, constitutive mutants are used
which do not require an inducer.
Submerged culture…
• Glucose represses the formation of some enzymes

(α-amylases). Thus the glucose concentration is
kept low.
• Either the glucose can be supplied in an incremental
manner or a slow metabolizable sugar (Lactose or
metabolized starch)
• Certain surfactants in the production medium
increases the yield of certain enzymes.
• Non- ionic detergents (eg. Tween 80, Triton) are
frequently used.
Amylase
• Amylase is an enzyme that catalyses the

hydrolysis of starch into sugars.
• Present in the saliva of humans
• Hydrolysis of Starch with amylase will first result in
the formation of a short polymer Dextrin and then
the disaccharide Maltose and finally glucose.
• Glucose is not as sweet as Fructose.
• Thus the next step would be the conversion of
Glucose to Fructose by the enzyme Glucose
isomerase.
Α-amylase producing strains
• Bacteria –
B. cereus,
B.subtilis,
B. amyloliquefaciens,
B. polymyxa,
B. licheniformis etc
• Fungi – Aspergillus oryzae, Aspergillus niger,
Penicillum, Cephalosporin, Mucor, Candida etc.
Applications
• Production of sweeteners for the food industry.

• Removal of starch sizing from woven cloth
• Liquefaction of starch pastes which are formed
during the heating steps in the manufacture of
corn and chocolate syrups.
• Production of bread and removal of food spots
in the dry cleaning industry where amylase
works in conjunction with protease enzymes.

Lipase
• Lipases are also called as Glycerol ester hydrolases

• They are a subclass of esterases
• It splits fats into mono or di- glycerides and fatty acids.
• They are extracellular enzymes
• Mainly produced by Fungi
• Eg: Aspergillus, Mucor, Rhizopus, Peniciilum etc
• Bacteria producing lipases include species of
Pseudomonas, Achromobacter and Staphylococcus.
• Yeasts like Torulopsis and Candida are also commercially
used.
Mode of action of lipase
• Enzyme production must be induced by adding oils

and fats.
• But in some cases the fats have effect on the lipase
production.
• Glycerol, a product of lipases action, inhibits lipase
formation.
• Lipases are generally bound to the cells and hence
inhibit an overproduction but addition of a cation
such as magnesium ion liberates the lipase and leads
to a higher enzyme titer in the production medium.
Applications
• Primarily marketed for therapeutic purposes

as digestive enzymes to supplement
pancreatic lipases.
• Since free fatty acids affect the odor and taste
of cheese, and the cheese ripening process is
affected by lipases, microbial affects during
the aging process can be due to lipase action.
• In the soap industry, lipases from Candida
cylindraceae is used to hydrolyze oils.
Pectinase
• Pectinase is an enzyme that breaks down pectin, a
polysaccharide found in plant cell walls.
• Pectic enzymes include Pectolyase, Pectozyme and
Polygalacturonase.
• Pectin is the jelly-like matrix which helps cement plant cells
together and in which other cell wall components, such as
cellulose fibrils, are embedded.
• Basic structure of a pectin consists of α-1,4 linked
Galactouronic acid with upto 95% of it’s carboxyl groups
esterified with methanol.
• Pectinase might typically be activated at 45 to 55 °C and
work well at a pH of 3.0 to 6.5.
Production Strains
• Aspergillus niger, A. wentii, Rhizopus etc

• Fermentation with Aspergillus Niger runs for
60-80 hours in fed batch cultures at pH 3-4
and 37o C using 2% sucrose and 2% pectin.

Applications
• Pectinase enzymes are commonly used in processes

involving the degradation of plant materials, such as
speeding up the extraction of fruit juice from fruit,
including apples.
• Pectinases have also been used in wine production
since the 1960s
• Helps to clarify fruit juices and grape must, for the
maceration of vegetables and fruits and for the
extraction of olive oil.
• By treatment with pectinase, the yield of fruit juice
during pressing is considerably increased.
Protease
• Protease (Mixture of Peptidases & Proteinases)

are enzymes that perform the hydrolysis of
Peptide bonds.
• Peptide bonds links the amino acids to give the
final structure of a protein.
• Proteinases are extracellular and Peptidases are
endocellular.
• Second most important enzyme produced on a
large scale after Amylase.
Alkaline Serine Proteases
• pH of the production medium is kept at 7.0 for satisfactory
results. Have serine at the active site
• Optimum temperature maintained is 30o to 40o C.
• Important producers are B. licheniformis, B.amyloliquefaciens, B.
firmus, B. megaterium, Streptomyces griseus, S. fradiae, S.
rectus and fungi like A. niger, A. oryzae, A.flavus.
• Enzymes used in detergents are chiefly proteases from bacillus
strains (Bacillopeptidases)
• Best known proteases are Subtilisin Carlsberg from B.
licheniformis & Subtilisin BPN and Subtilisin Novo from B.
amyloliquefaciens.
• These enzymes are not inhibited by EDTA (Ethylene diamine
tetraacetic acid) but are inhibited by DFP (Di isopropyl
fluorophosphate)
Proteases for the Use in Detergent industries
• Stability at high temperature

• Stability in alkaline range (pH- 9 to 11)
• Stability in association with chelating agents
and perborates
• But shelf life is affected in presence of surface
active agents.

Screening
• Because the enzymes should be stable in

alkaline conditions, screening for better
producers is done by using highly alkaline
media.
• It was found that B. licheniformis and B.subtilis
showed growth is the range of pH 6-7
• by new strains were found to grow even in
pH10-11.
• Genetic Manipulation can also be carried out.

• To prepare a suitable encapsulated product, a
wet paste of enzyme is melted at 50-70o C
with a hydrophobic substance such as
polyethylene glycol and then converted into
tiny particles.

Neutral Proteases
• They are relatively unstable and calcium,

sodium and chloride must be added for
maximal stability.
• Not stable at higher temperatures
• Producing organisms are B. subtilis, B.
megaterium etc
• They are quickly inactivated by alkaline
proteases.

Acid Proteases
• Similar to Mammalian pepsin

• It consists of Rennin like proteases from fungi
which are chiefly used in cheese production
• They are used in medicine, in the digestion of
soy protein for soya sauce production and to
break down wheat gluten in the baking
industry.

Applications
• Textile industry to remove proteinaceous

sizing.
• Silk industry to liberate silk fibers from
naturally occurring proteinaceous material in
• which they are embedded.
• Tenderizing of Meat
• Used in detergent and food industries.

Soil enzymes
• These enzymes, usually found in the soil, may have

significant effects on soil biology, environmental
management, growth and nutrient uptake in plants
growing in ecosystems.

Biosensors
Biosensors are electronic

monitoring devices that
make
use of an enzyme’s
specificity and the
technique of enzyme
immobilisation
Biosensors
Biosensors are electronic monitoring devices that make use of an
enzyme’s specificity and the technique of enzyme immobilisation
Amplifier Read-out
Transducer
Immobilised The enzyme The electrical

enzymes bind reaction brings signal is amplified
with specific about a change and gives a
molecules that is converted read-out on a
even when they into an electrical small display
are present signal by a screen
in very low transducer
concentrations

Biosensors
A biosensor has been developed for detecting
glucose in the blood of diabetics
Amplifier
Transducer
Glucose Glucose Glucose oxidase The current generated is

molecules oxidase oxidises any glucose proportional to the amount
in the blood present in the blood to of glucose present in the
release electrons – these sample and this is displayed
are detected by the as a digital read-out
transducer and converted
into an electrical current

The role of nanoparticles in enzyme technology
•Nanotechnology is the study of manipulating matter on an atomic scale.
•Nanotechnology refers to the constructing and engineering of the functional
systems at very micro level or we can say at atomic level.
•A Nanometer is one billionth of a meter, roughly the width of three or four
atoms.
• The average human hair is about 25,000 nanometers wide.
• The first ever concept was presented in 1959 by the famous professor of physics
Dr. Richard P.Feynman.
• Invention of the scanning tunneling microscope in 1981 and the discovery of
fullerene(C60) in 1985 lead to the emergence of nanotechnology.
• The term “Nano-technology" had been coined by Norio Taniguchi in 1974.

ENZYME IMMOBILISATION
• Application of nanomaterials as novel supporting

materials for enzyme immobilisation has generated
incredible interest in the biotechnology community.
• These robust nanostructured forms, such as nanoparticles,
nanofibres, nanotubes, nanoporous, nanosheets, and
nanocomposites, possess a high surface area to volume
ratios that can cause a high enzyme loading and facilitate
reaction kinetics, thus improving biocatalytic efficiency for
industrial applications.
• nanomaterials will become an integral part of sustainable
bioenergy production.
SINGLE ENZYME NANOPARTICLES (SEN)
• Enzyme lead short and brutal lives ,to increase the

enzymes longevity and versatility, a a team at
department of Energy’s Pacific Northwest , National
Laboratory in Richlad caged single enzyme to create
a new class of catalysts called SENs.
• The nanostructure protects the catalyst, allowing it to
remain active for several months.
• Kim and Grate , working in te W.R Wiley
Environmental molecular sciences laboratory
modified a common protein splitting enzyme called
alpha chymotrypsin.
ENHANCEMENT OF ENZYME ACTIVITY AND THERMOSTABILITY
• Study on Impaired Pectate Lyase from Attenuated Macrophomina

phaseolina in Presence of Hydroxyapatite Nanoparticle
• Hydroxyapatite nanoparticles (NP) can not only act as a chaperon
(by imparting thermostability) but can serve as a synthetic
enhancer of activity of an isolated extracellular pectate lyase
(APL) with low native state activity.
• The purified enzyme showed feeble activity at 50°C and pH 5.6.
However, on addition of 10.5 μg/ml of hydroxyapatite
nanoparticles (NP), APL activity increased 27.7 fold with a 51 fold
increase in half-life at a temperature of 90°C as compared to
untreated APL.
• The upper critical temperature for such compensation was
elevated from 50°C to 90°C in presence of NP.

ENZMET (ENZYME METALLOGRAPHY)
• EnzMet (Enzyme Metallography) is a new biological

labeling and staining method developed at
Nanoprobes.
• It uses a targeted enzymatic probe with a novel
metallographic substrate to provide a quantum leap
in staining clarity over conventional chromogenic
and fluorescent substrates.
• EnzMet™ has proven highly sensitive both for in situ
hybridization (ISH), where it readily visualizes
endogenous copies of single genes, and
immunohistochemistry (IHC) detection.

Enzyme Technology

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Enzyme Technology

Uploaded by

Copyright:

Available Formats

Enzymology and Enzyme Technology

Dr. Zekeria Yusuf 1

1. What are enzymes, nomenclature, classification,

Dr. Zekeria Yusuf 22

• Microbes are still the most common source of industrial enzymes.

• The microorganisms selected are usually cultured in large fermentation

• Micro-organisms have been used for thousands

• Given a suitable nutrient medium and the right conditions (temperature,

Dr. Zekeria Yusuf 28

Dr. Zekeria Yusuf 31

Dr. Zekeria Yusuf 32

Dr. Zekeria Yusuf 33

Dr. Zekeria Yusuf 91

Glucose oxidase Stability of dough

Dr. Zekeria Yusuf 92

Dr. Zekeria Yusuf 95

• where E, S, & P represent the enzyme, substrate, and product;

Dr. Zekeria Yusuf 135

Dr. Zekeria Yusuf 136

• Enzyme inhibitors are molecular agents that interfere with

Dr. Zekeria Yusuf 144

Dr. Zekeria Yusuf 152

Dr. Zekeria Yusuf 153

Dr. Zekeria Yusuf 157

Dr. Zekeria Yusuf 160

Dr. Zekeria Yusuf 161

1. Reversibility: deals whether the inhibition will

Dr. Zekeria Yusuf 163

Dr. Zekeria Yusuf 165

• bind to enzymes with noncovalent interactions such as

Dr. Zekeria Yusuf 167

Dr. Zekeria Yusuf 168

Dr. Zekeria Yusuf 170

Dr. Zekeria Yusuf 171

• In the early days, animal and plant sources largely

Enzymes are biological catalysts that fulfil their role

This specificity is one property of enzymes that

The value of using enzymes over inorganic catalysts in the

Enzymes are able to operate at room temperature, atmospheric

Enzymes are biodegradable and, unlike many inorganic

Micro-organisms are suitable for use in the large scale production of

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

• Micro-organisms are found in a wide variety of different habitats such that

• Micro-organisms have simple growth requirements and these can be

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

LABORATORY SCALE PILOT

PILOT PLANT – small scale

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

In the drinks industry, juice extracted from fruits

Pectinase is obtained from the fungus Aspergillus niger

Aspergillus niger produces pectinase as an extracellular enzyme

Dr. Zekeria Yusuf 187

Filtration or centrifugation to obtain

Aspergillus niger is grown in

Pure, powdered Dry and purify the crude

Dr. Zekeria Yusuf 189

Dr. Zekeria Yusuf 190

• Enzymes which are produced within the cell or

Dr. Zekeria Yusuf 191

Dr. Zekeria Yusuf 192

• Fermentation equipment used is the same as in the manufacture

• Glucose represses the formation of some enzymes

• Amylase is an enzyme that catalyses the

• Production of sweeteners for the food industry.