2. 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
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5. 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. 5
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23. 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.
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24. 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).
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26. 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
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27. • 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.
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28. Typical flowchart for enzyme production using fermentation process
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30. 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.
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32. • 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!
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91. • 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.
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92. Enzymes in baking industries/bakery
Glucose oxidase Stability of dough
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109. 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.
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114. 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.
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135. 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.
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144. 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.
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152. • 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.
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155. 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.
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157. 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.
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160. 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.
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161. Enzyme inhibitors are chemicals that can bind to enzyme
and either eliminate or drastically reduce their catalytic
ability.
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163. 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.
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165. 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.,
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167. 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.
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168. b. Uncompetitive Inhibition:
• the inhibitor binds only to the substrate-
enzyme complex, it should not be confused
with non-competitive inhibitors.
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169. 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.
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170. 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.
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171. 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.
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172. 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.
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174. 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.
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176. 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).
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178. 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.
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182. 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.
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183. 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
Enzyme Technology
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184. 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
Large Scale Production of Enzymes
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185. 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
SCREENING – choosing an
appropriate micro-organism
for the desired enzyme
INDUSTRIAL SCALE
FERMENTATION
The Biotechnological Process of Enzyme Production
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187. 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
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 Example
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188. PRODUCTION OF PECTINASE
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
Filtration or centrifugation to obtain
a cell-free system containing
pectinase in solution
Evaporate to concentrate
the enzyme
Precipitate the pectinase
out of the solution and
filter the solid
Dry and purify the crude
pectinase
Pure, powdered
pectinase
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190. 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.
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191. 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.
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192. 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.
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198. 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.
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199. 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.
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202. 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.
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209. 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.
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210. 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.
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211. 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.
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212. 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.
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213. 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.
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214. 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.
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215. 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.
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216. 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.
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217. 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.
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220. 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
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221. 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.
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222. 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.
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224. • 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.
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225. 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.
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226. 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.
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227. 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.
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228. 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.
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254. Biosensors are electronic
monitoring devices that
make
use of an enzyme’s
specificity and the
technique of enzyme
immobilisation
Biosensors
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Dr. Zekeria Yusuf
255. 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 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
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Dr. Zekeria Yusuf
256. A biosensor has been developed for detecting
glucose in the blood of diabetics
Glucose oxidase
oxidises any glucose
present in the blood to
release electrons – these
are detected by the
transducer and converted
into an electrical current
Transducer
Amplifier
The current generated is
proportional to the amount
of glucose present in the
sample and this is displayed
as a digital read-out
Glucose
molecules
in the blood
Glucose
oxidase
Biosensors
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Dr. Zekeria Yusuf
257. 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.
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258. 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.
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260. 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.
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261. 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.
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262. 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.
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