ERT211 BIOCHEMICAL ENGINEERING
Applied Enzyme Catalysis
Pn Syazni Zainul Kamal
School of Bioprocess Engineering
1)Survey some of the applications of enzymes
◦ Sources of enzyme
◦ Hydrolytic enzymes and applications
2)Examine immobilized enzyme catalyst
formulations
◦ which allow sustained, continuous use of the
enzyme.
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There are three major sources of enzyme
a) animal
b) plant
c) or microbial
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Although all living cells produce enzymes,
one of the three sources may be favored for a
given enzyme or utilization
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Some enzymes may be available only from
animal sources.
Enzymes obtained from animals may be
relatively expensive,
e.g., rennin obtain from calf's stomach,
◦ the value depend on demand of lamb or beef,
◦ and their availability.
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While some plant enzymes are relatively easy
to obtain
e.g., papain from papaya, bromelain from
pineapple, actinidin from kiwi fruit
◦ their supply is also governed by food demands
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Microbial enzymes are produced by methods
which can be scaled up easily
Recombinant DNA technology now provides
the means to produce many different
enzymes, including those not normally
synthesized by microorganisms or permanent
cell lines, in bacteria, yeast and cultured cells.
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Due to the rapid doubling time of microbes
compared with plants or animals
◦ microbial processes are attuned more easily to the
current market demands for enzymes.
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On the other hand,
◦ for use in food or drug processes, only those
microorganisms certified as safe may be exploited
for enzyme production.
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Although most of the enzymes used today
are derived from living organisms, they are
utilized in the absence of life
Example –
◦ extracellular enzymes,
◦ secreted by cells in order to degrade polymeric
nutrients into molecules small enough to permeate
cell walls.
◦ Grinding, mashing, lysing, or otherwise killing and
splitting
◦ intracellular enzymes,
◦ which are normally confined within individual cells.
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The enzyme kinetics study generally carried
out with the purest possible enzyme
preparations.
Such research involves
◦ the fewest possible number of substrates (one if
achievable)
◦ a controlled solution with known levels of activators
(Ca2+, Mg2+,pH etc.),
◦ cofactors,
◦ and inhibitors.
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Many useful industrial enzyme preparations are
not highly purified.
They contain a number of enzymes with different
catalytic functions and are not used with either a
pure substrate or a completely defined
synthetic medium.
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Also, the simultaneous use of several different
enzymes may be more efficient than sequential
catalysis by a separated series of the enzymes.
such enzyme preparations are kinetically more
simple than the integrated living organisms from
which they are produced
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Enzyme that catalyzes the hydrolysis of a
chemical bond
Hydrolytic enzymes are normally associated
with degradative reactions, (break down large
molecules into small molecules) e.g.,
◦ conversion of starch to sugar,
◦ proteins to polypeptides and amino acids,
◦ and lipids to their constituent glycerols, fatty acids
and phosphate bases
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3 major group of hydrolytic enzyme
Those involved in the hydrolysis of
◦ Ester (Esterase) – split ester into acid & alcohol
◦ Glycosidic (carbohydrase) – act on carbohydrate
◦ and various nitrogen bonds – act on proteins and
polypetides
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Enzymes are named according to the
chemical reactions they catalyze, rather than
according to their structure.
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Since One-enzyme – one-reaction uniqueness does
not generally exist,
Enzymes from different plant or animal sources which
catalyze a given reaction will not always have the
same molecular structure or necessarily the same
kinetics.
Consequently,
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maximum reaction rate,
Michaelis constant,
pH of optimum stability or activity,
and other properties –
depend on the particular enzyme source used.
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In macroscopic degradations such as
◦ food spoilage
◦ starch thinning,
◦ and waste treatment,
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Also in the chemistry of
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ripening picked green fruit
self-lysis of dead whole cells (autolysis),
desirable aging of meat,
curing cheeses,
preventing beer haze,
texturizing candies,
treating wounds,
and desizing textiles.
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In eucaryotes, hydrolases may be stored
inside the cell in membrane-enclosed
lysosome organelles, reside in the periplasm
in microbes like yeast,
or be secreted into the environment.
Most hydrolytic enzymes used commercially
are extracellular microbial products.
Carbohydrase
 Amylases - extensively applied enzymes
- can hydrolyze the glycosidic
bonds in starch and related
glucose-containing compounds
(eg. Cellulose).
*(glycosidic bond – join carbohydrates to
another group)
 There are three major types of amylases
◦ α-amylase
◦ b-amylase
◦ Amyloglucosidase/glucoamylase
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Starch contains straight-chain glucose
polymers called amylose and a branched
component known as amylopectin.
The branched structure is relative more
soluble than the linear amylose and is also
effective in rapidly raising the viscosity of
starch solution.
a(1-4) glycosidic linkage
between the C1 hydroxyl of
one glucose and the C4
hydroxyl of a second glucose
The b(1-4) glycosidic linkage
is represented as a "zig-zag"
line, but one glucose residue
is actually flipped over
relative to the other
Starch - polysaccharide of plant
- 2 polysaccharide occur together in starch
*amylose - α(1-4) glycosidic bond
*amylopectin – β(1-6) glycosidic bond
α-amylase
 The action of α-amylase reduces the
solution viscosity by acting randomly along
the glucose chain at α-1,4 glycosidic bonds
 α-amylase is often called the starchliquefying enzyme for this reason.
β-amylase
 b-Amylase can attack starch a-1,4 glycosidic
bond only on the nonreducing ends of the
polymer and always produces maltose when a
linear chain is hydrolyzed.
 Because of the characteristic production of the
sugar maltose, b-amylase is also called a
saccharifying enzyme.
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soluble mixture of starch and b-amylase yields
maltose and a remainder of dextrins (starch
remnants with 1,6- linkage on the end)
amyloglucosidase
 Another saccharifying enzyme, amyloglucosidase
(also called glucoamylase) attacks primarily the
nonreducing a-1,4 linkages at the ends of
starch, glycogen, dextrins, and maltose. (a-1,6
linkages are cleaved by amyloglucosidase at
much lower rates)
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Sequential treatment with a-amylase and
glucoamylase or enzyme mixtures are utilized
where pure glucose rather than maltose is
desired,
e.g., in distilleries and in the manufacture of
glucose syrups (corn syrup) and crystalline
glucose.
The sources of amylases are very numerous
 Amylases are produced by – microb, plant
e. g., 1) amylase produced by Clostridium
acetobutylicum which is clearly involved in the
microbial conversion of polysaccharides to butanol
and acetone.
2) amylase produced by Aspergillus niger, Penicillium
sp.
3) amylase from Bacillus used in clothing and
dishwasher detergent
(amylase from microb, not suitable to be used in food
industry)
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Application of Amylase
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Commercial amylase preparations used in
human foods are normally obtained from
grains,
e.g., barley, wheat, rye, oats, maize,
sorghum, and rice.
The ratio of saccharifying to liquefying
enzyme activity depends
◦ on the particular grain
◦ and upon whether the grain is germinated.
Application of Amylase
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In the production of malt for brewing, the
ungerminated seeds are exposed to a favorable
temperature and humidity so that rapid
germination occurs, with resulting large increase
in a-amylase.
The germinated barley is then kiln-dried slowly;
◦ this halts all enzyme activity without irreversible
inactivation.
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The dried malt preparation is then ground, and
its enormous liquefying and saccharifying power
is utilized in the subsequent yeast fermentation.
◦ to convert starches to fermentable sugars.
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Invertase hydrolyzes sucrose and polysaccharides
containing a b-D-fructofuranosyl linkage.
The hydrolyzed sucrose solution containing
fructose and glucose rotates a polarized light
beam in the direction opposite that of the
original solution.
The partially or completely hydrolyzed solution
allows two properties desirable in syrup and
candy manufacturing:
◦ a slightly sweeter taste than sucrose
◦ and a much higher sugar concentration before
hardening.
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Maltose
1. Maltose + H2O -*--> glucose + glucose
* = enzyme; in this case maltase
Enzymes end in -ase
Sucrose
Sucrose + H2O -*-> glucose + fructose
* = sucrase
Hydrolysis of Lactose
Lactose + H2O -*-> galactose + glucose
* = lactase
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Cellulose – polysaccharide consisting of a
linear chain of several hundred to over ten
thousand β-1,4 linked D-glucose units
Structural component of all plant cells from algae
to tree
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cellulase – enzyme that hydrolyze cellulose
◦ Trichoderma fungi are commonly used at the
present time.
◦ They are thoroughly developed and characterized at
present.
◦ There are three major classes of enzymes for
different substrates and products
1.Exo-b-1,4-cellobiohydrolase (CBH)
2.Endo-b-1,4-glucanase
3. b-glucosidase
3 steps of reaction catalyze by cellulase :
1)Breakage of non-covalent interactions
present in the crystalline structure of
cellulose by endo-β-1,4-glucanase
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2)Hydrolysis of individual cellulose fibers to break it
into smaller sugars (cellobiose) by exo-β-1,4cellobiohydrolase (CBH)
3)Hydrolysis of dissacharides or tetrasaccharides
into glucose by β-glucosidase
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Many other microorganisms including the
molds bacteria produce cellulases with
distinctive activities and properties. e.g.•
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Fusarium solani,
Aspergillus niger,
Penicillium funicolsum,
Sporotrichum pulverulentum,
Cellulomonas species,
Clostridium thermocellum,
and Clostridium thermosaccharolyticum
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Alcohol fermentation from biomass
Brewing
Waste treatment
Cereal processing
Pulp and paper industries
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Enzyme that catalyze the splitting of protein
into smaller peptide fractions and amino
acids by a process known as proteolysis
Eg. Proteinase + protein
polypeptides
polypeptidase + protein
amino acids
Some can detach the terminal amino acids
from the protein chain
i.e Exopeptidase – aminopeptidase,
carboxypeptidase A
Others attack internal peptide bonds of a
protein
i.e Endopeptidase – trypsin,pepsin,papain
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Animal, plant, microb
Trypsin – animal pancrease
Papain – papaya
Protease – Bacillus sp., Aspergillus sp.
1) Detergents
- Enzyme used in laundry aid as early 1913
- Protease-contain a mixture of bacterial
neutral-alkaline protease/lipase active at
pH6-10 and 30-60°C
- facilitate spot removal, so that it can be wash
easily
- Since one enzyme molecule can act on many
substrate (i.e., soil) molecules, a small
amount of enzyme added to a laundry
detergent can provide a big cleaning benefit
to the consumer
2) Meat tenderization
- eg. Bromelain and papain
- breaking the peptide bonds between amino
acids found in complex proteins
- Meat is held together by a complex protein
called collagen
- Meat is often tenderized before cooking, to
make it less tough and more suitable for
consumption
- If meat tenderizers are allowed to act for too
long, the meat can become squishy and lose
its special texture.
3) Tanning
- Making leather process from animal skin
- Ground pancreases contain digestive
proteases eg trypsins, lipases
- Use for – dehairing animal hides & removal of
noncollagen protein
- Environmental friendly rather than using
chemical
4) Dairy industries
- Coagulation of milk
- Rennin remove glucopeptide from soluble
calcium casein to yield paracaseinate
- Paracaseinate precipitate to form curd
- Curd further process to make cheese
5) Clinical & medical application
- eg. trypsin
- Reduce inflammation & swelling (internal
injuries & infection)
- Dissolve blood clots & extracellular protein
precipitates
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Cleave or synthesize ester bonds to yield an
acid and an alcohol
R1COOR2 + H2O
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R1COOH + R2OH
eg. Lipases – hydrolyze fats into glycerol &
fatty acids
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Most important enzyme in esterase group
Hydrolyze triglycerides into diglycerides,
monoglycerides, fatty acids and glycerols
Applications in food, detergent,
pharmaceuticals, leather, textile, cosmetic
1)Dairy industries
Lipase used for hydrolysis of milk fat
Current applications ; flavour enhancement
of cheese, acceleration of cheese ripening
(lipase degrade protein, fat, lactose)
Addition of lipase to cow’s milk, generate
flavour similar to that of ewe’s 0r goat’s
milk
2)Detergents
- Lipase add to detergents
- Remove fat and oils based stains
3) Lipase in oleochemical industry
- Before this used organic solvent and emulsifier
in oleochemical industry
- Now using immobilized lipase from Candida
cylindracea in production of soap
- Resulted in high productivity & continuous
running of the process
- Reduce cost for expensive equipment &
thermal energy
4) Meat processing industry
- to produce fat free meats
- Partial fat hydrolysis of the meat cut using
lipase
5) Pulp & paper industry
- Deinking – removal of ink process from surface
of paper
- Conventional method – used chemical to
remove ink ; cause water pollution & high cost
- Deinking by enzyme – Lipase used to remove
oil based ink
Mixture of enzymes may contain :
a) same general type = α- & β- amylase and
amyloglucosidase
b) Different type = found in pancreas extract
(trypsin, lipase, amylase)
 are often used more successfully than single
enzyme preparations
 eg. Blend of diff. amylase yields large amount of
saccharified starch suitable for yeast fermentation
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Hydrolysis enzymes applications dominate
past and present enzyme technology
Other enzyme processes currently serve
important function in food, pharmaceutical
and biochemical industries
Recently free or extracellular enzymes were
used in medicine
1)Lysozyme
 (in nasal mucus, saliva, tears)
 It hydrolyze mucopolysaccharides of bacterial
cell walls.
 Used as an antibacterial agent, treatment of
ulcer, skin disease
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2) Asparaginase
 Used as anticancer agent
 Enzyme that catalyze hydrolysis of asparagine
to aspartic acid
 Some cancer cells require asparagine
(nurient), their growth can be inhibited using
asparaginase
 Can be given to patient as intramuscular,
subcutaneous or intravenous injection (differ
from other chemo agent, no tissue irritation)
3)Penicillinase
 1st isolated from gram negative bacteria E.coli
 human beings do not produce penicillinase
 Remove allergenic form of penicillin from
allergic individuals
 Convert the drug into nonallergic form