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ERT211 BIOCHEMICAL ENGINEERING

Applied Enzyme Catalysis

Pn Syazni Zainul Kamal


School of Bioprocess Engineering
Inside the chapter
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.
Sources of enzyme
 There are three major sources of enzyme
a) animal
b) plant
c) or microbial
 Although all living cells produce enzymes,
one of the three sources may be favored for a
given enzyme or utilization
a) Animal sources
 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.
b) Plant sources
 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
c) Microbial enzyme
 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.
 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.
 On the other hand,
◦ for use in food or drug processes, only those
microorganisms certified as safe may be exploited
for enzyme production.
 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.
Enzyme Kinetic
 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.
Industrial enzymes
 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.
 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
Some industrially
important enzymes
Hydrolytic enzyme
 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
Hydrolytic enzymes
 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
 Enzymes are named according to the
chemical reactions they catalyze, rather than
according to their structure.
Classification of Hydrolytic
Enzymes
 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,
◦ maximum reaction rate,
◦ Michaelis constant,
◦ pH of optimum stability or activity,
◦ and other properties –
depend on the particular enzyme source used.
Application of hydrolytic
enzymes
 In macroscopic degradations such as
◦ food spoilage
◦ starch thinning,
◦ and waste treatment,
 Also in the chemistry of
◦ 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.
Application of Hydrolytic Enzymes
 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.


Hydrolysis of starch
 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
 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.
Glucose Structure
The b(1-4) glycosidic linkage is
a(1-4) glycosidic linkage
represented as a "zig-zag" line,
between the C1 hydroxyl of but one glucose residue is
one glucose and the C4 actually flipped over relative to
hydroxyl of a second glucose the other
Starch - polysaccharide of plant
- 2 polysaccharide occur together in starch
*amylose - α(1-4) glycosidic bond
*amylopectin – β(1-6) glycosidic bond
Amylopectin Structure
Hydrolysis of starch

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 starch-

liquefying enzyme for this reason.


Hydrolysis of starch
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.
 soluble mixture of starch and b-amylase yields

maltose and a remainder of dextrins (starch


remnants with 1,6- linkage on the end)
Hydrolysis of starch
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)

 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.
Sources of amylase
 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)
Common Application of Amylase
Preparation
Application of Amylase
 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
 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.
 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.
 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.
Hydrolysis of Disaccharides
 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
Hydrolysis of Cellulose
 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
Hydrolysis of cellulose
 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
Hydrolysis of cellulose
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

2)Hydrolysis of individual cellulose fibers to break it


into smaller sugars (cellobiose) by exo-β-1,4-
cellobiohydrolase (CBH)

3)Hydrolysis of dissacharides or tetrasaccharides


into glucose by β-glucosidase
Hydrolysis of Cellulose
Cellulase sources
 Many other microorganisms including the
molds bacteria produce cellulases with
distinctive activities and properties. e.g.-
• Fusarium solani,
• Aspergillus niger,
• Penicillium funicolsum,
• Sporotrichum pulverulentum,
• Cellulomonas species,
• Clostridium thermocellum,
• and Clostridium thermosaccharolyticum
Applications of cellulase
 Alcohol fermentation from biomass
 Brewing
 Waste treatment
 Cereal processing
 Pulp and paper industries
Proteolytic enzymes
 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
Sources of proteolytic
enzyme
 Animal, plant, microb
 Trypsin – animal pancrease
 Papain – papaya
 Protease – Bacillus sp., Aspergillus sp.
Application of proteolytic
enzyme
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
Esterase
 Cleave or synthesize ester bonds to yield an
acid and an alcohol

R1COOR2 + H2O R1COOH + R2OH

 eg. Lipases – hydrolyze fats into glycerol &


fatty acids
lipase
 Most important enzyme in esterase group
 Hydrolyze triglycerides into diglycerides,

monoglycerides, fatty acids and glycerols


 Applications in food, detergent,

pharmaceuticals, leather, textile, cosmetic


Applications of lipase
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


Enzyme mixtures
 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


Other applications of enzyme in
solution
 Hydrolysis enzymes applications dominate
past and present enzyme technology
 Other enzyme processes currently serve

important function in food, pharmaceutical


and biochemical industries
Medical applications of enzymes
 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


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
Thank You