Download Hydrolytic Enzymes

Dr. A.K.M. Shafiqul Islam
School of Bioprocess Engineering
15.01.2010
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Survey some of the applications of enzymes
◦ Enzymatic hydrolysis and concept: Starch and
Cellulose
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Examine immobilized enzyme catalyst
formulations
◦ which allow sustained, continuous use of the
enzyme.
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There are three major sources of enzyme
• Plant
• animal
• 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
◦ 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.
 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.
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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.
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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Hydrolytic enzymes are normally associated
with degradative reactions, 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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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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The three groups of enzymes.
Those involved in the hydrolysis of
◦ ester,
◦ glycosidic,
◦ and various nitrogen bonds.
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Enzymes are named according to the
chemical reactions they catalyze, rather than
according to their structure.
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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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Many hydrolases are directed to specific
compartments separated from the cytoplasm by
membranes.
This serves the purpose of protecting essential
cytoplasmic bipolymers from degradation.
Example,
Gram-positive bacteria secrete a variety of
hydrolases into their environment. With their
double membrane outer envelope, gramnegative bacteria have available the periplasmic
space which safely stores a variety of hydrolases.
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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.
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Amylases are extensively applied enzymes
which can hydrolyze the glycosidic bonds in
starch and related glucose-containing
compounds.
There are two major types of amylases◦ a-amylase
◦ b-amylase
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
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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.
The action of a-amylase reduces the solution
viscosity by acting randomly along the glucose
chain at a-1,4 glycosidic bonds
a-amylase is often called the starch-liquefying
enzyme for this reason.
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b-Amylase can attack starch a-1,4 bonds 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)
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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.
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The sources of amylases are very numerous.
Amylases are produced by a number of
bacteria and molds –
◦ e. g., amylase produced by Clostridium
acetobutylicum which is clearly involved in the
microbial conversion of polysaccharides to butanol
and acetone.
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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.
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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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For cellulase
◦ 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
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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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Cellulose structure e.g., crystallinity, specific
surface area and degree of polymerization are
important
Cellulose structure can be altered by a variety
of pretreatments such as ball or compression
milling, g-irradiation, pyrolysis, and acidic or
caustic chemicals.
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Cleave or synthesize ester bonds to yield an
acid and an alcohol
Anaerobic waste digestion
Meat processing
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Attack nitrogen-carrying compounds,
particularly proteins
Dry cleaning
Detergents
Meat processing
Cheesemaking