Enzymes
• materials, nutraceuticals, pharmaceuticals, monitoring devices,
bulk chemicals, and fine chemicals
• A number of enzymatic processes have already replaced
conventional chemical processes.
• The application of enzymes in some processes has helped to
develop processes unavailable to conventional chemistry.
Introduction
• key roles in numerous biotechnology products and processes that are
commonly encountered in the form of food and beverages, cleaning
supplies, clothing, paper products, transportation fuels, pharmaceuticals,
and monitoring devices
• the most frequently used enzymes in biotechnology are
which catalyze the breakdown of molecules.
hydrolases,
• Enzymes can display regio- and stereospecificity, properties that have
been exploited for asymmetric synthesis and racemic resolution.
• Chiral selectivity is employed to prepare enantiomerically pure
pharmaceuticals, agrochemicals, chemical feedstocks, and food additives.
• industrial enzyme market has expanded at a rate of about 10% annually,
microbial enzymes have largely replaced the traditional plant and animal
enzymes, and most of them are produced recombinantly.
• DNA technology has been used to modify substrate specificity and improve stability
properties of enzymes for increasing yields of enzyme-catalyzed reactions.
Introduction
• used in metabolic engineering of cellular metabolism to increase yields
of fermentation products
• Enzymes are environmentally friendly; they work under moderate
conditions of temperature, pH, and pressure, their catalyzed reactions
rarely form wasteful side products, and the proteins themselves are
biodegradable and generally pose no threat to the environment.
Classification
They are protein catalysts, accelerate chemical reactions, the transfer of
electrons, atoms, or functional groups, by several orders of magnitude while
maintaining high fidelity of reaction trajectories and producing low levels of
side-reaction products.
may require organic (e.g., coenzymes such as flavin mononucleotide and thiamine
pyrophosphate) or inorganic (e.g., Mg2þ) cofactors that, like the enzyme itself,
are not consumed in the catalyzed reactions.
Nomenclature
Assigned longer systematic names and four-part classification
numbers
(EC number, ‘enzyme commission number’) by the
enzyme nomenclature system maintained by the
International Union of Biochemistry and Molecular Biology and
the International Union of Pure and Applied Chemistry (IUPAC)
Classification
Depending on the type of reaction catalyzed, enzymes are divided into six main classes:
1. Oxidoreductases. Transfer of electrons from one substrate molecule to another
(e.g., dehydrogenases, reductases, oxidases).
2. Transferases. Transfer of functional group from one substrate molecule to another
(e.g., glycosyl transferases, acetyl transferases, and aminotransferases).
3. Hydrolases. Transfer of functional group from substrate to water (e.g., glycoside
hydrolases, peptidases, esterases).
4. Lyases. Elimination of functional group from substrate with the formation of double
bonds. Thus, bonds are cleaved using a different principle than hydrolysis (e.g., pectate
lyases break glycosidic linkages by b-elimination).
5. Isomerases. Transfer of groups from one position to another in the same molecule
(e.g., glucose isomerase).
6. Ligases. Addition of function group to substrate usually coupled with ATP hydrolysis
(e.g., glycine–tRNA ligase).
Industrial Uses
1. Starch conversions
• Production of glucose syrup
• Production of high fructose corn syrup
• Production of high maltose conversion syrups
• Production of cyclodextrins
• Production of ethanol
2. Lignocellulosic Biomass conversions
• Cellulose conversion
• Hemicellulose conversion
• Lignin conversion
Industrial Uses
3. Enzymes in the Production of Functional Oligosaccharides
and Other Neutraceuticals
4. Enzymes in the Modification of Fats and Oils
5. Enzymes in the Animal Feed Industry
6. Enzymes in the Pulp and Paper Industry
7. Enzymes in the Fruit Juice Processing Industry
8. Enzymes in the Meat and Fish Processing Industry
9. Enzymes in the Dairy Industry
10.Enzymes in Detergents
11. Enzymes in the Leather Industry
12.Enzymes in the Production of Bulk and Fine Chemicals
13.Analytical Applications of Enzymes
14.Enzyme-Replacement Therapy
1. Starch conversions
Starch contains about 15–30% amylose and 70–85% amylopectin.
Enzymes have largely replaced the use of strong acid and high temperature
to break down starchy materials.
Three types of enzymes are involved in starch bioconversion:
1. endo-amylase (a-amylase, EC 3.2.1.1), [Bacillus lichiniformis, Bacillus
subtilis, and Bacillus amyloliquefaciens and fungi such as Aspergillus oryzae]
2. exo-amylases
• glucoamylase or glucan 1,4-a-glucosidase, EC 3.2.1.3 [Endomycopsis,
Aspergillus, Penicillium, Rhizopus, and Mucor];
• b-amylase, EC 3.2.1.2) [Bacillus megaterium, Bacillus cereus, Bacillus
polymyxa, Thermoanaerobacter thermosulfurogenes, and Pseudomonas sp.]
3. debranching enzymes (pullulanase, EC 3.2.1.41; isoamylase, EC
3.2.1.68). [Aerobacter aerogenes and isoamylase is produced by Pseudomonas
amyloderamosa.]
In amylose these are linked  -(1, 4)-, with the ring oxygen atoms all on
the same side.
In amylopectin about one residue in every twenty or so is also linked (1,6)- forming branch-points.
 -1,4
hydrolyzes the -1,4glycosidic bonds in starch
from the nonreducing
ends, generating maltose.
The enzyme is unable to
bypass the -1,6 linkages
and
leaves
dextrins,
known as b-limit dextrins
Hydrolyse internal (endo) a- 1,4
but not a- 1,6 producing maltooligosaccharides
Enzymatic hydrolysis of amylose
cleaves glucose units from the nonreducing end
of starch and it can hydrolyze both -1,4 and 1,6 linkages of starch, slower
Enzymatic hydrolysis of amylopectin
Dextrins: A group of low-molecularweight carbohydrates produced by the
hydrolysis of starch.
Dextrins are mixtures of linear a-(1,4)linked D-glucose polymers starting with
an a-(1,6) bond.
Amylases break starch into sugars
All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds.
-Amylases
Both the salivary and pancreatic amylases are α-Amylases. They are Ca
metalloenzymes,completely unable to function in the absence of calcium.
They act at random locations along the starch chain hence faster than
b-amylases
Working from the non-reducing end, β-amylase catalyzes the hydrolysis
of the second α-1,4 glycosidic bond, cleaving off two glucose units
(maltose) at a time. During the ripening of fruit, β-amylase breaks
starch into sugar, resulting in the sweet flavor of ripe fruit. Both are
present in seeds; β-amylase is present prior to germination, whereas αamylase and proteases appear once germination has begun.
Enzymes for starch conversion
a-amylase randomly hydrolyse a-1,4 linkages in both amylose and
amylopectin to yeild mixture of glucose, maltose, maltotriose and
series of a-limit dextrins.
b-amylase sometimes used in place of a-amylase. They hydrolyze
alternate a-1,4 linkages and yield maltose residues and b-limit dextrins
Glucoamylase hydrolyses a-1,3. a-1,4 and a-1,6 linkages but is less
efficient than a-amylase. Major role is to break cross links of
amylopectin resulting in complete breakdown to glucose. Generally used
to reduce CHO content of beers. Industrially obtained from fungus
Aspergillus niger.
Glucose isomerase is used for conversion of glucose obtained after
processing to fructose.
Pullulanase (pullulan -1,6-glucanohydrolase) or isoamy- lase (glycogen
-1,6-glucanohydrolase) cleaves the -1,6- linked branch points of
starch and produces linear amylosaccharides of varying lengths.
1.
2.
3.
4.
Production of glucose syrup
Production of high fructose corn syrup
Production of high maltose conversion syrups
Production of cyclodextrins
1. Production of D-glucose from starch by acid hydrolysis (chemical)
produces undesirable bitter sugar (gentiobiose), and the inevitable
formation of salt (from subsequent neutralization with alkali) and
coloring materials.
With the discovery and development of thermostable a-amylase from
an enzymatic process has replaced
the acid hydrolysis process.
Bacillus licheniformis,
liquefaction and saccharification
Typically, glucose syrups (DE 97–98) having 96% glucose contain
2–3% disaccharides (maltose and isomaltose) and 1–2% higher
saccharides.
Dextrose equivalent (DE) is a measure of the amount of reducing sugars present in a sugar product,
relative to glucose, expressed as a percentage on a dry substance basis (DS). an estimate of the
percentage reducing sugars present in the total starch product
GLUCOSE ---- FURCTOSE
ISOMERIZATION
2. HFCS
Glucose isomerase (also known
as xylose isomerase, EC 5.3.1.5)
is an example of the highly
successful
application
of
enzyme biotechnology to an
industrial process that has no
commercially
viable
route
through
conventional
chemistry.
Chemical
isomerization
of
glucose to fructose at high pH
and high temperature leads to
undesirable
side
products,
some of which are colored and
have off flavors.
Enzymecatalyzed
isomerization (at moderate pH
and temperature) does not
form undesired side products.
DNA TECHNOLOGY FOR INCR
IN THERMAL STABILITY
Streptomyces, Bacillus, Arthobacter, and Actinoplanes
3. Various maltose-containing syrups are used in the brewing, baking, soft
drink, canning, confectionery, and other food industries.
There are three types of maltose-containing syrups:
1. high-maltose syrup (DE 35–50, 45–60% maltose, 10–25% maltotriose,
0.5–3% glucose),
2. extra high-maltose syrup (DE 45–60, 70–85% maltose, 8–21%
maltotriose, 1.5–2% glucose), and
3. high conversion syrup (DE 60–70, 30–47% maltose, 35–43% glucose, 8–
15% maltotriose).
liquefaction and saccharification, as in the production of glucose.
However, in this process, the liquefaction reaction is terminated when the
DE reaches about 5–10 since a low DE value increases the potential for
attaining high maltose content.
maltogenic amylase such as b-amylase, b-amylase with pullulanase or
isoamylase, or a fungal a-amylase at pH 5.0–5.5 and 50–55o C.
Production of ethanol
The process of making ethanol from starch involves three basic steps:
(1) preparation of the glucose feedstock,
(2) fermentation of glucose to ethanol, and
(3) recovery of ethanol.
Enzymes have major role in preparation of feedstock:
Corn kernels contain 60–70% starch
Wet milling process
corn is steeped in acidic water solutions (corn steep liquor) and the oil, protein,
and fiber fractions are successively removed as products leaving the starch
fraction.
Enzymatic liquefaction and saccharification of the starch fraction are then
carried out for the production of glucose
Glucose is fermented by the traditional yeast Saccharomyces cerevisiae to
ethanol, which can be recovered by distillation.
Dry grinding process
lower capital investment required in comparison to that of wet mills.
In the typical dry grind process, corn is mechanically milled to a coarse
flour. Oil, protein, and fiber fractions are not isolated
liquefaction, enzymatic saccharification using glucoamylase
fermentation using the conventional yeast are carried out simultaneously.
The addition of protein-splitting enzymes (proteases) releases soluble
nitrogen compounds from the fermentation mash and promotes growth of
the yeast, decreasing fermentation time.
The residue left after fermenting the sugars is known as distiller’s grains,
which is used as animal feed.
Milled grain
steam
Gelatinized material
cool
-amylase
Liquefied material Bacillus amyloliquefaciens
Glucoamylase
Saccharified material
Glucose isomerase
Fructose
Yeast
Fermentation
Alcohol
Industrial production of alcohol and fructose from starch
2. Lignocellulosic Biomass conversions
Various agricultural residues (straws, hulls, stems, cobs, stalks), deciduous
and coniferous woods, municipal solid wastes (paper, cardboard, yard
debris, wood products), waste from the pulp and paper industry, and
energy crops (switchgrass, miscanthus).
These materials are structurally diverse and compositions vary widely
(cellulose, 35–50%;
hemicellulose, 20–35%;
lignin, 10–25%;
proteins, oils, and
ash, 3–15%).
Native lignocellulosic biomass is resistant to enzymatic hydrolysis
hence Pretreatment is required like
steam explosion, dilute acid, concentrated acid, alkali, SO2, alkaline
peroxide, ammonia fiber expansion, and organic solvents.
• hemicellulose to simple sugars (xylose, arabinose, and
other sugars) and acids (acetic, glucuronic), which
are water-soluble.
• insoluble residue contains cellulose and lignin.
• The lignin can be extracted with solvents such as
ethanol, butanol, or formic acid.
• Alternatively, enzymatic hydrolysis of cellulose with
lignin present produces glucose, and the residues are
lignin plus any unreacted materials.
Cellulose (C6H10O5)n
•Structural component of cell wall of green plants, many algae and fungi. Some bacteria
secrete it to form biofilms.
•It is the most common organic compound on earth.
•33% plant matter is cellulose. Cotton is 90% cellulose and wood is 50% cellulose.
•Industrially, cellulose is obtained from wood pulp and cotton to produce cardboard and
paper and derivatized to make cellophane and rayon.
•Cellulose can be digested in the gut of ruminants and termites with the help of symbiotic
bacteria (Trichonympha, which produces cellulases). Humans cannot digest cellulose but
acts as dietary fiber and hydrophilic bulking agent for faces.
•The major combustible component of non-food energy crops is cellulose, with lignin
being second.
•Some bacteria can convert cellulose into ethanol which can then be used as a fuel .
•Cellulose is crystalline, strong, and resistant to hydrolysis, cellulose contains only
anhydrous glucose residues with beta configuration.
Structural Unit
Cellulose is a linear polymer of β-(1,4)-D-glucopyranose units in 4C1 conformation. The fully
equatorial conformation of β-linked glucopyranose residues stabilizes the chair structure,
minimizing its flexibility (for example, relative to the slightly more flexible α-linked
glucopyranose residues in amylose).
Functionality
Cellulose has many uses as an anticake agent, emulsifier, stabilizer, dispersing
agent, thickener, and gelling agent but these are generally subsidiary to its most
important use of water holding capacity.
Water cannot penetrate crystalline cellulose but dry amorphous cellulose absorbs
water becoming soft and flexible. Some of this water is non-freezing but most is
simply trapped.
Less water is bound by direct hydrogen bonding if the cellulose has high
crystallinity but some fibrous cellulose products can hold on to considerable water
in pores and its typically straw-like cavities; water holding ability correlating well
with the amorphous (surface area effect) and void fraction (that is, the porosity).
As such water is supercoolable, this effect may protect against ice damage.
Cellulose can give improved volume and texture particularly as a fat replacer in
sauces and dressings but its insolubility means that all products will be cloudy.
Lignocellulose: structural support system for all terrestrial plants
Lignocellulosics
Primary
cellulosics
Plants harvested for
cellulosic
content,
structural use or feed
Cotton, timber, hay
Agricultural
waste
cellulosics
Plant material that
remain
after
harvesting
and
processing
Straw,
corn,
rice
hulls,
sugarcane
baggase,
animal
manure,
timber residues
Plant biomass comprises
of Lignin, hemicellulose
and cellulose combined in
different proportions
Municipal
Waste
cellulosics
Waste
paper
discarded
products
and
paper
Lignin
•Complex chemical compound most commonly derived from wood and an integral part
of the cell walls of plants.
•one of the most abundant organic polymers on Earth, superseded only by cellulose,
employing 30% of non-fossil organic carbon and constituting from a quarter to a
third of the dry mass of wood
LIGNIN: 3D, GLOBULAR, IRREGULAR, INSOLUBLE, HIGH MW POLYMER MADE
OF PHENYLPROPANE SUBUNITS
NO CHAINS OF
HYDROLYSABLE
REPEATING
UNITS
OR
BONDS
THAT
ARE
EASILY
In plants lignin is bonded to hemicellulose and wraps around fibres composed of
cellulose
Gives rigidity, resistance to mechanical stress and microbial attack
Fungus: Phanerochaete chrysosporium
HEMICELLULOSE:SHORT
CHAIN
HETEROGENOUS
POLYMERS
CONTAN BOTH HEXOSES (C6: GLU, MAN, GAL) AND PENTOSES (C5:
XYL, ARA)
Difference between hemicellulose and cellulose
Hemicellulose is amorphous, random and easily hydrolysed by acid or
base or hemicellulases. It consists of shorter chains - 500-3000
sugar units, hemicellulose is a branched polymer. Composed of
hexoses and pentoses.
Cellulose is crystalline, strong and resistant to hydrolysis.
Contain 7,000 - 15,000 glucose molecules per polymer and is
unbranched. Composed of anhydrous glucose units joined by b-1,4.
Major hemicelluloses are
1. Xylans (backbone of b-1,4 xylans with side chains to ara, glucoronic acid,
arabinoglucoronic acid) present in hardwoods
2. Mannans (glucomannans, galactomannans, arabinogalactans) present in
softwoods
Trichoderma sp., Aspergillus sp., Fusarium sp., and Bacillus sp
Enzymes for cellulose hydrolysis
Fungus: Trichoderma reesei
Cellulomonas fimi
Aspergillus
Endo-1,4-b-glucanse: hydrolyzes b-1,4 linkages b/w adj glu mocs
(cellulase, EC 3.2.1.4)
Exo-1,4-b-glucanase: degrades nicked cellulose chains from non reducing
ends and produced glucose, cellobiose (2 glu units) and cellotriose (3 glu
units)
1,4-b-Cellobiohydrolase; type of exoglucanase removes units of 10 or
more glu residues from non reducing ends (found in cellulolytic fungi)
b-glucosidase or cellobiase converts cellobiose and cellotriose to glucose
cellobiose
Crystalline region
Enzymatic
biodegradation
of cellulose
Amorphous region
Endoglucanase
Removal
of
oligosacc.
from reducing ends
cellobiose
cellotriose
Exoglucanase
Exoglucanase
Cellobiohydrolase
glu
Endoglucanase
b-glucosidase
b-glucosidase not only produces glucose from
cellobiose but also lowers cellobiose inhibition,
allowing the cellulolytic enzymes to function more
efficiently.
However, like b-glucanases, most b-glucosidases
are subject to product (glucose) inhibition.
Cellulose
Inhibits
Feedback Inhibitor of
cellulose hydrolysis
Feedback Inhibitor of
cellobiose
Cellobiose
Inhibits
Glucose
Fermentation
To increase rate and extent of degradation , addition of
b-glucosidase
Decrease
amount
of
cellobiose which prevents
end product inhibition of
exo and endo
Cloning of gene in host cell
b-glucosidase enhances enzymatic utilization of cellulose