ENZYMES - They are inactive at 0°C, and denatured at 65°
ENZYMES
- An enzyme is a substance that acts as a
catalyst in living organisms, regulating the rate
at which chemical reactions proceed without
itself being altered in the process.
- Enzymes catalyze all aspects of cell
metabolism. This includes the digestion of
food, in which large nutrient molecules (such
as proteins, carbohydrates, and fats) are
broken down into smaller molecules; the
conservation and transformation of chemical
energy; and the construction of cellular
macromolecules from smaller precursors.
WHAT ARE ENZYMES MADE OF?
- A large protein enzyme molecule is composed
of one or more amino acid chains called
polypeptide chains. The amino acid sequence
determines the characteristic folding patterns
of the protein’s structure, which is essential to
enzyme specificity.
- If the enzyme is subjected to changes, such
as fluctuations in temperature or pH, the
protein structure may lose its integrity
(denature) and its enzymatic ability.
- Bound to some enzymes is an additional
chemical component called a cofactor, which
is a direct participant in the catalytic event and
thus is required for enzymatic activity. A
cofactor may be either a coenzyme—an
organic molecule, such as a vitamin—or an
inorganic metal ion. Some enzymes require
both.
- All enzymes were once thought to be proteins,
but since the 1980s the catalytic ability of
certain nucleic acids, called ribozymes (or
catalytic RNAs), has been demonstrated,
refuting this axiom.
- Enzymes are the complex protein molecules,
often called biocatalysts, which are produced
by living cells. They are highly specific both in
the reactions that they catalyze and in their
choice of reactants, which are known as
substrates.
- Each enzyme has a temperature range in
which a maximal rate of reaction is achieved.
This maximum is known as the temperature
optimum of the enzyme. The optimum
temperature for most enzymes is about 98.6°F
(37° C)
C.
- They have molecular weights ranging from
10,000 to 2,000,000
PROPERTIES OF ENZYMES
CATALYTIC PROPERTY
- They are active in very small quantities. A
small amount of enzyme is enough to convert
a large quantity of substrate. The enzymes
remain unchanged after the reaction. The
turnover number of enzymes ranges from 0.5
to 600000.
- Turn over number is the number of substrate
molecules converted by one molecule of
enzymes per second when its active site is
saturated with substrate.
SPECIFICITY
- Enzymes are very specific in their action.
Particular enzymes act on particular
substrates only. Enzymes are also specific to
a particular type of reaction
REVERSIBILITY
- Most of the enzymes catalysed reactions are
reversible. The reversibility of the reaction
depends upon the requirements of the cell. In
some cases, there are separate enzymes for
forward and reverse reaction. Some enzyme
catalysed reactions are not reversible.
SENSITIVENESS TO HEAT AND TEMPERATURE
AND pH
- Enzymes are very sensitive to heat and
temperature. They are thermolabile. The
correct temperature for the utmost activity is
termed optimum temperature.
- Enzymes will be inactive at very low
temperatures; this is the reason for preserving
food and vegetables in the refrigerator. The
enzymatic activity increases with the increase
in temperature up to a certain level. At higher
temperature (60-70° Celsius), the enzyme is
destroyed or denatured. The optimum pH of
most endoenzyme is pH 7.0 (neutral pH).
However, digestive enzymes can function at
different pH. For example, salivary amylase
act best at pH 6.8, pepsin act best at pH2 etc
CHEMICAL NATURE OF ENZYMES
- Many enzymes are inactive when they are
produced. Their inactive form is called
proenzyme or zymogen.
- Enzymes are very unstable compounds mostly
soluble in water, dilute glycerol, NaCl and
dilute alcohol.
- Enzymes combine with their substrates to
form a temporary enzyme-substrate complex.
- Many enzymes need cofactors in order to
function, and it's function may slow down or
stopped by inhibitors.
- Enzymes often occur in combination with
organic and inorganic substances that have an
important part in catalytic action. Nonprotein
compounds are called coenzymes; inorganic
ions are called an activator.
- A coenzyme is defined as an organic molecule
that binds to the active sites of certain
enzymes to assist in the catalysis of a
reaction. More specifically, coenzymes can
function as intermediate carriers of electrons
during these reactions or be transferred
between enzymes as functional groups
- Enzymes often occur in combination with
organic and inorganic substances that have an
important part in catalytic action. Non-protein
compounds are called coenzymes; inorganic
ions are called an activator.
- A coenzyme is defined as an organic molecule
that binds to the active sites of certain
enzymes to assist in the catalysis of a
reaction. Examples of coenzymes are
nicotineamideadenine dinucleotide (NAD),
nicotineamide adenine dinucelotide phosphate
(NADP), and flavin adenine dinucleotide
(FAD).
- Enzyme activators are molecules that bind to
enzymes and increase their activity. Some
examples of enzyme activators are calcium
and magnesium ions, calmodulin, EDTA,
EGTA, fructose 2,6-bisphosphate,
hexokinase-1, and, glucokinase
- Both are collectively called cofactors which are
bound to the protein part of enzyme tightly. In
this case, cofactor is termed as prosthetic
cofactor/group.
CLASSIFICATION OF ENZYMES
BASIS OF ACTIVITY
OXIDOREDUCTASES
- catalyze the transfer of electrons from one
molecule (the oxidant) to another molecule
(the reductant)
TRANSFERASES
- catalyze the transfer of a group of atoms, such
as amine, carboxyl, carbonyl, methyl, acyl,
glycosyl, and phosphoryl from a donor
substrate to an acceptor compound
HYDROLASES
- Catalyze the cleavage of a covalent bond
using water. Types of hydrolase include
esterases, such as phosphatases, that act on
ester bonds, and proteases or peptidases that
act on amide bonds in peptides
LYASES
- catalyzes the breaking (an elimination
reaction) of various chemical bonds by means
other than hydrolysis (a substitution reaction)
and oxidation, often forming a new double
bond or a new ring structure
ISOMERASES
- convert a molecule from one isomer to another
LIGASES
- catalyze the joining of two large molecules by
forming a new chemical bond
BASIS OF SITE OF ACTION
ENDOENZYMES
- An endoenzyme, or intracellular enzyme, is an
enzyme that functions within the cell in which it
was produced. Because the majority of
enzymes fall within this category, the term is
used primarily to differentiate a specific
enzyme from an exoenzyme.
EXOENZYMES
- An exoenzyme, or extracellular enzyme, is an
enzyme that is secreted by a cell and
functions outside that cell. Exoenzymes are
produced by both prokaryotic and eukaryotic
cells and have been shown to be a crucial
component of many biological processes
 BASIS OF NATURE OF SUBSTRATE
THE AMYLOLYTIC ENZYMES OR
CARBOHYDRATES
DIASTASE AND AMYLASE
- a form of alpha-amylase that acts on starch
(amylose and amylopectin) and breaks it down
to simple sugars such as maltose and dextrins
MALT DIASTASE
- an enzyme found in malt produced during the
germination of the seeds. It is effective in
converting starch into maltose and eventually
transforms into sugar
INVERTASE OR SUCRASE
- enzymes present in yeast and in the intestinal
mucosa of animals that catalyze the hydrolysis
of cane sugar, or sucrose, to the simple sugar
glucose and fructose
THE AMYLOLYTIC ENZYMES OR
CARBOHYDRATES
MALTASE
- catalyzes the hydrolysis of the disaccharide
maltose to the simple sugar glucose; enzyme
is found in plants, bacteria, and yeast; in
humans and other vertebrates it is thought to
be synthesized by cells of the mucous
membrane lining the intestinal wall
ZYMASE
- catalyzes the fermentation of sugar into
ethanol and carbon dioxide. It occurs naturally
in yeasts. Zymase activity varies among yeast
strains. Zymase is also the brand name of the
drug pancrelipase.
DIASTASE
Synonyms: Amylase, Maltin, Diastase of malt
Description: Amorphous powder, obtained from the
infusion of malt
Color: Yellowish white
Odor: Faint characteristics
SOURCES
BARLEY (HORDEUM VULGARAE)
Barley is grown throughout the world wherever the
climate is favorable. Malt or malted barley is dried ,
artificially germinated barley grain. To prepare malt,
heaps of barley grain are kept wet with water in a
warm room and allowed to germinate until the caulicle
protrude. The grain then is quickly dried. The enzyme
diastase in the moist warm grains converts the starch
to maltose, thereby stimulating the embryo to grow.
The embryo dies when the grain is dried. Dry malt
resembles barley, but is more crisp, has an agreeable
odor, and has a sweet taste. It contains 50% to 70% of
maltose; 2% to 25% of dextrin; 8% of proteins;
diastase; and a peptase enzyme. Malt is extensively
used in the brewing and alcohol industries.
USES
- as a digestant
- in the production of predigested starchy foods
and also for the conversion of starch to
fermentable sugars in fermentation and
brewing industries
IDENTIFICATION TEST
ARSENIC
- 0.25 gm of diastase is placed in a platinum,
quarts, or porcelain crucible.
- Add 10 ml of Magnesium Nitrate in ethyl
alcohol and ignite it. Then reduce to ash by
heating at 450-550 degrees Celsius
- If carbeneous matter persist, it is wetted with
minute amount of Nitric acid, further treated
and heated at 450-550 degrees Celsius.
- After cooling, add 3mL HCl to residue, which
is dissolved by heating in water bath.
- When the test is carried out with arsenic it
should NMT 4ppm.
HEAVY METALS
- Take 0.5 g of diastase in porcelain, carbonized
by heating.
- Add 2 mL of HNO3 and 5 drops of H2SO4;
heat until white smoke disappears.
- Reduced to ash by further heating at
450-550°C
- Cool.
- 2 ml of HCl is added and evaporated in the
water bath
- 3 drops of HCl and 10 ml hot water are added
to the residue, heated for 2 minutes.
- Cool.
- Add 1 drop of Phenolphthalein indicator, then
ammonia solution is added until the color of
the solution becomes pale red.
- Solution is transferred to Nessler cylinder by
rising water.
- 50 mL test solution prepared by 2 mL of
diluted Acetic acid and water.
- For heavy metals NMT 40ppm
 - Color Solution: 2 mL HNO3, 5 drops of
H2SO4, 2 mL HCL and evaporate to dryness
in the crucible.
- 3 drops of HCl are added to the residue,
transferred into another Nessler cylinder.
- Finally, add 2 mL of lead standard solution, 2
mL Acetic acid, water added, and makeup to
50 mL.
LEAD
- 0.8 g of diastase slowly carbonized by heating
temperature below 550 degrees Celsius.
- 20 mL of dilute HNO3 is added to ash, boil for
5 minutes. Filter, adn wash the residue with
water
- Detection of lead NMT 10ppm.
COLIFORM GROUP
- Diastase tested by Microbe test method for in
General Test Method for Food Code.
- It should contain 30 or less per 1 g of the
product.
SALMONELLA
- Diastase tested by Microbe test method for in
General Test Method for Food Code.
- It should result to Negative.
PEPSIN (SUS SCROFA LINNE. VAR DOMESTICUS
GRAY)
Family name: Suidae
- Pepsin is a proteolytic enzyme obtained from
glandular layer of the fresh stomach of the hog
(pig).
- The generic name SUS is from the Greek
meaning hog; SCROFA from Latin meaning
breading; and DOMESTICUS meaning
household.
DESCRIPTION
- Pepsin occurs as lustrous, transparent, or
translucent scales, as granular or spongy
masses ranging in color from light yellow to
light brown, or as fine white or cream colored
amorphous powder. It is free from offensive
odor and has a slightly acid or slaine taste.
PREPARATION
- Mucous membrane is scraped from the
stomach and placed in acidified water at 37
degrees Celsius for 2 hours (PEPSINE and
PEPTONE)
- Filter and add sodium or ammonium salt until
it becomes half saturated. Pepsin is separated
while peptone remain.
- Precipitates by addition of alcohol.
- Collect and dry
USES
- It is a component of rennet used to curdle milk
during the manufacturing of cheese.
- Used to modify and provide whipping
properties to soy protein and gelatin.
- To make precooked cereals into instant hot
cereals.
- For flavoring food and beverages in the
leather industry to remove residual hair and
tissue from a hide.
- In preparation of F2 fragments from antibodies
TRYPSIN
Synonyms: Protinase, Tripsin, Procine, Peptidyl
peptide, hydrolase
Family name: Bovidae
- obtained from the mammalian pancreas of ox
Bos taurus
- available in amorphous powder
Color: White to whitish-yellow
Solubility:
Soluble in water;
insoluble in alcohol, chloroform, and ether.
Taste: Acrid
PREPARATION
- Purification occurs by affinity chromatography
on benzamidine or aprotinin.
- Recombinant trypsin has been expressed
many systems including E. Coli,
Saccharomyces, cerevisiae, and Pichia
pastoris.
IDENTIFICATION TEST
- Dilute 1 mL of sulfur solution in 100 mL of
water
- In a depression in a white-spot plate, mix 1.0
mL of the solution with 0.2 mL of tosylarginine
methyl ester hydrochloride solution
- A reddish-violet color appear in 3 minutes.
USES
- Trypsin is given to patients who lack enzyme
for digestion
- Given with combination with bromelain and
rutin for treatment of osteoarthritis.
- Apply directly on wounds and ulcer to remove
dead tissue and improve healing
PAPAIN
Synonyms: Papayotin, Vegetable pepsin, Arbuz,
Nematolyt, Caroid, Tromasin, Velardon, Vermizym
Family name: Caricaceae
- It is green and dried leaves of Carica papaya
L.
- Cultivated in Sri lanka, Tanzania, Hawaii and
Florida
- 5-6 m in height, bearing 30 cm length, and 5
kg in weight.
PREPARATION
- mostly found in the latex of the fruit (stem,
leaves, petioles)
- latex is collected but making 2-4 incisions,
about 1/8 inches deep
- incision makes early the morning with an
interval of 3-7 days
- latex is collected on non-metallic container or
on cloth
- dry as soon as possible either by sun drying or
by heating above 38°C
- final product should be creamy white
- sealed in an airtight container to prevent loss
of activity
- Activity can be preserved by adding 10%
common salt or 1% solution of formaldehyde
before drying
- yield of papain from latex is about 20%
(20-250 grams per tree)
- highly active product is obtained by dissolving
the commercial product in H2SO4, H2S ppts
in alcohol
- commercial papain is adulterated with
arrowroot starch, dried milk of cactus, gutta
percha, rice flour, and pepsin (process:
Adulteration)
PROPERTIES
- occur as white or greyish-white, slightly
hygroscopic powder
- partially soluble in water and glycerol; may
digest about 35 times its weight of lean meat
- best grades digestion of 200-300 times their
weight of coagulated egg albumin in alkaline
media
- temperature range from 60-90 degrees
Celsius; optimum point of 65 °C
- best pH is 5.0, but also work in alkaline and
neutral media activated by reduction (HCN,
H2S); deactivated by oxidation (H2O2)
CHEMICAL CONSTITUENTS
- peptidase I, rennin, clotting enzyme pectase,
chymopapain
- contains 15.5% nitrogen and 1.2% sulfur
- crystalline papain is most suitable in range of
5-7 pH and readily destroyed at 30 °C below
pH 2.5 and above pH 12
IDENTIFICATION TEST
- reacted with gelatin solution at 80°C in the
presence of activating cysteine chloral hydrate
solution for one hour
- solution is cooled for 4 degrees Celsius for
long time
USES
- to prevent adhesions in sloughing and infected
wounds, internally, as protein digestant,
anthelmintic, to relieve episiotomy (incision of
the vagina), and used in the treatment of
dyspepsia (digestive), intestinal and gastric
disorder
- in treatment of diphtheria
- used in digestive mixtures, liver tonic, reducing
enlarged tonsils, in preventing of
post-operating adhesions, carbuncles (boil,
spot) and eschar (dry scar) burns
- preparation of toothpaste, cosmetics, in
tanning industries for bating skin and hides
- anti-inflammatory
PANCREATIN
Synonyms: Diastase vera, Pandrotanon, Zyapanor
Family name: Suidae and Bovidae
- extracted from pancreas of certain animals like
hogs sus scrofa
- fine amorphous powder
Color: White to cream
Odor: Faint characteristics
PREPARATION
- Pancreatic α- amylase
- used to prepare pancreatin
- prepared by fractional precipitation of aqueous
extract from hog or bovine pancreas
- the technical enzyme preparation was used
principally for textile sizing because of low
stability
IDENTIFICATION TEST
- 50 mg of substance in test tube, add 2-3 mg of
resorcinol and 1 mL of H2SO4, shake, heat at
130 degrees Celsius for 5 minutes then cool
 - dilute with 5 mL H2O and add hydroxide
solution, drop wise
- to make alkaline solution, cool 7 dilute to 10
mL greenish blue fluorescence under UV lamp
USES
- used to treat pancreatic exocrine insufficiency
(PEI) attributed to cystic fibrosis , chronic
pancreatitis nutritional formula of tablets,
capsules , or powders
- preparation of leather hides and meat
tenderizer.
BROMELAIN
Synonyms: Stem Bromelain, pineapple stem
Family name: Bromeliaceae
- proteolytic enzyme obtained from the stem
and ripened fruit of Pineapple (ananas
comosus)
Color: buff colored powder
Odor: odorless
Taste: acrid
PREPARATION
- fresh stems were collected and washed with
H2O2; peeled and cut into pieces
- juice is collected from fresh pineapple stem
and fruit by homogenization in the presence of
sodium acetate buffer solution and filtered
- 500 mL of filtrate is collected
- benzoic acid or sodium benzoate is added as
preservative at 1 gm/kg concentration
- filtrate was called crude extract.
- bromelain is added in the crude extract, then
by centrifugation for 10 minutes at 2000 rpm,
4000 rpm, and 6000 rpm at 4 degrees Celsius,
a supernatant solution was collected
IDENTIFICATION TEST
- in a 0.2 g of sample, add 1 g of anhydrous
sodium carbonate and heat slowly to
carbonize
- cool then add 5 mL of H20, stir and filter right
after
- acidify slightly the filter with dilute HNO3, heat
in water bath for 5 minutes, and cool
- now, add silver nitrate which yields yellow ppts
which is insoluble in diluted HNO3 or
ammonia.
- separate precipitates and strong ammonia
with shaking
- separate liquid and acidified in diluted HNO3,
yield white turbidity.
USES
- angina, dysmenorrhea, and other CVS
disorders
- arthritis, athletic injuries, bronchitis, burn
debridement, cancer, digestive disorder,
pancreatic insufficiency, thrombophlebitis
FICIN
Synonyms: Ficus Proteinase
Family name: Moraceae
- found in latex of plant Ficus
- commercial latex from the fig tree (with fruit
and shrub); Ficus glabrata or ficus carcia
- micro granular powder
- optimum temperature is 50-55 degrees
Celsius; optimum pH is 5.0 to 7.5
- partially soluble in water, and insoluble in
organic solvents
Color: white to yellowish
PREPARATION
- crystalline ficin was prepared by Walti (1938)
-- this fraction formed by self-digestion of the
enzymes during fractionation
- the crude enzyme was inactivated by sodium
tetrathionate, and after gel filtration
- Jones and Glazer separate latex into 5
components which is quite similar in moisture
weight 25,000-26,000 (Lucien)
- Figs contain protease; an average green fig
contain 100-150 mg commercial ficin
- sundried figs retain about 12% or their original
protease activity.
IDENTIFICATION TEST
- neutralize 1 mL of sample with NaOH and 2
drops of sample, 1 mL of FeCl3 which results
to deep reddish-orange change to yellowish
orange upon addition of the mineral acid
USES
- In beer and alcohol industry
- hydrolyzation of proteins and meat processing
- baking industry
- ophthalmology; for cleaning contact lens
- antineoplastic agent, digestive aid, treatment
for arthritis
UROKINASE
Synonyms: U-plasminogen, Urinary plasminogen
activator
- obtained from human urine; also obtained in
E. coli by rDNA technique
 - available in form of lyophilized white powder
which is soluble in water
- converts plasminogen to plasmin
- degrade fibrin and other plasma proteins
PREPARATION
- produced by E. coli by rDNA technique
- First pro urokinase than converted into
plasmin (kallikrein)
- Directly found in the purification of human
urine
- Adsorbent used-silica and kaolin
- Further purification- precipitations of NaCl or
ethanol of by chromatography
- Human urokinase needs sterile filtration,
septic filling, or freeze drying
USES
- Coronary thrombosis
- Myocardial infarction
- Pulmonary embolism
- Venous thrombosis
STREPTOKINASE
- culture filtrates of β-haemolytic Streptococci
group C
- shows activity on plasminogen into proteolytic
enzyme
- plasmin carries out degradation of fibrin clots,
fibrinogen, plasma proteins
- it is purified bacterial protein with about 484
amino acid residue
PREPARATION
- available as sterile, friable solid or white
powder
- water-soluble at pH 7 (maximum activity)
- higher concentration solution stable for 6
hours at 4 degrees Celsius
IDENTIFICATION TEST
- it is bacterial protein with half life of 23
minutes; its anisolylated plasminogen activator
complex (APSAC) has a higher half life of 6
hours
- its activity is determined by Silverstein
- it is based on the observation that
streptokinase-human plasma complex can
hydrolyze the artificial subtract
USES
- as a fibrinolytic agent to help the removal of
fibrin thrombi (thrombus clot) from the arteries
- in thromboembolic disorder, lysis of arterial
thrombus, acute coronary thrombosis, deep
vein thrombosis, pulmonary emboli
HYALURONIDASE
Synonym: Spreading factor
- observed in the testes and semen
- specifically depolymerizes hyaluronic acid,
therefore enhancing the permeability of the
connective tissues
- acts as the disperse cells of corona radiate
about the newly ovulated ovum, thus largely
facilitating entry of the sperm.
- reduce the viscosity of tissue cement and
increase the rate of intramuscular and
subcutaneous injectables in humans
- it is a group of enzymes like 4-
lykanohydrolase, hyaluronate 3-3 glycano
hydrolase and hyaluronate lyase
- they are mucopeptides composed of
alternating N-acetyl glucosamine and
glucuronic acid.
PREPARATION
- it is a sterile, dry soluble enzyme preparation
which is prepared from testes and semen by
fractional precipitation of aqueous extract and
further by dialysis
- sterilization by filtration and lyophilization
- it is odorless, white to yellow in color; highly
soluble in water but insoluble in organic
solvent
IDENTIFICATION TEST
- acid mucopolysaccharide, histochemically,
using colloidal iron stain
- determined by digestion of a serial section
with hyaluronidase prior to staining
USES
- enhance absorption rate
- reduce discomfort caused by intramuscular or
subcutaneous injections
- also used in hypodermolysis
PENICILLINASE
Synonym: β-lactamase
- a bacterial enzyme, bacillus species and
certain strains of staphyloccocus
- contains β-lactam ring
- include penicillin derivative
- inhibits bacterial cell wall synthesis
PREPARATION
- Obtained for B. subtilis and B. cereus
- It is divided into 2 class:
- PENICILLIN AMIDASE / ACYLASE
- Amidase attack on acyl group
attached to the nucleus that is why it
is called acylase
- Amidase is more specific with Pen- V
&K
- β-lactamase
- Act on basic nucleus itself
- More specific with the Pen- G & X and
lesser with Pen- V

USES
- Inactivation of penicillin
- Antigen-antibody reaction

IDENTIFICATION TEST
- Each strip emarginated with benzylpenicillin
and pH indicator, bromocresol purple
- Positive produce penicilloic acid
- Causes fall in pH
- Purple to yellow