Enzymes are biological catalysts that speed up chemical reactions. This document discusses several important enzymes including amylolytic enzymes that break down starches and sugars, lipolytic enzymes that break down fats, proteolytic enzymes that break down proteins, and oxidizing enzymes involved in oxidation reactions. Specific enzymes discussed include pepsin, trypsin, rennin, papain, pancreatin, and hyaluronidase. These enzymes are obtained from animal sources like the stomach and pancreas and are used to treat digestive deficiencies or aid in various medical applications.

1. ENZYMES
A protein with catalytic properties due to its
power of specific activation
Enzymes are biological catalysts.

2. Important ENZYMES
1. The amylolytic enzymes
2. Lipolytic enzyme
3. Proteolytic enzymes
4. Oxidizing enzymes

3. Amylolytic ENZYMES
1. DIASTASE and AMYLASE
Salivary Diastase or Ptylin
Pancreatic diastase or amylopsin
2. Malt diastase  starch to maltose
3. Invertase or sucrase sucrose to glucose &
fructose
4. Maltase maltose to glucose

4. 5. Zymase monosaccharides to CO2 and alcohol
6. Emulsin  amygdalin to glucose, HCN and
benzaldehyde
7. Myrosin  Sinalbin, Sinigrin and other
glycoside

5. Proteolytic enzymes
1. Pepsin  Proteins into proteoses & Peptons
2. Trypsin  proteoses & Peptons to
polpeptides and amino acids
3. Erepsin  proteoses & Peptons to amino
acids
4. Rennin  Curdles soluble casein of milk
5. Papain  meat tenderize proteoses &
Peptons to polpeptides and amino acids r

6. OXIDIZING Enzymes
1. Peroxidases  Oxidation reaction
2. Thrombin  Fibrinogen to Fibrin
3. Zymase Monosaccharide spliting enzyme
but works by oxidation

7. MALT EXTRACT
BARLEY is dried grain of Hordeum vulgare
Graminae
Cultivated through out the world wherever climate
is favourable
Malt or Malted Barley
Dried, artificially germinated barley grain
Grains kept wet with water in warm room
germinate till caulicle protrudes  dried quickly

8.  Enzyme diastase Converts starch to maltose
 Stimulate embryo formation which is killed
by drying
 Dry malt resembles barley & has agreeable
odour and sweet taste
 Contains sugar maltose 50-70%
 Dextrins 2-15%
 Proteins 8%
 Diastase and peptase emzyme
 Used in Brewing & alcohol industries

9. Malt extract
 Extracting malt with hot water at 60C.
 Concentracted at 60C under pressure
 Malt extract is mixed with 10% glycerine
 It contains dextrin, maltose, glucose and
amylolytic enzyme
 Starch  soluble sugars (5times of its own
weight)

10. Uses
 Malt extract is used as easily digested
nutritive and as an aid in starch digestion.
 15g
 Diastase 50times starch  sugars
 Lactase converts lactose galactose and
glucose. It is used in pt having lactose
intolerance

11. Papain
 Phytoenzyme
Biological source
Dried and purified latex of the fruit of
Carica papaya family Caricaceae
Geographical source
Indigenous to tropical America
Cultivated in Sri Lanka, Tanzania, Hawaii, Florida

12. Plant description
 Plant attains height of 5-6 meters
 Fruit grows to a length of about 12 inches
(30cm)
 Weight of fruit is about 5 Kg

13. Collection procedure
 The epicarp of the fruit adheres to the orange
colour, fleshy sarcocarp, which surrounds the
central cavity.
 Cavity contains a mass of nearly black seeds
 Full grown but unripe fruits are subjected to
shallow incisions on the 4 sides.
 Latex flow for few seconds and then soon
coagulates
 Latex is then collected

14.  After collection coagulated lumps are
shredded and dried by sun or by artificial heat
 Later methods yield better grades of crude
papain
 Incisions & collections are made at weekly
intervals as long as the fruit exudes the latex.
 Crude papain is purified by dissolving in
distilled water and then precipitating with
alcohol

15. Papain enzymes
 Papain is referred to as “Vegetable Pepsin”
because it contain enzyme similar to that of
Pepsin. However unlike Pepsin, Papain acts in
acid, neutral and alkaline media.
 Papain contain one or more proteolytic
enzymes because a single sample of papain
yield variable result with the type of protein
used

16.  Papain contains one or more proteolytic
enzymes, Peptidase-I, converts proteins
dipeptides and polypeptides
 Rennin like coagulating enzyme that act on
milk casein
 An Amylolitic enzyme
 A Clotting enzyme similar to Pectase Pectin
 pectic acid and methyl alcohol
 An enzyme that act on fat

17. Actions and uses of Papain
 Papain differ in strength with method of
preparation
 It is used as meat tenderizer  It can digest
35 times of its own weight of lean meat
 It is used extensively in tenderization of beef

18.  Best grade of papain can digest 300 times of
its own weight of egg albumin
 It is used as protein digestant as it has same
action on protein as that of Pepsin
 Used to relieve the symptoms of episiotomy

19. Pepsin
 Animal enzyme
 Proteolytic enzyme
Biological source
Enzyme obtained from the glandular layer of the
fresh stomach of various animals like Pig, sheep
or calf. Commonly from pig,
Sus scrofa family Suidae
Sus is from Greek word Us meaning HOG

20. Preparation of pepsin
 Pepsin is prepared by using stomach linings
 The mucous membrane is separated from the
stomach by the process of stripping or it is
scrapped off
 Then it is placed in acidified water for
autolysis at 37ºC for 2 hours
 The liquid so obtained consists of both pepsin
and peptone

21.  It is then filtered and sodium or ammonium salts
are added to the liquid till it is half saturated
 At this point only the pepsin separates out in the
form of precipitates, and the peptone remains in
solution
 The precipitates are collected and subjected to
dialysis for the separation of salts
 Remaining amount of pepsin if any in the
aqueous solution is precipitated by the addition
of alcohol into it.

22.  Concentrated solution containing precipitates,
is poured on glass plates to dry  forming
Scale pepsin
 Or concentrated solution is carefully
evaporated in vacuum  forming spongy
pepsin

23. Description of enzyme
 Pepsin is lustrous, transparent or translucent
scales or occurs as granular or spongy masses.
 Colour ranges between light yellow to light
brown or as fine white or cream coloured
amorphous powder
 It is free from offensive odour and has a
slightly acid or saline taste.
 It show optimal activity at a pH 1.8 to 3.5
 Reversibly inactive at pH 7 to 8

24. Standard pepsin
 Pepsin digests not less than 3000 and not
more than 3500 times its weight of coagulated
egg albumin.
 Commercial pepsin, especially spongy pepsin
is often 4-5 times more active than that of
medicinally used.
 A pepsin of higher digestive power may be
reduced to standard pepsin by admixture with
a pepsin of lower grade or with lactose.

25.  Pepsin best act at a temperature 40ºC and pH
2-4
 Pepsin denatured at 70ºC and in the presence
of alcohol.
 Pepsin can be stored at 2-8ºC for about 1-2
years

26. Uses
 pepsin is used in deficiency of gastric secretion
 Pepsin is administered to assist digestive digestion
in combination with pancreatin.
 It is proteolytic enzyme is administered after meals
and followed by a dose of hydrochloric acid, its
Usual dose is 500mg
 It is used in laboratory analysis of various proteins.
 It is used in preparation of cheese and other protein
containing food

27. Pancreatin
 Animal enzyme
 Pancreatin is a mixture of several digestive
enzymes. It is composed of
 Amylase (hydrolyses starches to oligo &
disaccharides maltose)
 Lipase (hydrolyses triglycerides to fatty acids
and glycerols)
 Protease (Trypsin hydrolyses proteins to
oligopeptides)

28. Biological source
Pancreatin is produced by the exocrine cells of
the pancreas of Pig or Hog,
Sus scrofa Family Suidae
or from Pancreas of Ox or calf
Bos taurus Family Bovidae

29. Preparation method
 The Pancreas is a gland that lies directly
inside the posterior wall of the abdomen.
 Fresh glands are minced and extracted by
similar methods as employed in the
manufacture of Pepsin

30. Standard pancreatin
 It is cream coloured amorphous powder with a
faint, characteristic but not offensive odour.
 Pancreatin contain in each mg,
 Not less than 25 USP units of amylase activity
 Not less than 2 USP units of lipase activity
 Not less than 25USP units of protease activity

31. USP units
 One USP unit of amylase activity is contained
in the amount of pancratin that digests 1mg of
the dry USP potato starch standard
 One USP unit of lipase activity is contained in
the amount of pancratin that liberates 1µEq of
acid per minute at a pH 9 and 37ºC.
 One USP unit of protease activity is contained
in the amount of pancratin that digests 1mg of
casein under specific conditions

32. Properties
 It show optimal activity in neutral or faintly
alkaline solution
 More than traces of mineral acids or large
amounts of alkali hydroxide render pancreatin
inert, and excess of alkali carbonates inhibits
its action

33. Pancreatin – uses
 Pancreatin is a mixture of several digestive
enzymes. This mixture is used to treat
conditions in which pancreatic secretions are
deficient, such as surgical pancreatectomy,
pancreatitis and cystic fibrosis.
 As digestive aid for invalids
 Enteric coated granules are used to treat infants
with celiac disease and related pancreatic
deficiencies,. Usual dose is 325mg – 1g as
tablets, capsules or granules

34.  Routine cancer eradication
 As is mixture of enzymes but proteolytic
enzyme play role in cancer eradication
 Cancer is often disease of protein metabolism
 Pancreatin enzyme cancer defence mechanism
 Effected by protein rich foods at inappropriate
time or in excessive amount
 Body need 12 hrs time free of protein
consumption for proper working of this
defence system

35.  Pancreatin become inactive in acid or with
alcohol contact
 cancer continuously produce acids
 Many cosmetic contain acid or
alcoholspecial concern with the skin cancer

36. Pancrealipase
 Is same as Pancreatin but is more concentrated
form of Pancreatin
 Pancrealipase contain in each mg,
 Not less than 100 USP units of amylase activity
 Not less than 24 USP units of lipase activity
 Not less than 100USP units of protease activity
Thus the lipase activity is increased by 12folds,
but the amylase and protease activity only 4 folds
when compared to Pancreatin

37. Biological source
Pancreatin is produced by the exocrine cells of
the pancreas of Pig or Hog,
Sus scrofa Family Suidae
or from Pancreas of Ox or calf
Bos taurus Family Bovidae

38. Uses
 Employed as digestive aid
 It increase intestinal absorption of fat used
to treat steatorrhea
 Usual dose is 8000 to 24000 USP units
 Dose is determined on the basis of clinical
evaluation according to pancreatic
insufficiency

39. Rennin
 Animal enzyme
 Protease enzyme
 Milk curdling enzyme
Biological source
Rennin is partially purified enzyme obtained
from the glandular layer of the stomach of the
calf, Bos taurus
Family : Bovidae

40. Rennin (RENNET)
Three main sources,
 Bos taurus  animal source
 Withania coagulans  plant source
 Rhizomucor miehei  microbial source

41. Preparation method
 Rennin is prepared by macerating the minced
glandular layer of the digestive stomach of the
calf in 0.5% sodium chloride solution.
 Filtration is then carried out
 Filtrate is acidified with hydrochloric acid
 Then saturation is carried out with sodium
chloride
 Which precipitates the Rennin
 Rennin is then separated and dried and
powdered

42. Standard Rennin
 Rennin occur as a yellowish white powder or
as yellowish grains or scales
 It has characteristic and slightly saline taste
 It has peculiar odour
 Standard Rennin can coagulate approximately
25000 times its own weight of fresh cow’s
milk

43. Uses
 Rennin is used to coagulate milk, thus
rendering it more digestible for convalescents
 It is ingredient in Rennin and pepsin elixir
called pepsin essence
 Protease enzyme that curdles the casein in
milk, helping young mammals digest their
mothers' milk.
 Its principal used is in the manufacture of
cheese, mainly for coagulation of milk

44.  Used to separate milk into solid curds used for
cheese making and liquid whey

45. Important enzyme
 Trypsin
Proteolytic enzyme from pancreas gland of Ox
Used for debridement of necrotic and pyogenic
surface lesions
Wound and ulcer cleanser
 Chymotrypsin
Proteolytic enzyme from pancreas of Ox
Ophthalmic solution

46. Hyaluronidase (mucolytic enzyme)
 The hyaluronidases are a family of enzymes that degrade
hyaluronic acid. It is found in human testes, in bacterial cultures as
by product or in the heads of leeches and snake venoms
 By catalysing the hydrolysis of hyaluronic acid, hyaluronidase
lowers the viscosity of hyaluronic acid, thereby increasing
tissue permeability.
 It is, therefore, used in medicine in conjunction with other drugs to
speed their dispersion and delivery.
 Common applications are ophthalmic surgery, in combination
with local anesthetics.
 It also increases the absorption rate of parenteral fluids given
by interstitial or subcutaneous infusions (hypodermoclysis).
 It is an adjunct in subcutaneous urography for improving
resorption of radiopaque agents

47.  Streptokinase & streptodornase
 Thrombolytic enzyme
 Streptokinase breakdown fibrin whereas streptodornase
affects deoxyribonucleic acid and desoxyribo-nucleo-
protein, which is chief constituents of pus and necrotic
tissue, therefore these are used wherever, clotted blood or
fibrinous or prulent accumulations appear following injury
to the tissue.
 The enzymes are used in 4:1 treptokinase:treptodornase
 Usual oral dose is 10000 units of streptokinase in
combination with 2500 units of streptodornase 4times/ day
 For IM administration 5000:2500 units twice a day are
used

48. Streptokinase
 Streptokinase is used for its indirect thrombolytic activity
 It acts on the endogenous fibrinolytic system by converting
plasminogen to plasmin(proteolytic enzyme) or to fibrinolysin
 Fibrinolysin degrades fibrin clots
 Used for massive pulmonary emboli and for extensive thrombi
of the deep veins in adults
 Streptokinase is administered through continuous IV infusion
@ 10000 units per hour for 24 to 72 hours
 It should be used by only the physician who is expert of
managing thrombotic diseases
 It should not be used to treat superficial thrombophlebitis

49. Urokinase
 Isolated from human urine
 Alternate of the streptokinase for the
treatment of massive pulmonary emboli
 Have less chances of allergic reaction due to
human origin
 Administered through IV infusion @ 4400
untis/kg body weight per hour for 12 hours

50. Fibrinolysin
 It is present in blood serum as a protease and in plasma
as the inactive precursor plasminogen
 It is prepared commercially by activating blood serum
with streptokinase
 It act on the protein portions of the dead tissues,exudates
and blood clots found in the wounds, ulcers & burns
 It is used to treat the blood clots within the
cardiovascular system
 It is used to treat Phlebothrombosis, thrombophlebitis
and pulmonary emboli
 It is administered by IV infusion

51. Sutilains
 Proteolytic enzyme
 Obtained from Bacillus subtilis
 It is cream colour powder
 Standard enzyme has not less than 2.5million
USP units of casein proteolytic activity per
gram
 It is used in topically in the form of ointment
for wound debridement

52. Collagenase
 It is obtained from the culture of Clostridium
histolyticum
 It cleaves collagen
 It is used topically to debride the dermal ulcers and
severely burned areas
 Enzyme is deactivated by heavy metals so care should
be exercised in this regard
 In case of bacteremia, Burrow’s solution should be
used to stop the action of enzyme
 Used in the form of ointment containing 250 units of
collagenase activity per gram

53. Deoxyribonuclease
 Nucleolytic enzyme
 Obtained form the pancreas of bovine origin
 It is active in dry form
 It catalysis cleavages of giant molecules of
DNA (deoxyribonucleic acid) into numerous
small fragments called polynucleotides
 Thus it act against the devitalized tissues in
purulent states
 It is available in combination with fibrinolysin

54. L -Asparaginase
 Obtained from Escherichia coli (E. coli)
 It is used to treat acute leukemia in children
 Its anticancer effect is attributed to the fact that this
enzyme catalysis the conversion of Asparagine to
aspartic acid and ammonia, there by depleting the
asparagine, which result in the death of cells that
requires exogenous sources of this amino acids for
their survival (mostly cancer cell or leukemic cell).
 Clinical effectiveness of the drug is due to the
requirement for L-asparagine differences of the normal
cells and the cancer/neoplastic cells

55.  It is used in combination with other
anticancers like prednisolone and vincristine
 Usual dose IV is 1000 units per kg body
weight daily or IM 6000 units per square
meter of body surface area at 3 day interval
 A number of adverse effects have been noted
with L-Asperaginase use, like allergic
reaction, anaphylactic reaction

56. Properties of enzymes

57. Chemical reactions
 Chemical reactions need an initial input of energy =
THE ACTIVATION ENERGY
 During this part of the reaction the molecules are
said to be in a transition state.

58. Reaction pathway

59. Making reactions go faster
 Increasing the temperature make molecules move
faster
 Biological systems are very sensitive to temperature
changes.
 Enzymes can increase the rate of reactions without
increasing the temperature.
 They do this by lowering the activation energy.
 They create a new reaction pathway “a short cut”

60. An enzyme controlled pathway
 Enzyme controlled reactions proceed 108 to 1011 times faster
than corresponding non-enzymic reactions.

61. Enzyme structure
 Enzymes are
proteins
 They have a
globular shape
 A complex 3-D
structure
Human pancreatic amylase
© Dr. Anjuman Begum

62. The active site
 One part of an enzyme,
the active site, is
particularly important
 The shape and the
chemical environment
inside the active site
permits a chemical
reaction to proceed
more easily
© H.PELLETIER, M.R.SAWAYA
ProNuC Database

63. Cofactors
 An additional non-
protein molecule that is
needed by some
enzymes to help the
reaction
 Tightly bound cofactors
are called prosthetic
groups
 Cofactors that are bound
and released easily are
called coenzymes
 Many vitamins are
coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors
Jmol from a RCSB PDB file © 2007 Steve Cook
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS
IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

64. The substrate
 The substrate of an enzyme are the reactants
that are activated by the enzyme
 Enzymes are specific to their substrates
 The specificity is determined by the active
site

65. The Lock and Key Hypothesis
 Fit between the substrate and the active site of the enzyme is
exact
 Like a key fits into a lock very precisely
 The key is analogous to the enzyme and the substrate
analogous to the lock.
 Temporary structure called the enzyme-substrate complex
formed
 Products have a different shape from the substrate
 Once formed, they are released from the active site
 Leaving it free to become attached to another substrate

66. The Lock and Key Hypothesis
Enzyme may
be used again
Enzyme-
substrate
complex
E
S
P
E
E
P
Reaction coordinate

67. The Lock and Key Hypothesis
 This explains enzyme specificity
 This explains the loss of activity when
enzymes denature

68. The Induced Fit Hypothesis
 Some proteins can change their shape
(conformation)
 When a substrate combines with an enzyme, it
induces a change in the enzyme’s conformation
 The active site is then moulded into a precise
conformation
 Making the chemical environment suitable for the
reaction
 The bonds of the substrate are stretched to make the
reaction easier (lowers activation energy)

69. The Induced Fit Hypothesis
 This explains the enzymes that can react with a
range of substrates of similar types
Hexokinase (a) without (b) with glucose substrate
http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

70. Factors affecting Enzymes
 substrate concentration
 pH
 temperature
 inhibitors

71. Substrate concentration: Non-enzymic reactions
 The increase in velocity is proportional to the
substrate concentration
Reaction
velocity
Substrate concentration

72. Substrate concentration: Enzymic reactions
 Faster reaction but it reaches a saturation point when all the
enzyme molecules are occupied.
 If you alter the concentration of the enzyme then Vmax will
change too.
Reaction
velocity
Substrate concentration
Vmax

73. The effect of pH
Optimum pH values
Enzyme
activity Trypsin
Pepsin
pH
1 3 5 7 9 11

74. The effect of pH
 Extreme pH levels will produce denaturation
 The structure of the enzyme is changed
 The active site is distorted and the substrate
molecules will no longer fit in it
 At pH values slightly different from the enzyme’s
optimum value, small changes in the charges of the
enzyme and it’s substrate molecules will occur
 This change in ionisation will affect the binding of
the substrate with the active site.

75. The effect of temperature
 Q10 (the temperature coefficient) = the increase in
reaction rate with a 10°C rise in temperature.
 For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with every
10°C rise in temperature)
 Enzyme-controlled reactions follow this rule as they
are chemical reactions
 BUT at high temperatures proteins denature
 The optimum temperature for an enzyme controlled
reaction will be a balance between the Q10 and
denaturation.

76. The effect of temperature
Temperature / °C
Enzyme
activity
0 10 20 30 40 50
Q10 Denaturation

77. The effect of temperature
 For most enzymes the optimum temperature is about
30°C
 Many are a lot lower,
cold water fish will die at 30°C because their
enzymes denature
 A few bacteria have enzymes that can withstand very
high temperatures up to 100°C
 Most enzymes however are fully denatured at 70°C

78. Inhibitors
 Inhibitors are chemicals that reduce the rate of
enzymic reactions.
 The are usually specific and they work at low
concentrations.
 They block the enzyme but they do not
usually destroy it.
 Many drugs and poisons are inhibitors of
enzymes in the nervous system.

79. The effect of enzyme inhibition
 Irreversible inhibitors: Combine with the
functional groups of the amino acids in the
active site, irreversibly.
Examples: nerve gases and pesticides,
containing organophosphorus, combine with
serine residues in the enzyme acetylcholine
esterase.

80. The effect of enzyme inhibition
 Reversible inhibitors: These can be washed
out of the solution of enzyme by dialysis.
There are two categories.

81. The effect of enzyme inhibition
1. Competitive: These
compete with the
substrate molecules for
the active site.
The inhibitor’s action is
proportional to its
concentration.
Resembles the substrate’s
structure closely.
Enzyme inhibitor
complex
Reversible
reaction
E + I EI

82. The effect of enzyme inhibition
Succinate Fumarate + 2H++ 2e-
Succinate dehydrogenase
CH2COOH
CH2COOH CHCOOH
CHCOOH
COOH
COOH
CH2
Malonate

83. The effect of enzyme inhibition
2. Non-competitive: These are not influenced by the
concentration of the substrate. It inhibits by binding
irreversibly to the enzyme but not at the active site.
Examples
 Cyanide combines with the Iron in the enzymes
cytochrome oxidase.
 Heavy metals, Ag or Hg, combine with –SH groups.
These can be removed by using a chelating agent such
as EDTA.

84. Applications of inhibitors
 Negative feedback: end point or end product
inhibition
 Poisons snake bite, plant alkaloids and nerve
gases.
 Medicine antibiotics, sulphonamides,
sedatives and stimulants