Transcript ENZYME

WHAT ACTUALLY IS BROMELAIN…??
 Mixture of protein digesting enzymes known as proteolytic
enzymes or proteases – include several other substances as
well
 either of two proteases extracted from plant family
bromeliaceae i.e.,
Stem bromelain - EC 3.4.22.32
Fruit bromelain - EC 3.4.22.33
 May also refer to a combination of those enzymes along
with other compounds produced in an extract
 Referred to as sulfhydryl proteases since a free sulfhydral
group of a cysteine side chain is essential
 The other substances typically include peroxidase, acid
phosphatase, protease inhibitors, and calcium
temperature
Optimal temperature
Deactivation temperature
40-60 °C
50-60 °C
above65 °C approx.
α HELIXES
AND
HELICAL
TURNS
PEEK IN TO THE PAST…
 First isolation  Vicente Marcano in 1891 from fruit of
pineapple.
 In 1892, Chittenden, Joslin and Meara investigated the
matter fully and named it ‘Bromelin’
 Later, Bromelian was introduced and orignally applied to any
protease from any member of family Bromeliaceae.
 In 1957 first introduced as therapeutic supplement
 Pioneer research  at Hawaii but recent  in countries in
Asia, Europe and Latin America.
 Germany has recently taken a great interest in bromelian
research.
 13th most widely used herbal medicine in Germany.
IT COMES FROM…
 Pineapple plant (Ananas sp.)
 Stem  most common commercial source
 Traditionally as a medicinal plant among natives of
South and Central America.
 Produced in Thailand, Taiwan, and other tropical parts of
the world where pineapples are grown.
 Prepared from stem part of pineapple after harvesting
the fruit.
ROLE OF THE STUD…
 Bromelain
bloods fibrolytic activity and kininogen
and bradykinin serum and tissue levels as well as reduce
excretion of proinflammatory cytokines and chemokines
 Also effects prostaglandin synthesis
 Inhibits fibrinogen synthesis
 Directly degrades fibrin and fibrinogen
 cleave at Lys-, Ala-, Tyr-, Gly Is activated by cysteine, bisulfite salt, NaCN, H2S, Na2S,
and benzoate.
 inhibited by Hg++, Ag+, Cu++, a-1-antitrypsin, estatin
A&B, Iodoacetate, TLCK, TPCK
PAY BACK TIME…
 product name  ‘Ananase’
 Various uses in Folk medicine
 Explored as a potential healing agent in alternative medicine.
 Work by blocking some proinflammatory metabolites when applied
topically
 Used for reducing swelling
 Involved in the migration of neutrophils to the site of acute
inflammation.
 Used for treating arthiritis
 When used in conjunction with trypsin and rutin is as effective as
prescribed analgesics in the osteoarthiritis management.
 Meat tenderizing
WHAT ELSE…??
Other effects include:

Hay fever

Treating a bowel condition that includes swelling and ulcer ulcerative
colitis

Removing dead and damaged tissue after a burn debridement

Preventing the collection of water in the lung pulmonary edema

Relaxing muscles

Improving the absorption of antibiotics

Preventing cancer

Shortening labor

Help the body in reducing fats

Supplement may effect heart rate

systemic enzyme therapy
DIASTASE
Alpha
amylase
 Diastase are any one of a group of enzymes which
catalyses the breakdown of starch into maltose.
 first enzyme discovered.
diastase
 It was extracted from malt solution in 1833 by Anselme
Payen and Jean-François Persoz, chemists at a French
sugar factory.
Gamma
Beta
 The name "diastase"
Greek (diastasis)
amylase comes from theamylase
(a parting, a separation)
ALPHA AMYLASE
 EC NUMBER: 3.2.1.1 is 1,4-a-D-Glucan
glucanohydrolase
• Ph-5.9-6.6


Conditions
ALTERNATIVE NAME : glucogenase
• Temperature opt
for
=37’C
Location: it is secreted in saliva
and pancreas, found in
amylase
humans and other animals food reserve of fungi/
 Acts on starch, glycogen and related polysaccharides
and oligosaccharides in a random manner; reducing
groups are liberated in the
• alpha-configuration.
Chloride ion and
Metal ions
and
activators
 Causes hydrolyses alpha-bonds
of largeion
alpha-linked
bromide
polysaccharides, such as starch and glycogen, yielding
• Most effective.
glucose and maltose.
STRUCTURE

679 amino acid residues with a molecular weight of 75112 residues
 It has 3 domains A B C
 DOMAIN A: These domains are generally found on all α-amylase
enzymes. The A domain constitutes the core structure, with a (β/α)8barrel.

DOMAIN B :consists of a sheet of four anti-parallel β-strands with a
pair of anti-parallel β-strands. Long loops are observed between the βstrands. Located within the B domain is the binding site for Ca2+-Na+Ca2+.
 DOMAIN C consisting of eight β-strands is assembled into a globular
unit forming a Greek key motif. It also holds the third Ca2+ binding site
in association with domain A
 ACTIVE SITE:
 Positioned on the C-terminal side of the β-strands of the (β/α)8-barrel
in domain A is the active site. The catalytic residues involved for the
BSTA active site are Asp234, Glu264, and Asp331
AMYLOSE IN
STARCH
GLUCOSE RESIDUE CLEAVED BY
INDUSTRIAL APPLICATION:
 used in ethanol production to break starches in grains
into fermentable sugars.
 detergents, especially dishwashing and starch-removing
detergents.
 in textile weaving, starch is added for warping.
 -Amylase is used for the production of malt, as the
enzyme is produced during the germination of cereal
grains
 Checking out pancerititis the amylase levels are
measured in the pancertic cells.
Trypsinogen is activated by removal of a
terminal hexapeptide to yield singlechain β-trypsin. Limited autolysis
produces other active forms having two
or more peptide chains bound by
disulfide bonds. Predominant forms are
*α-trypsin, having two peptide chains and
*β-, a single chain
What is Alkaline Phosphatase?
 Alkaline phosphatase (EC 3.1.3.1)comprises a group
of enzymes that catalyze the hydrolysis of phosphate
esters in an alkaline environment, generating an
organic radical and inorganic phosphate.
 This has many isoenzymes including
 Intestinal (ALPI),
Chromosome 2
 Placental (ALPP)
 Liver/bone/kidney (ALPL)
Chromosome 1
 It belongs to Alpha and Beta class of proteins
STRUCTURE
 Alkaline phosphatase is a glycoprotein mainly
parallel beta sheets
 Core has 3 layers: a/b/a.
 In general, alkaline phosphatase is a dimer
containing nearly identical subunits which each
have two molecules of zinc and one molecule of
magnesium ion.
 One molecule of zinc is tightly bound, giving the
structure stability and the other is loosely bound
which provides for the catalytic activity.
General Mechanism
I
Alkaline phosphatase binds substrate. Only
the phosphate moiety of the substrate
binds specifically to the enzyme.
II
A nucleophile attacks the phosphorous atom of
the phosphate and induces cleavage of the
oxygen-phosphorous bond.
At alkaline pH, a nucleophilic hydroxide
(from an active site water molecule) attacks
the covalently bound phosphate,
The noncovalently
bound phosphate
IV
molecule is slowly released from the
enzyme.
III
Properties AND FUNCTION
This enzyme was partially purified and studied by Kunitz
(1960)
It is a hydrolase enzyme found in bacteria and mammals
Optimum pH: 8 – 9
Activators: Mg2+
Wide specificity
Inhibitors: acidic pH, chelators of the metal ions, urea and
high levels of Zn2+
The property of dephosphorylation allows for uses in
molecular biology, in pasteurization and in nature by bacteria.
It catalyses the following reaction
A phosphate monoester + H(2)O
an alcohol + phosphate
Alkaline Phosphatase Test
One of the most important functions of alkaline phosphatase is
as an indicator for disease.
High levels
of alkaline
phosphatase
• bones, liver, bile system or
malignancies,blood,
reproductive tissues and GI
tract
Low levels
of alkaline
phosphatase
• hypophosphatasia,malnutrit
ion, celiac disease,
magnesium and zinc
deficiency, anemia
PEPSIN Classification
EC number 3.4.23.3
Member of the aspartate protease family
First animal enzyme to be discovered
Second to be crystallized
Discovery – Theodor
Schwann
Northrop
Structure:
Two aspartate molecules at the active site
Three sulphide bridges
“A tricky business”

PEPSINOGEN - primary
structure has an additional 44
amino acids

Released by chief cells in the
stomach

HCL causes activation
Pepsinogen → pepsin
TARGETS:

(Amide
autocatalysis
in aromatic
acidic env)
bonds of
amino acids like tryptophan,
phenylalanine and tyrosine
Phenylalanine
Tryptophan
Activity and Stability:
Temperature: 37°C-42°C
pH: 1.5 – 2
Stable until pH 8- can be reactivated upon
re- acidification
Deficiency:
Imbalance in pH
Inability to digest
protein
PAPAIN
Papaya Proteinase I
Cysteine protease
hydrolase
Enzyme
extraction
papaya fruit is first slit at the neck.
latex that drips is either collected in a container or
left to dry on the fruit
latex that is collected is further allowed to dry into a
crude format.
material needs to undergo a purification process to
get rid of all the contaminating products present in it.
Family & structure
 Source: present in papaya (Carica
papaya) and mountain
papaya(Vasconcellea
cundinamarcensis).
 Cysteine
protease (EC 3.4.22.2) enzyme
 Family: members found in
baculovirus, eubacteria, yeast, and
practically all protozoa, plants and
mammals, lysosomal or secreted
 contains 345 amino acid residues, and
consists of a signal sequence (1-18),
a propeptide (19-133) and the mature
peptide (134-345). The amino acid
numbers are based on the mature
peptide. The protein is stabilised by
three disulfide bridges.
Mechanism of action
 mechanism by which it
breaks peptide
bonds involves
deprotonation of Cys-25 by
His-159
 1. Deprotonation of thiol in
cysteine by basic histidine
 2. Nucleophilic attack by
deprotonated cysteine on
substrate carbonyl atom
applications
 The main function of the papain enzyme is to aid
in digestion and to promote effective digestive
health. This is done by breaking down all the
protein in the body for easy digestion.
 The papain enzyme as a meat tenderizer has
been used for many years. Since it is a
proteolytic enzyme that tenderizes meat, it also
acts as a clarifying agent in many food industry
processes.
 It is used in treatment of stings that are
administered by jellyfish, bees, wasps or insects
by breaking down the toxin and the venom.
 It boosts the immune system and is seen to be
beneficial in food allergies and tumors
Introduction
(Cellulase)
Cellulase refers to an entourage of
enzymes produced chiefly by fungi,
bacteria and protozoans that catalyze
cellulolysis (i.e. the hydrolysis of
cellulose).
However, there are also cellulases
produced by a few other types of
organisms, such as some termites and
the microbial intestinal symbionts of
other
termites.
Several
different kinds of cellulases are
known, which differ structurally and
mechanistically.
Complete vs. incomplete cellulases
 Some species of fungi and bacteria are able to
exhaustively digest crystalline cellulose in pure culture
are said to have complete or true cellulases.
 The majority of organisms that produce cellulases can
only hydrolyze the cellulose in their diets to certain
extent. they are known as incomplete cellulases.
 These cellulases unable to digest cellulose exhaustively
can still generate sufficient amount of glucose for their
producers. Endogenous cellulases of termites belong to
this category.
Types of reactions/
Classification
General types of cellulases based on the
type of reaction catalyzed:
1. Cleaves internal bonds at Endocellulase
(EC 3.2.1.4) randomly amorphous sites
that create new chain ends.
2. Cellobiase (EC 3.2.1.21) or betaglucosidase hydrolyses the exocellulase
product into individual monosaccharides.
3. Cellulose phosphorylases depolymerize
cellulose using phosphates instead of
water.
Uses
1. Cellulase is used for commercial food
processing in coffee.
2. It performs hydrolysis of cellulose during
drying of beans.
3. Furthermore, cellulases are widely used in
textile industry and in laundry detergents.
4. They have also been used in the pulp and
paper industry for various purposes, and they
are even used for pharmaceutical applications.
5. Cellulase is used in the fermentation of
biomass into bio fuels, although this process is
relatively experimental at present.
6. Cellulase is used as a treatment for
phytobezoars, a form of
cellulose bezoars found in the human stomach.
Succinyl coenzyme A
synthetase
Succinyl coenzyme
A synthetase is an
enzyme
that catalyzes the
reversible reaction
of succinylCoA to succinate.
Source
Bacteria
e.g.E.coli
Mammals
Chemical Reaction
 Succinyl CoA synthetase catalyzes the following reversible
reaction:
 Succinyl CoA + Pi + NDP ↔ Succinate + CoA + NTP
Succinyl CoA
succinate
Mechanism
The enzyme facilitates coupling of the conversion
of succinyl CoA to succinate with the formation of
NTP from NDP and Pi.
The first step involves displacement of CoA from
succinyl CoA by a nucleophilic inorganic phosphate
molecule to form succinyl phosphate. The enzyme
then utilizes a histidine residue to remove the
phosphate group from succinyl CoA and generate
succinate.
Finally, the phosphorylated histidine transfers the
phosphate group to a nucleoside diphosphate,
which generates the high-energy carrying
nucleoside triphosphate.
Mechanism
Uses
Succinyl-CoA synthetase plays a key role in
 the citric acid cycle
 ketone metabolism
 heme synthesis
Urokinase
sources
SOURCE ORGANISM



Human urine.
much lower
concentrations in human
plasma.
Other organism may
include rat, mouse, yeast
etc.
SOURCE TISSUE




Ovary
produced by kidney
cells.
produced by a variety
of tumor cells and
involved in the formation
of tumor metastasis.
Phagocytic cells
MOLECULAR CHARACTERISTICS






411-residue protein
three domains: serine protease domain, kringle domain
and growth factor domain.
synthesized as a prourokinase or single-chain urokinase
form ; activated by proteolytic cleavage.
The two resulting chains are kept together by
disulphide bond.
found in multiple molecular sizes. Low molecular weight
(33-KDa) and high molecular weight (57-KDa).
Urinary Urokinase contained predominantly the LMW
form
REACTION CATALYSED
PLASMINOGEN + H20  PLASMIN


Specific cleavage of Arg-Val bond in plasminogen
to form plasmin.
Reaction type: Hydrolysis of peptide bond
USES
used clinically for therapy of thrombotic
disorders
 used in medicine to dissolve blood clots.
 employed
in clinical medicine in the
treatment of acute myocardial infarction
and arterial blood clots in the legs and
arms.
 Used in peritoneal dialysis.





Luciferase is a generic term for the class of
oxidative enzymes used in bioluminescence
62 kDa molecular weight
pH optimum of 7.8
A variety of organisms regulate their light
production using different luciferases
 bacteria
 Fireflies
 Jack-O-Lantern mushroom
 Metridia longa (marine copepod)
 Dinoflagellate, etc

1.
2.
The chemical reaction catalyzed by firefly
luciferase takes place in two steps:
luciferin + ATP → luciferyl adenylate + PPi
luciferyl adenylate + O2 → oxyluciferin + AMP +
light

The protein structure of
firefly luciferase consists
of two compact domain
 The
N-terminal
domain
 The C-terminal
domain





gene report and detection
RNAi system research
interaction between proteins
cell analysis
detection of Microorganism
Asparaginase
About protein
• A tetrameric protein composed of four identical subunits, each
subunit contains 326 amino acid residues. The two threonine
residues present at the active site are required for activity.
Molecular Weight:
Monomer: 36.8 kDa
Optimal pH:
8.0- 8.6
ADVANTAGES
• Treatment of acute lymphoplastic
leukemia.
• Aspariginase can be used as a food
processing aid to reduce the
formation of acrylamide, a
suspected carcinogen, in starchy
food products.
DISADVANTAGES
• anaphylaxis
• pancreatitis
• coagulopathy
POTENTIAL
• fusion protein of asparaginase-TTP-CETPC could
also be useful for the development of a vaccine
against atherosclerosis.