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

WHAT ACTUALLY IS BROMELAIN…??

temperature 40-60 °COptimal temperature 50-60 °CDeactivation temperature above65 °C approx.Effective pH 4.0-8.0Optimal pH 4.5-5.5Molecular weight 28.4 kD

β COIL

S

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

Diastase are any one of a group of enzymes which catalyses the breakdown of starch into maltose.

first enzyme discovered.

It was extracted from malt solution in 1833 by Anselme Payen and Jean-François Persoz, chemists at a French sugar factory.

The name "diastase" comes from the Greek (diastasis) (a parting, a separation)

diastase

Alpha amylase

Beta amylase

Gamma amylase

ALPHA AMYLASE

EC NUMBER: 3.2.1.1 is 1,4-a-D-Glucan glucanohydrolase

ALTERNATIVE NAME : glucogenase

Location: it is secreted in saliva and pancreas, found in 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.

Causes hydrolyses alpha-bonds of large alpha-linked polysaccharides, such as starch and glycogen, yielding glucose and maltose.

• Ph-5.9-6.6• Temperature opt

=37’CConditions for amylase

• Chloride ion and bromide ion

• Most effective.Metal ions

and activators

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)8-barrel.

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 β-strands. 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 AMYLASE

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.

ENZYME: TRYPSIN

*TYRPSINOGEN*

HISTORY & SOURCE***1876, first named by

Kuhne who described the proteolytic activity of this pancreatic enzyme.

1931, Norothrop and Kunitz purified trypsin by crystallization.

1974, three dimensional structure was determined

PANCREAS

TRYPSINOGEN TRYPSIN

BOVINE PANCREAS

expresses two forms of trypsin:

dominant cationic form

minor anionic form

These protein sequences share

72% identity, while their coding

regions share 78% identity.

CONVERSION***

TRYPSINOGEN TRYPSIN

{Ph 9.3} {ph 10.5}

Trypsinogen is activated by removal of a terminal hexapeptide to yield single-chain 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

REACTION CATALYZED***

Process catalyzed by trypsin

*Trypsin Proteolysis*

{Trypsinisation}

Trypsin is considered as an endopeptidase*

Cleavage occurs within the

polypeptide chain rather than at the terminal

amino acids located at the

ends of polypeptides.

TRYPSIN

SERINE PROTEASE

HYDROLYZE PROTEINS

USED FOR*** TISSUE DISSOCIATION

Mitochondria isolation

IN VITRO STUDIES OF PROTEINS

Various hemagglutination procedures

DNA FINGERPRINTING

Environmental monitoring

REDUCTION OF CELL DENSITY IN TISSUE CULTURE

Cleavaging fusion proteins

GENERATING GLYCOPEPTIDES FROM PURIFIED GLYCOPROTEINS

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

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.

STRUCTURE

General Mechanism

Alkaline phosphatase binds substrate. Only the phosphate moiety of the substrate binds specifically to the enzyme.

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 molecule is slowly released from the enzyme.

III

IIIIV

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 – 9Activators: Mg2+

Wide specificityInhibitors: 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

One of the most important functions of alkaline phosphatase is as an indicator for disease.

• bones, liver, bile system or malignancies,blood, reproductive tissues and GI tract

High levels of alkaline

phosphatase

• hypophosphatasia,malnutrition, celiac disease, magnesium and zinc deficiency, anemia

Low levels of alkaline

phosphatase

Alkaline Phosphatase Test

PEPSIN ClassificationEC 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

PEPSINOGEN - primary

structure has an additional 44

amino acids

Released by chief cells in the

stomach

HCL causes activation

Pepsinogen → pepsin

( autocatalysis in acidic env)

“A tricky business”

TARGETS:

Amide bonds of aromatic

amino acids like

tryptophan, phenylalanine

and tyrosine

Tryptophan Phenylalanine

Temperature: 37°C-42°CpH: 1.5 – 2Stable until pH 8- can be reactivated upon re- acidification

Activity and Stability:

Imbalance in pHInability to digest protein

Deficiency:

PAPAINPapaya Proteinase ICysteine protease hydrolase

Enzyme extraction

material needs to undergo a purification process to get rid of all the contaminating products present in it.

latex that is collected is further allowed to dry into a crude format.

latex that drips is either collected in a container or left to dry on the fruit

papaya fruit is first slit at the neck.

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.

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.

Complete vs. incomplete cellulases

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 beta-glucosidase hydrolyses the exocellulase product into individual monosaccharides.

3. Cellulose phosphorylases depolymerize cellulose using phosphates instead of water.

Uses1. 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 succinyl-CoA 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 Source

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

bacteriaFirefliesJack-O-Lantern mushroomMetridia longa (marine copepod)Dinoflagellate, etc

Chemical reaction

The chemical reaction catalyzed by firefly luciferase takes place in two steps:

1. luciferin + ATP → luciferyl adenylate + PPi

2. luciferyl adenylate + O2 → oxyluciferin + AMP + light

Mechanism

Structure

The protein structure of

firefly luciferase consists of two compact domain

The N-terminal domain

The C-terminal domain

USES

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.