BROMELAIN Mixture of protein - PowerPoint Presentation

Slide1

BROMELAINSlide2

Mixture of

protein

digesting enzymes known as

proteolytic enzymes or proteases – include several other substances as welleither of two proteases extracted from plant family bromeliaceae i.e., Stem bromelain - EC 3.4.22.32 Fruit bromelain - EC 3.4.22.33May also refer to a combination of those enzymes along with other compounds produced in an extractReferred to as sulfhydryl proteases since a free sulfhydral group of a cysteine side chain is essentialThe 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

COILS

α

HELIXES AND HELICAL TURNS Slide3

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 supplementPioneer 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.Slide4

IT COMES FROM…

Pineapple plant (

Ananas

sp.)Stem  most common commercial sourceTraditionally 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. Slide5

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 chemokinesAlso effects prostaglandin synthesisInhibits fibrinogen synthesisDirectly degrades fibrin and fibrinogencleave at Lys-, Ala-, Tyr-, Gly- Is activated by cysteine, bisulfite salt, NaCN, H2

S, Na2S, and benzoate.inhibited by Hg++, Ag+, Cu++, a-1-antitrypsin,

estatin A&B, Iodoacetate, TLCK, TPCK Slide6

PAY BACK TIME…

product name



‘Ananase’Various uses in Folk medicineExplored as a potential healing agent in alternative medicine. Work by blocking some proinflammatory metabolites when applied topicallyUsed for reducing swellingInvolved in the migration of neutrophils to the site of acute inflammation. Used for treating arthiritisWhen used in conjunction with trypsin and rutin is as effective as prescribed analgesics in the osteoarthiritis management.

Meat tenderizingSlide7

WHAT ELSE…??

Other effects include:

Hay fever

Treating a bowel condition that includes swelling and ulcer ulcerative colitisRemoving dead and damaged tissue after a burn debridementPreventing the collection of water in the lung pulmonary edemaRelaxing musclesImproving the absorption of antibioticsPreventing cancer Shortening laborHelp the body in reducing fatsSupplement may effect heart ratesystemic enzyme therapySlide8

DIASTASE ENZYME.Slide9

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) Slide10

ALPHA AMYLASE

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

glucanohydrolase

ALTERNATIVE NAME : glucogenaseLocation: 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.Slide11

STRUCTURE

679 amino acid residues with a molecular weight of 75112 residues

It has 3 domains A B CDOMAIN 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 Asp331Slide12

AMYLOSE IN STARCH

GLUCOSE RESIDUE CLEAVED BY AMYLASESlide13

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 grainsChecking out pancerititis the amylase levels are measured in the pancertic cells.Slide14

ENZYME:

TRYPSIN

*TYRPSINOGEN*Slide15

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.Slide16

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 chainSlide17

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 PROTEINSSlide18

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 Slide19

ALKALINE

PHOSPHATASESlide20

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

includingIntestinal (ALPI), Chromosome 2Placental (ALPP)Liver/bone/kidney (ALPL) Chromosome 1

It belongs to Alpha and Beta class of proteinsSlide21

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.

STRUCTURESlide22

General Mechanism

I

II

III

IVSlide23

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 + phosphateSlide24

One of the most important functions of alkaline

phosphatase

is as an indicator for disease.

Alkaline

Phosphatase

TestSlide25

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 Slide26

Structure:

Two aspartate molecules at the active site

Three sulphide bridgesSlide27

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 Slide28

Temperature: 37°C-42°C

pH: 1.5 – 2

Stable until pH 8- can be reactivated upon re- acidification

Activity

and Stability

:

Imbalance in pH

Inability to digest protein

Deficiency:Slide29

PAPAIN

Papaya

Proteinase

ICysteine protease hydrolaseSlide30

Enzyme extractionSlide31

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 secretedcontains 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.Slide32

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 histidine2. Nucleophilic attack by deprotonated cysteine on substrate carbonyl atomSlide33

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 tumorsSlide34

CellulaseSlide35

Introduction

(source)

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.Slide36

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

cellulasesSlide37

Other Names

Other names for '

endoglucanases' are: endo-1,4-beta-glucanase, carboxymethyl cellulase (CMCase), endo-1,4-beta-D-glucanase, beta-1,4-glucanase, beta-1,4-endoglucan hydrolase, and celludextrinase. The other types of cellulases are called exocellulases.Slide38

Types of reactions/ Classification

General types of cellulases based on the type of reaction catalyzed:Cleaves internal bonds at Endocellulase (EC 3.2.1.4) randomly amorphous sites that create new chain ends.Cellobiase (EC 3.2.1.21) or beta-glucosidase hydrolyses the exocellulase

product into individual monosaccharides.

Cellulose phosphorylases depolymerize cellulose using phosphates instead of water.Slide39

Uses

Cellulase is used for commercial food processing in coffee.

It performs hydrolysis of cellulose during drying of beans.

Furthermore, cellulases are widely used in textile industry and in laundry detergents. They have also been used in the pulp and paper industry for various purposes, and they are even used for pharmaceutical applications. Cellulase is used in the fermentation of biomass into bio fuels, although this process is relatively experimental at present. Cellulase is used as a treatment for phytobezoars, a form of cellulose bezoars found in the human stomach.