biochem 1 [enzyme]

8/3/2019 Biochem 1 [Enzyme]

1/68

CHAPTER 1

ENZYME

By:

BIOPROCESS AND BIOSYSTEM ENG.TECH.


2/68

OBJECTIVES

Objectives :

To understand the concept of biocatalyst (enzyme)To study kinetic of enzyme

o stu y t e n t on n react on cata ystTo study the activity and stability of the enzyme

in enzyme activity

Overview the a lication of free and immobilizedenzyme in industrial application


3/68

OUTLINEOUTLINE Introduction to Enzymes

Simple Enzyme Kinetic Enzyme Reactor with Simple Kinetics

Inhi i i n f Enz m r i n

Other influence on enzyme activity

Industrial Application of Enzyme.


4/68

Enzyme


5/68

Enzyme are

usually proteinsproteins of highhigh molecularmolecular weightweight15000several million Daltons that act as a

catalystcatalyst..

chemicalchemical reactionreaction without undergoing apermanent chemical change.

speedspeed upup reactionsreactions by providing an alternative

Because enzymes do not affect the relative,do not affect equilibriumequilibrium of a reaction


6/68

Most chemicalchemical catalystscatalysts catalyzed a wide range. .

,catalyzingcatalyzing specificspecific reactionsreactions only. This specificityis due to the sha essha es ofof thethe enz meenz me moleculesmolecules.

Specific,Specific, versatileversatile and very effective biologicalcatalyst resulting in much higher reaction rates

as compared to chemically catalyzed reactions.

.


7/68

nzyme ers rom or nary c em ca ca a ysin several important aspects:

Higher reaction rates

Milder reaction conditions

Capacity for Regulation


8/68

Many enzymes consist of a proteinprotein andand aa nonnon--proteinprotein (called the cofactorcofactor).

The proteinsproteins in enzymes are usually globularglobular. The intra- and intermolecular bondsbonds that hold

proteins in their secondary and tertiary

temperaturetemperature andand pHpH.

This affects shapesshapes and so the catalyticcatalytic activityactivity

of an enzyme.

.


9/68

Cofactors ma be:

organicorganic groupsgroups that are permanently bound tothe enzyme (prosthetic groups)

cationscations - positively charged metal ions

active site of the enzyme, giving an intensepositive charge to the enzyme's protein e.g

g, , , e e cor anicor anic moleculesmolecules usuall vitamins or made

from vitamins (coenzymes), which are not

permanently bound to the enzyme molecule,-complex temporarily


10/68

Holoenzyme non-polar group

apoenzyme

+

Co-factor


11/68

..

molecule that has just the right shape and

moleculesmolecules..

is called the substrate.-

'route'. The enzyme and substrate form a

reactionreaction intermediateintermediate.. Its formation has a lowerlower activationactivation energyenergy than

the reaction between reactants without a

catalyst.


12/68

k2

k-

k1E + S ES E + P

Enzyme-substrate complexBy weak forces

-hydrogen bonding and-van deer Waals forces


13/68

Uncatalysed Enzyme-catalysed-

e

n

er

Intermediate

reactants

g

y formed

between

enzyme and exergonicone or more

reactant

molecules

reaction

Click to see how an enzyme is involved in an enzyme-catalysed reactionCourse of reaction


14/68

no enzymepresent

e

n

e

present

rg

y

n erme a e :

enzyme/reactant 1

+reactant 2

reactant 2

products+enzyme

+

enz me

Course of reaction Replay Close window


15/68

Reaction rofile

transition state

eactivationener E

bondsbreaking

bondsn

er

forming

reactants

y

exergonicreact on

Course of reaction Replay Close window


16/68

Lock and key hypothesis

This is the simplest model to representhow an enzyme works. The substrate

sim l fits into the active site to form a

reaction intermediate.


17/68


18/68

In this model the enzyme moleculec anges s ape as e su s ra e mo ecu esgets close. The change in shape is

n uce y e approac ng su s ra emolecule. This more sophisticated modelre es on t e act t at mo ecu es are

flexible because single covalent bonds arefree to rotate.


19/68

Enzyme are named by adding the suffixsuffix aseaseto t e en o t e su strate:

catalyzes urea decomposition)Or

alcohol dehydrogenase (catalyzes the

oxidative dehydrogenation of an alcohol).


20/68

There are sixsix majormajor classesclasses of reaction.

ese un s orm e as s or e nzymeCommission (EC) system for classifying

enzymes based on the reactions they.


21/68


22/68

These activities are usually measured interms of the activity unit (U) which is defined

as the amount which will catalyze thetrans ormat on o m cromo e o t esubstrate per minute under standard

con ons.

Another unit of enzyme activity has been

recommended. This is the katal (kat) whichis defined as the amount which will catalyzethe transformation of one mole of substance

per second (1 kat = 60 000 000 U).


23/68


24/68

Kinetic of sim le enz me-catal zed

reactions are often referred to as

--kineticskinetics..

More complicated enzyme-substrate

systems.

An enzyme solution has fixed number of

.


25/68

At high substrate concentrations, allese s es may e occup e y

substrates or the enzymeenzyme isis saturatedsaturated. Saturation kinetics can be obtained from

a reversiblereversible stepstep for enzyme-substrate

step of the ESES complexcomplex.


26/68

k2k1

k-1E + S ES E + P

][Pd 12dt

= rate o pro uct ormat on or su strateconsumption in mole/l.s

The rate of variation of the ES complex is:

][ESd

211

dt 2


27/68

nce t e enzyme s not consume , t econversion equation on the enzyme yields:

[E] = [Eo] [ES]3

The ra id e uilibrium assum tion

rapid equilibrium between the enzyme and

e uilibrium coefficient to ex ress ES interms of [S]


28/68

][1

1'

ESkKm == 4

Since [E] = [Eo] - [ES], if enzyme is conserved, then:

][][

'SK

ESm

o

+= 5

][][

'2

SKSK

k

dt

v

m

m

m

o

+=

+==

Vm = k 2[Eo]


29/68

V = maximum forward velocit of thereaction

Vm change with addition of enzyme

Km Michaelis-menten constant

Low value of K su est that the enz mehigh affinity for the substrate


30/68

assum tion

For batch reactor close s stem

Initial substrate concentation reatl

exceeds the initial enzyme concentration

[Eo] was small, d[ES]/dt 0


31/68

1][ES =8

21

]][[][

21 kk

SEES o

+=

9

1k

]][[][2

SEkPdo

==10

][21 Skkdt

++

1


32/68

][SVv m= 11

m +

Km is (k-1+k2)/k1 and Vm is k2[Eo]


33/68

Michaelis-Menten t e Kinetics

Double-reciprocal Plot( Lineweaver-Burkplot)

Eadie-Hofstee plot

Batch Kinetics


34/68

Double-reci rocal Plot

(Lineweaver BurkPlot)

11 Km=

][SVVv mm


35/68

Eadie-Hofstee

Plot

][SKVv mm=

H W lf


36/68

Hances-WoolfPlot

1][ KS

VVv mm+=

B t h Ki ti


37/68

Batch Kinetics

][][ SVSd m

][SKdt m +

integration to yie :

][ln][][ SKSSV omo ==tt

plot of 1/t ln[So]/[S] versus {[So]-[S]}/t results in ane o s ope - m an ntercept o m.


38/68


39/68

enzymes and reduce their activity.

ese compoun s are nown o e

enzyme inhibitors.

Irreversible

reversible


40/68

Irreversible

Irreversible inhibitors such as heavyheavy metalsmetals

lead cadmium mercur and others form astable complex with enzyme and reduce

uch enzyme inhibitioninhibition may be reversedreversed only

by using chelatingchelating agentsagents such as EDTA(ethylenediaminetetraacetic acid) and citrate.


41/68

reversible

Reversible inhibitors may dissociate moreeas y rom e enzyme a er n ng.

inhibitions are competitivecompetitive, noncompetitivenoncompetitivean uncompe ve n onsuncompe ve n ons.

cases.


42/68

enzyme

]['

,

SKv

appm

m

+=

)1(''

,I

mappm KKK +=


43/68

Competitive Inhibition


44/68


45/68


46/68


47/68

Uncompetitive inhibitors bind to the ES complexbind to the ES complex

on y an ave no a n ty or t e enzyme tse .

][, SVv

appm=

][,

SKappm

+


48/68

inhibitions in some enzymatic reactions which

Vm

+1

'Km

'

][SVVv m

m

m

+=


49/68

Influences on Enzyme Activity

pH Temperature

[Substrate, product and enzyme][Salt]


50/68

Certain enzyme have ionicionic groupsgroups onon theirtheir


51/68

Certain enzyme have ionicionic groupsgroups onon theirtheir

act veact ve s tess tes an t ese on c groups must e n asuitablesuitable formform (acid(acid oror basebase) to function.

Change in pH may also alteralter thethe threethree

.

,

a certain pH range. The pH of the medium mayaffectaffect thethe maximummaximum reactionreaction rate,rate, KKmm andands a ys a y oo ee enzymeenzyme.

n some cases e su s ra e may con a n on con cgroupsgroups and the pH of the medium affects the

.


52/68

Effect of pH


53/68


54/68


55/68

-increaseincrease withwith temperaturetemperature up to a certain

.

Above a certain temperature, enzymeactivitactivit decreasesdecreases with tem eraturebecause of enzyme denaturationdenaturation (physical(physical

dama edama e


56/68


57/68

res r c onres r c on o enzyme mo yenzyme mo y n a xe space

Important advantagesenzyme reutilizationreutilization and elimination of

enz me recover and urification rocess

and may provide a better environmentbetter environment forenzyme activity.

Product purityProduct purity is usually improved andeffluent handling problems are minimizedby immobilization.


58/68

Criteria used in the selection of supportsupportmaterialmaterial are:

The bindinbindin ca acitca acit of the su ortmaterial which is a function of chargedensit functional rou s orosit and

hydrophobicity of the support surface.

StabilityStability and retentionretention ofof enzymaticenzymaticactivity which is a function of functionalgroups on support material andmicroenvironmental conditions.


59/68

Entrapment Surface Immobilization

Adsor tionphysical enclosure of

enz mes in a smallspace.

Covalent binding

Matrix entrapment

Entra ment CriteriaEntrapment


60/68

Entra ment Criteria

methods

MatrixMatrix entrapmententrapment Matrix material:o Polymeric material such as Ca-alginate,

agar, -carrageenin, polyacryamide and

o Solid matrices such as activated carbon,porous ceramic and diatomaceous earth

o Can be particle, a membrane or fiber.

Process:

enzyme solution is mixed with polymersolution before polymerization takes

p ace.o Polymerized gel-containing enzyme is

either extruded or a tem late is used toshape the particle from a liquid polymer-

enzyme mixture.

MembraneMembrane Matrix:


61/68

en rapmenen rapmen em rane o ny on, ce u ose,

polysulfone and polyacrylate

Hollow fiber units have been used to

entrap an enzyme solution between thin,semi permeable membranes.Configuration other than hollow fibers

permeable membrane is used to retainhi h molecular wei ht com ounds(enzyme) which allowing smallmolecular weight compounds (substrateor product) access to the enzyme.


62/68

Inherent Enzyme leakage into solution,

enzymeentra ment

,reduced enzyme activity and

stabilit

and lack of control environmentalconditions

Adsorption Surface Immobilization


63/68

e attac ment o enzymes on t e sur ace o support part c e

by weak physical forces such as van der Waals or dispersionforces.The active site of the adsorbed enzyme is usually unaffectedand nearly full activity is retaining upon adsorption.

uppor ma er a use:Inorganic material such as alumina, silica, porous glass,ceramic, diatomaceous earth, cla and bentoniteOrganic material such as cellulose (CMC,DEAE-cellulose),starch, activated carbon.

,The surfaces of the support materials may need to bepretreated (chemically or physically) for effectiveimmobilization.

Advantages of Adsorption

Desorption of enzyme when strong hydrodynamic forces are

presence since binding forces are weak.

Covalent binding


64/68

formationEnzyme molecules bind to support material via certain functionalgroup suc as am no, car ony , y roxy an su y ry groups.These functional groups must not be in the active site.

The active site of enzyme was block by flooding the enzymesolution with a competitive inhibitor prior to covalent binding.Functional groups on support material are usually activated byusin chemicals rea ents such as c ano ens bromide, carbodiimideand glutaraldehyde.

The retention of enzymes on support surfaces by covalent bondformationSupport type:o --OH

--o --COOH

o Support containing anhydridesBinding groups on the protein molecules are usually side groups(R)

or the amino or carboxyl groups of the polypeptide chain.


65/68

Name Application


66/68

Amylase Starch hydrolysis, glucoseproduction

Glucoamylase Saccharification of starch,glucose production

Trypsin Meat tenderizer, beer hazeremoval

Papain Digestive aid, meattenderizer, medicalapp ca ons

Pepsin Digestive aid, meat

Rennet Cheese manufacturing


67/68

fructose

en c nase egra a on o pen c n

Glucose oxidase Glucose gluconic acid,r e egg manu ac ur ng

Lipases Hydrolysis of lipids,flavoring and digestive aid

Invertase H drol sis of sucrose forfurther fermentation

,hydrolysis of pectin

e u ose e u ose y ro ys s


68/68

~The End~

biochem 1 [enzyme]

Documents