CHAPTER 1

ENZYME
By: FAZLENA HAMZAH BIOPROCESS AND BIOSYSTEM ENG.TECH.

OBJECTIVES
Objectives : To understand the concept of biocatalyst (enzyme) To study kinetic of enzyme To T study the i hibi i i reaction catalyst d h inhibition in i l To study the activity and stability of the enzyme To study the performance of immobilized techniques in enzyme activity Overview the application of free and immobilized pp enzyme in industrial application

OUTLINE
Introduction to Enzymes Simple Enzyme Kinetic Enzyme Reactor with Simple Kinetics Inhibition of Enzyme reactions Other influence on enzyme activity Immobilized Enzyme Industrial Application of Enzyme.

Enzyme

Enzyme are usually proteins of high molecular weight ( (15000<MV>several million Daltons) that act as a ) catalyst. catalyst. a substance which increases the rate of a chemical reaction without undergoing a permanent chemical change. speed up reactions by providing an alternative reaction pathway of lower activation energy. energy. Because enzymes do not affect the relative energy between the products and reagents they reagents, do not affect equilibrium of a reaction

Most chemical catalysts catalyzed a wide range of reactions They are not usually very selective reactions. selective. In contrast enzymes are usually highly selective selective, catalyzing specific reactions only. This specificity is due to the shapes of the enzyme molecules. p y molecules Specific, versatile and very effective biological p y g catalyst resulting in much higher reaction rates as compared to chemically catalyzed reactions under ambient condition condition. More than 2000 enzymes are known known.

Enzyme diff E differs from ordinary chemical catalyst f di h i l t l t in several important aspects:
Higher reaction rates
Milder reaction conditions

Greater Reaction Specificity

Capacity for Regulation

Many enzymes consist of a protein and a nonnonprotein (called the cofactor cofactor). The proteins in enzymes are usually globular globular. The intra- and intermolecular bonds that hold proteins in their secondary and tertiary structures are disrupted by changes in temperature and pH pH. This affects shapes and so the catalytic activity of an enzyme. .

Cofactors may be: y organic groups that are permanently bound to the enzyme (prosthetic groups) cations - positively charged metal ions (activators), which temporarily bind to the active site of the enzyme, giving an intense positive charge to the enzyme's protein e.g Mg, Zn, Mn, Mg Zn Mn Fe etc organic molecules, usually vitamins or made g , y from vitamins (coenzymes), which are not permanently bound to the enzyme molecule, but combine with the enzyme substrate enzyme-substrate complex temporarily

Holoenzyme

Enzyme containing a non-polar group

apoenzyme
+

Co-factor

How enzyme work

Enzymes have an active site. This is part of the site. molecule that has just the right shape and functional groups to bind to one of the reacting molecules. molecules. The reacting molecule that binds to the enzyme is called the substrate. An enzyme-catalyzed reaction takes a different enzyme catalyzed 'route'. The enzyme and substrate form a reaction intermediate. intermediate. Its formation has a lower activation energy than the reaction between reactants without a catalyst.

E +

k1 k-1 1

ES

k2

E + P

Enzyme-substrate complex By weak forces -hydrogen bonding and -van deer Waals forces

Reaction profiles: uncatalysed and enzyme-catalysed enzyme catalysed

Uncatalysed reaction Enzyme-catalysed reaction

e n e r g y reactants

Intermediate formed between y enzyme and one or more reactant molecules

exergonic reaction products

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

no enzyme present

e n e r g y

enzyme present Intermediate : I t di t enzyme/reactant 1 reactant 1 + reactant 2 + enzyme + reactant 2 products + y enzyme Course of reaction
Replay Close window

Reaction profile p
transition state (or activated complex)

e n e r g y

bonds breaking

activation energy, Ea gy,

bonds forming

reactants

exergonic reaction ti

products

Course of reaction

Replay Close window

Lock and key hypothesis

This is the simplest model to represent how an enzyme works. The substrate py simply fits into the active site to form a reaction intermediate.

Lock and key Model

Induced fit hypothesis

In this model the enzyme molecule changes shape as th substrate molecules h h the b t t l l gets close. The change in shape is 'induced' b th approaching substrate 'i d d' by the hi b t t molecule. This more sophisticated model relies on th f t th t molecules are li the fact that l l flexible because single covalent bonds are free to rotate.

Naming
Enzyme are named by adding the suffix ase to the end of the substrate: h d f h b such as urease (urease is the enzyme which catalyzes urea decomposition) Or to the reaction which is catalyzed such as alcohol dehydrogenase (catalyzes the oxidative dehydrogenation of an alcohol). y g )

There are six major classes of reaction which enzymes catalyze catalyze. These units f Th it form th b i f th E the basis for the Enzyme Commission (EC) system for classifying and assigning index numbers to all enzymes based on the reactions they catalyze. catalyze

Enzyme unit
These activities are usually measured in terms of the activity unit (U) which is defined as the amount which will catalyze the transformation of 1 micromole of th t f ti f i l f the substrate per minute under standard conditions. diti Another unit of enzyme activity has been recommended. This is the katal (kat) which ( ) is defined as the amount which will catalyze the transformation of one mole of substance per second (1 kat = 60 000 000 U).

Simple Enzyme Kinetics

Enzyme Kinetics
Kinetic of simple enzyme-catalyzed p y y reactions are often referred to as MichaelisMichaelis-menten kinetics or saturation kinetics. kinetics. More complicated enzyme-substrate interaction can take place in biological systems. An enzyme solution has fixed number of active sites to which substrates can bind bind.

At high substrate concentrations, all these sites may b th it be occupied b i d by substrates or the enzyme is saturated saturated. Saturation kinetics can be obtained from a simple reaction scheme that involved a reversible step for enzyme-substrate complex formation and a dissociation step of the ES complex. complex

k1 E + S k-1 ES

k2 E + P

d [ P] v= = k 2 [ ES ] dt
V = rate of product formation or substrate f d f i b consumption in mole/l.s The rate of variation of the ES complex is:

d [ ES ] = k1 [ E ][ S ] k 1 [ ES ] k 2 [ ES ] dt

Since the enzyme i not consumed, the Si h is d h conversion equation on the enzyme yields:

[E] = [Eo] [ES]

The rapid equilibrium assumption p q p

rapid equilibrium between the enzyme and substrate to form an [ES] complex q p equilibrium coefficient to express [ ] in [ES] terms of [S]

k 1 [ E ][ S ] K = = k1 [ ES ]
' m

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

[ E o ][ S ] [ ES ] = ' K m + [S ]

[ E o ][ S ] Vm [ S ] d [ P] = k2 v= = ' dt K m + [S ] K m + [S ]
Vm = k 2[Eo]

Vm = maximum forward velocity of the y reaction Vm change with addition of enzyme Km Michaelis-menten constant Low value of Km suggest that the enzyme gg y high affinity for the substrate

The quasi-steady state assumption p

For batch reactor (close system) Initial substrate concentation greatly g y exceeds the initial enzyme concentration [Eo] was small, d[ES]/dt 0

k1 [ E ][ S ] [ ES ] = k 1 + k 2
[ E o ][ S ] [ ES ] = k 1 + k 2 + [S ] k1

k 2 [ E o ][ S ] d [ P] v= = k 1 + k 2 dt + [S ] k1

10

Vm [ S ] v= K m + [S ] [S
Km is (k-1+k2)/k1 and Vm is k2[Eo]

11

Determining rate parameters for Michaelis-Menten type Kinetics yp

Double-reciprocal Plot( Lineweaver-Burk plot) Eadie-Hofstee plot Hanes-Woolf Plot Batch Kinetics

Doublereciprocal Plot (Lineweaver Burk Plot)

Km 1 1 = + v Vm Vm [S ]

Eadie-Hofstee Plot

v v = Vm K m [S ]

Hances-Woolf Plot

[S ] K m 1 = + [S ] v Vm Vm

Batch Kinetics

Vm [ S ] d[S ] v= = dt K m + [S ]
integration to yield: ld

[S o ] [S ] K m [S o ] = Vm = ln t t [S [S ]
plot of 1/t ln[So]/[S] versus {[So]-[S]}/t results in a line f l li of slope -1/Km and i t 1/K d intercept of Vm. t f

Inhibition of Enzyme Reaction

Inhibited Enzyme Kinetic

Certain compounds may bind to enzymes and reduce their activity. These compounds are k Th d known t b to be enzyme inhibitors.
Irreversible Inhibitor reversible

Irreversible
Irreversible inhibitors such as heavy metals ( (lead, cadmium, mercury and others) form a , , y ) stable complex with enzyme and reduce enzyme activity. Such enzyme inhibition may be reversed only S by using chelating agents such as EDTA (ethylenediaminetetraacetic acid) and citrate.

reversible
Reversible inhibitors may dissociate more easily f il from th enzyme after binding. the ft bi di The three classes of reversible enzyme inhibitions are competitive noncompetitive competitive, and uncompetitive i hibiti d titi inhibitions inhibitions. The substrate may act as an inhibitor in some cases.

Competitive
Compete with substrate for the active site of the enzyme

Vm [S ] [S v= ' K m ,app + [ S ]
' K m , app

' K m (1 +

[I [I ] ) KI

Competitive Inhibition

Noncompetitive
Inhibitors bind on sites other than the active sites and reduce enzyme affinity t th substrate. it d d ffi it to the b t t Noncompetitive enzyme inhibition can be d ib d d described as f ll followed:

v=

Vm ,app
' Km 1 + [S ] [S

Vm ,app =

Vm [I ] 1 + K I

Uncompetitive
Uncompetitive inhibitors bind to the ES complex only and h l d have no affinity f th enzyme it lf ffi it for the itself.

v=

Vm,app [ S ] K
' m , app

+ [S ]

Substrate Inhibition
High substrate concentrations may cause inhibitions in some enzymatic reactions which known as substrate inhibition

v=

Vm
' Km 1 + [S ] [S

' Km 1 1 1 = + v Vm Vm [ S ]

Influences on Enzyme Activity

pH

Temperature

[Salt]

[Substrate, product and enzyme]

pH

Certain enzyme have ionic groups on their active sites and th ti it d these i i groups must b i a ionic t be in suitable form (acid or base to function. base) Change in pH may also alter the three dimensional shape of the enzyme enzyme. For these reason enzymes are only active over reason, a certain pH range. The pH of the medium may affect the maximum reaction rate, Km and stability of th enzyme t bilit f the enzyme. In I some cases th substrate may contain i i the b t t t i ionic groups and the pH of the medium affects the affinity of the substrate to the enzyme enzyme.

Effect of pH

Temperature

Effect of Temperature

Rate of enzyme catalyzed reaction enzyme-catalyzed increase with temperature up to a certain limit. limit Above a certain temperature, enzyme y p activity decreases with temperature because of enzyme denaturation (physical damage) g )

Immobilization of Enzyme

Immobilization of Enzyme
restriction of enzyme mobility i a fi d space t i ti f bilit in fixed Important advantages enzyme reutilization and elimination of enzyme recovery and p y y purification p process and may provide a better environment for enzyme activity. y y Product purity is usually improved and effluent handling problems are minimized by immobilization.

Criteria used in the selection of support material are: The binding capacity of the support g p y pp material which is a function of charge density, functional groups, porosity and y, g p , p y hydrophobicity of the support surface. Stability and retention of enzymatic activity which is a function of functional groups on support material and microenvironmental conditions.

Method of Immobilization

Entrapment
physical enclosure of enzymes in a small y space.

Surface Immobilization Adsorption p

Covalent binding
Matrix entrapment

membrane entrapment

Entrapment p methods

Criteria

Entrapment

Matrix entrapment Matrix material: o Polymeric material such as Ca-alginate, agar, -carrageenin, polyacryamide and collagen o Solid matrices such as activated carbon, porous ceramic and diatomaceous earth o Can be particle, a membrane or fiber. Process: o When immobilizing in a polymer matrix, enzyme solution is mixed with polymer solution before polymerization takes place. l o Polymerized gel-containing enzyme is either extruded or a template is used to p shape the particle from a liquid polymerenzyme mixture.

Membrane entrapment t t

Matrix: Membrane of nylon, n lon cellulose, cellulose polysulfone and polyacrylate Process: Hollow fiber units have been used to entrap an enzyme solution between thin, semi permeable membranes. Configuration other than hollow fibers are possible but in all cases a semi permeable membrane is used to retain high molecular weight compounds (enzyme) which allowing small molecular weight compounds (substrate or product) access to the enzyme.

Inherent problems in enzyme p entrapment

Enzyme leakage into solution, significant diffusion limitations limitations, reduced enzyme activity and y stability and lack of control environmental conditions

Adsorption

Surface Immobilization

The tt h Th attachment of enzymes on th surface of support particle t f the f f t ti l by weak physical forces such as van der Waals or dispersion forces. The active site of the adsorbed enzyme is usually unaffected and nearly full activity is retaining upon adsorption. Support material use: Inorganic material such as alumina, silica, porous glass, y ceramic, diatomaceous earth, clay and bentonite Organic material such as cellulose (CMC,DEAE-cellulose), starch, activated carbon. Ion exchange resins such as Amberlite Sephadex and Dowex Amberlite, The surfaces of the support materials may need to be pretreated (chemically or physically) for effective immobilization. Advantages of Adsorption Desorption of enzyme when strong hydrodynamic forces are presence since binding forces are weak.

Covalent binding The retention of enzymes on support surfaces by covalent bond formation Enzyme molecules bind to support material via certain functional group such as amino, carbonyl, h d h i b l hydroxyl and sulfhydryl groups. l d lfh d l These functional groups must not be in the active site. The active site of enzyme was block by flooding the enzyme solution with a competitive inhibitor prior to covalent binding. Functional groups on support material are usually activated by using chemicals reagents such as cyanogens bromide, carbodiimide g g y g and glutaraldehyde. The retention of enzymes on support surfaces by covalent bond formation Support type: o --OH o --NH2 o --COOH o Support containing anhydrides Binding groups on the protein molecules are usually side groups(R) or the amino or carboxyl groups of the polypeptide chain.

Industrial Application

Name Amylase Glucoamylase Trypsin Papain

Pepsin Rennet

Application pp Starch hydrolysis, glucose production Saccharification of starch, glucose production Meat tenderizer, beer haze removal Digestive aid, meat tenderizer, medical applications li ti Digestive aid, meat tenderizer Cheese manufacturing

Glucose isomerase Penicillinase P i illi Glucose oxidase Lipases Invertase Pectinase Cellulose

Isomerization of glucose to fructose Degradation of penicillin D d ti f i illi Glucose gluconic acid, dried d i d egg manufacturing f t i Hydrolysis of lipids, flavoring and digestive aid Hydrolysis of sucrose for y y further fermentation Clarification of fruit juices juices, hydrolysis of pectin Cellulose hydrolysis

~The End~