ChEn 5751 - Spring 2007

Outline
Enzyme applications Enzyme kinetics Enzyme reactors Immobilized enzymes Enzyme engineering

Biocatalysis ChEn 5751

Prof. Wei-Shou Hu

Enzyme Biocatalysis
Advantages of enzyme (or whole cell) biocatalysis: Substrate specificity
Selectively use a substrate in a mixture of feed

Mechanism of catalysis (proteolysis) by chymotrypsin (a serine protease)

Substrate flexibility (non-specificity)

Can often be used to catalyse a reaction on a similar, but non-native, substrate
Transition State Peptide bond is cleaved

Relatively mild reaction conditions, environmentally friendly (green) Minimal side reactions (comparing to high temperatures or living cell processes) Rigioselectivity (stereoselectivity)

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Binding site provides the specificity of enzymes

Selectivity of Proteases

Chymotrypsin (prefer aromatic side chain)

Trypsin (prefer charged side chain)

Introduction to Enzyme Technology

Classes of Enzymes EC 1 Oxidoreductase EC 2 Transferase EC 3 Hydrolase EC 4 Lyase Catalysed Reaction Oxidation reduction Group transfer Hydrolysis (i) (ii) EC 5 Isomerase EC 6 Ligase (Synthetase) Removal of group leaving double bond Addition of group to double bond

Isomerization Joining of two molecules coupled with clearage of a pyrophosphate bond of ATP or other NTP

EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Major Categories Given Trivial Enzyme Classification

Dehydrogenase Hydroxylase Kinase Mutase Oxidase Oxygenase Phosphatase Phosphorylase Synthetase Thiokinase Transferase

iy

Industrial Enzyme Application Examples

Carbohydrate processing Isomerase Cellulases and lactase -amylase -amylase glucose isomerase amylase glucoamylase Example: corn starch glucose fructose

hydroxylation using O2 as donor transfer of phosphate from nucleotide of triphosphate migration of phosphate in the same molecules

oxidize with O as a donor break c-c bond with incorporation of oxygen hydrolysis of phosphate from oxygen

Protease

Glucose-fructose mixture Trypsin Acid (Rennets) Neutral Alkaline Alkaline (detergents)

High fructose corn syrup

Glucose isomerase is a common name. The original enzyme is xylose isomerase, the natural substrate is xylose. Its nonspecificity allows it to become a major industrial enzyme.

Lipase

Example: Use lipase for transesterification; converting the fatty acids in cheaper glycerides to coconut butter-like glycerides.

Others Analytical Pharmaceutical Biological research; PCR, restriction engineering

Sources of Enzymes
Plant enzymes
e.g., papain This production node cannot respond to short-term changes in market demand
Energy source Material sources (precursor, substrate)

Fermentation
cells

Cell

metabolites from energy metabolism product

Animal enzymes
Pancreatic trypsin and lipase Rennet (also called rennin or chymosin (EC 3.4.23.4), from cowstomach, added to milk to
cause coagulation)

Biocatalysis (Enzyme Processes) Free or immobilized enzymes Or immoiblized cells (dead cell or resting cells)

Urokinase

Plasminogen activator Factor VIII

substrate

Microbial enzymes
Drawbacks: need approval, historical check up and toxicity safety testing

product

Recombinant heterologous enzymes

Hosts: E. coli, Bacillus

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Why use enzyme? Why not just use cells 1. Restrict the number of reactions. especially to eliminate down stream of the product 2. Stabilize the enzyme, whole cell cannot last long, enzymes can last long. Eznyme turn over number is high i.e. the same amount of enzyme catalyses more conversions 3. Can use conditions not suitable for cell growth (i) high substrate concentration (ii) organic phase (iii) inhibitory substrate (vi) higher temperature (vii) breaking permeability barrier
Where enzymes are not used? 1. Few multiple enzyme reactions are in practice 2. Energy Intensive, except in very small reaction, like PCR 3. long pathways

Enzymes are mostly simple reactions, involving few reaction steps. In fact, most involve just a single step

A + B E1 A B + C
Some use multiple enzymes in series of reactor
E1 A + B C

C is often H2O

E2 C D

or use single reactor

E1 A + B C E2 C D

Some other cases, use mutliple reactions, one for cofactor regeneration

E1 A + B C + B '

C is the product B is the cofactor

B '+ D B + D '
E2

Activities of Enzymes
I. Enzymes described by units, usually not by mass or mole The internationally-recognised unit of enzyme activity is the Katal (abbreviated to kat). It is defined as the enzyme activity which transforms 1 mole of substrate per second under optimal conditions. In practice, few enzyme suppliers use the Katal, preferring instead units that reflect the preferences of their typical customers. For example, bakers are familiar with amylase activity expressed in 'SKB units', whereas brewers traditionally refer to amylase activity (which they call diastatic activity) in 'Degrees Lintner'. To obtain these various measures of enzyme activity, different assay methods have been used, and unfortunately there is no means of comparing dissimilar tests without conducting further practical work. units of some enzymes, especially ones defined by suppliers, can be very ill-defined for some classes

Physical Factors Affecting Optima

Activity: measure initial rates at a set of physical conditions. II. Stability: expose the enzyme under different physical conditions over a range of time an then adjust the conditions back to the reference, state for activity measurement. I. Operational stability II. Storage stability (shelf life) Effect of pH, temperature on enzyme activity They affect Km, rmax and stability. However, in applied enzymology the effect is often expressed in practical and quantitative way. Data is usually expressed in residual activity.
activity Enzyme - Active site - Substrate binding site

Optimal range

pH or Temperature

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Michalis-Menten Kinetics
E+S k1
k2 ES E + P

The same enzyme at two different concentrations

Two regions of reaction can be approximated -Zero order region:

k1
d [P] = k2 [ ES ] = r dt
d [ ES ] = k1[ E ][ S ] k1[ ES ] k2 [ ES ] dt

Since total amount of enzyme (i.e. the sum of substrate bound and free enzyme) is constant,

r rmax

[ E ] + [ ES ] = [ E ]0
where [E]0 is the total enzyme concentration. At a stage that the ES concentration can be assumed to be at a quasi-steady state:

([ s ] >> K m )
-First order region:

k1[ ES ] = k1[ E ][ S ] k2 [ ES ] = k1{[ E ]0 [ ES ]}[ S ] k2 [ ES ] k1[ ES ] = k1[ E ]0 [ S ] [ ES ]{k1[ S ] + k2 }

[ ES ]( k1 + k1[ S ] + k2 ) = k1[ E ]0 [ S ]

r rmax

[s] [Km ]

Two enzymes at the same rmax but differ in Km

([ s] < km )
Michaelis-Menten kineticsa saturation type kinetics

k2 [ ES ](

k1 + k2 + [ S ]) = k2 [ E ]0 [ S ] k1

r=

k2 [ E0 ][ S ] k [ E ][ S ] r [S ] = cat 0 = max k1 + k2 K M + [S ] K M + [S ] + [S ] k1

Determination of kinetic parameters

A. Double reciprocal plot:

Some notes on Michaelis-Menten kinetics

Michaelis-Menten kinetics are saturation type kinetics: the rate reaches a limiting value eventually Biological reactions like A B are not majority
Many involve more than one substrate and one product A+B C+D Many reactions are reversible (more reversible reactions in energy metabolism than irreversible reactions)

K 1 1 1 = + m r rmax rmax [ s ]
Plot

1 1 vs. r [ s]
Intercept on y axis
1 rmax

Km Slope: rmax

Saturation type kinetics are appropriate to approximate the reaction kinetics, even though the may not be sounds mechanistic representations.

B. Regressional analysis

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Other reaction mechanisms-example

E+S k1 k 1 ES k2 k 2 E+P

Reversible Reactions
The equations are also in the handouts for recitation 4

Use a similar approach as in Michalis-Menten kinetics, the total enzyme concentration is constan
[ E ] = [ E ]0 [ ES ]

E+S

k1 k1

ES

k2 k2

P + EAc E
k3

By material balance:

d [ ES ] = k1[ E ][ S ] + k 2 [ E ][ P] ( k 2 + k 1 )[ ES ] dt

= k1{[ E0 ]1 [ ES ]}[ S ] + k2 {[ E ]0 [ ES ]}[ P] (k2 + k1 )[ ES ]

Collect all terms with [ES], the equation becomes:

[ ES ] = k1[ E0 ][ S ] + k2 [ E0 ][ P] k2 + k1 + k1[ S ] + k2 [ P ]
r = k2 [ ES ] k2 [ E ][ P ]

k 2 k3 [S ] ( k 2 + k3 ) kcat[ E ]0 [ S ] = r= k 2 k3 Km + [S ] + [S ] ( k 2 + k3 ) [ E ]0
K M = ks k3 k 2 + k3 kcat = k 2 k3 k 2 + k3 Ks = k1 k1

The net reaction rate is the sum of forward and reverse reaction.

= =

k2 {k1[ E0 ][ S ] + k2 [ E ]0 [ P]} k2 {[ E0 ] [ ES ]}[ P ] k2 + k1 + k1[ S ] + k2 [ P] k1k2 [ E0 ][ S ] k 1 k2 [ E ]0[ P] k1 + k2 + k1[ S ] + k2 [ P]

When [P] approaches zero, the reaction rate becomes:

r= k1k2 [ E ]0[ S ] k [ E ] [S ] = 2 0 k1 + k2 + k1[ S ] k1 + k2 + [ S ] k1

It has the same form as the Michalis-Menten kinetics.

r= k 1k 2[ E ]0[ P ] k 1[ E ]0[ P] = k 1 + k 2 + k 2[ P] k 1 + k 2 + [ P ] k2

The same holds true for the case that [S] approaches zero: The negative sign can come from the definition of r. The expression for r can be rewritten:
r= k1k2 k k [ E ]0 [ S ] 1 2 [ E ]0 [ P ] k1 + k 2 k1 + k2 k [S ] k2 1+ 1 [ P] + k1 + k2 k1 + k2

In general Michaelis-Menten kinetics still applies but the expression of KM and kcat differ.

We define:

k1 + k 2 = kms k1

k1k 2 = ks k1 + k2

k1 + k 2 = kmp k2

k1k2 = kp k 1 + k2

Reversible Reactions (cont)

Many industrially important enzyme reactions are reversible, e.g. glucose isomerase, esterase, lipase. The general expression and its simplified form are k1 k2 k3 k1 k2 E+S ES E+P E+S ES EP E+P k -1 k -2 k -1 k -2 k -3 The reaction rate is expressed as

Operating Equation for Reversible Reactions

k1k2 k 1k 2 k 1 + k2 k2
The reversible reaction equation is difficult to apply, since there are many parameters. Operationally one may use the following

K eq = K ms =

r=

k1k2 [ E0 ][ S ] k 1 k2 [ E ]0[ P ] k2 [ E0 ][ S ] k 1[ E ]0[ P] = k 1 + k 2 [ S ] [ P ] k1 + k2 + k1[ S ] + k2 [ P ] + + k1 k2 k1

r =

rmax f = k2 [E ]0 rmax r = k 1[E ]0

K m + [S Se ]

rmax [S Se ]

[P ] rmaxf rmaxr [S ] K eg r = rmaxf [P ] K ms rmaxr + rmaxr [S ] + K eg

Se is the equilibrium concentration. Se is concentration dependent, thus the apparent half saturation constant Km is also concentration dependent

P.39 Biotechnology 2nd ed. Vol 9 Enzyme, Biomass, Food and f, eds. H.-J. Rehm and G. Reed. (1991

This equation is essentially the same as one shown in previous slide

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Multi-substrate reactions
A+ B C+D

Depending on the mechanism the reaction equations differ Common practice to use an empirical form of saturation type kinetics

r=

rmax [ A][ B ] [( K ma + [ A])( K mb + [ B ])

Inhibitions in Enzyme Reactions

Competitive
Inhibitor competes for substrate binding site Km is affected, not rmax
1/r

[I]

1/s 1/r

Non-competitive
Inhibitor binds some where else, but affects active site rmax is affected, not Km

[I]

1/s 1/r

Uncompetitive
Both rmax & Km are affected

[I]

1/s

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Enzyme stability
Two kinds of stability should be considered: storage stability and operational stability. The kinetics of enzyme inactivation are described as first order: dE = k d E dt where kd is the inactivation rate constant, and E is the concentration of active enzyme.

High Yield Enzymatic Process

Two scenarios: a) So (feed substitute concentration) >>S>>Km

[E ] = [E o ]Exp( k d t )
The expression thus describes a exponential decay of enzyme activities. This is a simplified view. Recalling reaction rate is k [E ][S ] r = cat K m + [S ] the inactivation may affect either kcat or Km .

So , the yield (So-S)/So is high, but is still in zero order reaction kinetics regime, then it is fine to use well mixed continuous reactor

b) So>>S~Km The yield is high, but the reaction rate will be far from the maximum, if well mixed continuous reactor is used

Batch Enzyme Reactor

r= dS kcat E S = dt Km + S

Plug Flow Reactor

At the steady state
kEoS F dS = -V A dl Km + S

Batch process is used when the enzymes is very active and inexpensive. It is used in low concentration, in order to not contaminate the product and does not need to be recovered.

Integrated from l=0 to l=L; and from S=So to S; (V=AL)

CSTR
V kEoS dS = FSo FS V dt Km + S
V kEoS dP V = FP + dt Km + S

S V kEoSo = ( So S ) + K m ln o F K m + So S

at steady state/0=F(So-S)-rv

r =

For a high yield, S has to be small. Unless S0 >>S>>Km, the reaction rate will be far below rmax. So, CSTR is not used in enzyme reactions.

kE S F (So S ) o V Km + S

However, enzyme is often expensive, and needs to be retained in the reactor for long-term use. So, employ immobilized enzymes.

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Example:
Glucose isomerase (a natural substrate is probably xylose, (km glucose)/(km xylose)=100, from Streptomyces (M.W. 43kd)
K cat = 2 x10
2 mol mid . min

Enzyme and Cell Immobilization

Why? Enzyme Re-use
allows for a continuous process (in packed bed reactor) allows for a batch recirculation process, so that enzyme catalysts can be used at the end of the batch process

kmapp=0.5mM, feed 40 wt% glucose, at 60% can convert 55% of glucose to fructose; the final product contains 0.55:0.41:0.04 (fructose: glucose: other acidic side product).
2 x102 mol / mol.min x60 min x
0.2 g

mol 180 g = 50.4 g glu cos e x g .enz.hr 43, 000 g mole

400 g x0.55 x 0.2 genzy.hr / l = 21.5hr

l 50.4 g use enzyme in the feed kcat is too low. Most industrial enzymes are not used to catalyze reactions involving their natural substrates.

Increased enzyme concentration, especially in a packed bed process

Methods of Immobilization
Entrapment:
enzymes and gel precursor or monomer are mixed, then gel is allowed to form, or to polymerize from monomer, thus entrapping the enzyme (cells)

Entrapment- physical retention of catalyst Use non-covalent gel:

Frequently used for cell catalyst Enzyme + soluble polymer
gel formation

Adsorption
Enzymes are added to porous polymer matrix and adsorbed to the internal surfaces through different interactions (charge, hydrostatic, etc.)

entrapped enzyme

Covalent Binding:
enzymes are added to porous matrix and covalently linked to the matrix

Example: alginate, agar, agarose, collagen, K-caragelnan

Membrane retention
enzymes are either entrapped in one compartment and retained in the reactor be selection of the pore size of the membrane, or enzymes are covalently bound to the membrane.

Covalent-gel:

Enzyme +monomer

polymerization

entrapped enzyme

Example: acrylamide (becomes polyacrylamide)

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Adsorption
Very easy to perform, enzymes are adsorbed by electrostatic or ionic interaction The matrices are often those used in adsorption chromatography, such as DEAE-cellulose DEAE-sephadex, carboxyl methyl cellulose silica gel Over time, some enzymes are lost

Immobilization by covalent bond

Matrix

Functional group

Amino group or carboxylic group

On enzyme

Form covalent bond Matrix Enzyme

Reaction rate limiting vs. Mass Transfer limiting

i. Reaction rate is limiting the use of low catalyst (enzyme) concentration.
high r S0

Reaction-Diffusion in Immobilized Biocatalysts

Assume spherical pellet, treat cells and gels uniformly. The reaction rate of substrate consumption in the biocatalyst, i.e. the amount of substrate consumed per unit biocatalyst volume per unit time, is described as

ii.

Mass transfer is limiting the use of high catalyst concentration.

low r Substrate concentration

dS = rs dt
For the reactions involving cell growth, the substrate consumption rate is

rs =

maxSx Yx / s ( K s + S )

where x is biomass concentration in the catalyst, with units of g cells per unit volume of pellet or biocatalyst. For enzymatic reactions, it can be written as

S is high enough in the pellet to maintain zero order or 1st order reaction.

Near the center, there isnt enough S to carry out the reaction at a reasonably fast rate.

rs =

(Ks + S )

rmaxS

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

We perform a shell balance by taking a control volume between r and r+dr. The balance equation on substrate s is:

dS dS 2 2 = rs 4 r 2dr Ds dr 4 r r Ds dr 4 r redr Divide both sides by(4 dr )and by assuming that Ds is constant, we obtain
dS Ds r 2 dr
r + dr

We define an average reaction rate in the pellet. The total reaction rate in the catalyst, i.e. the product of average reaction rate and the catalyst volume, equals the total rate of diffusion of substrate into the catalyst at the surface, which is the product of total surface area and the diffusion rate at the catalyst surface

rs =

Ap ds Ds Vp dr

r =R

r 2

dS dr

dr Take the limit

r = r r2 s

where Ap is the total surface area per biocatalyst and Vp is the volume of the biocatalyst. Here we define an effectiveness factor

=
d 2S 2 dS Ds 2 + = rs r dr dr

and dr 0 rewrite the equation

rs ( so )Vp

rsVp

Ds

d dr

2 dS 2 r dr = rs r

The effectiveness factor is basically the observed rate for the whole pellet over the rate which would have been obtained if there is no mass transfer limitation within the biocatalyst, i.e. there is no concentration gradient in catalyst. Recall that the reaction rate for cell growth is described as

To solve the equation we use two boundary conditions. First assume that concentration profile at the center of the biocatalyst is symmetrical

rs =

max sx Yx / s (K s + s )

dS = 0 @ r=0 dr
Take the condition that the concentration of substrate at the surface is the same as that in the bulk. In other words, assuming that there is no external mass transfer limitation, so

For enzymatic reaction, the equation is very similar. As a first approximation (assume so>>Ks), we assume the reaction is in the zero order kinetics region

rs =

max x
Yx / s

Substitute into the equation

s(R) = S0

d 2 s 2 ds max x + Ds = dr r dr Yx / s

Now we define dimensionless variables for both substrate concentration and the radius

s= s
2

so

r=r

Effectiveness Factor and Thiele Modules

Replace s and r with their dimensionless counter parts.

m xs R 2 s xs R 2 xR 2 d s 2 rs R 2 s s + = = max = = max = 2 2 s o K s Yx / s D s 1 + s r D s S o K s + s Yx / s Ds S o k s / s o + s Yx / s Ds s o dr 1+ s Ks

: effectiveness factor
:Thiele modulus

x m / Yx / s K s where = R is called Thiele modulus Ds

The boundary conditions now become

s = o Ks

is expressed in terms of kinetic parameters Use and : observable Thiele modules

R r = ( )2 obs 3 Di S o

s
R

r =1

=1
so

ds dr

r =0

=0

2, Thiele modulus, can be rewritten as

3

So : substrate concentration on the

surface

max x
Yx / s K s RDs s o

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Example:
Consider the conversion of penicillin N to isopenicillin N using immobilized enzyme catalysts. The turnover number is

Co-factors in enzyme reactions

40% of all enzymes require co-factors Oxidoreductase all require co-factor Common co-factors:
NAD+/NADH NADP+/NADPH FAD/FADH2 Vitamin B6, B12 Lipoic acid ATP/ADP CoA

k s = 10

nmoles g . protein hr

Km=0.30 nM So=50 nM D=5x10-6 cm2/s Diameter of the catalyst pellets is 1mm with an enzyme concentration in beads of 100g/l.
10 nmole mmole 10 1hr 10 g 50 x50 x100 l nmole gproteinhr 60 min l = 60 = 16 Mole nMole l min 50.3 0.30m + 50 l .l

Reaction

V0 =

kES0 = km + S 0

A+ B C + B' B' B
(regeneration of cofactor)

Ap

6 6 = = = 60cm 1 V d 0.1cm

So = 167 km
16 nmole 1l 1 min 1 nmole 2 x x cm 2 16 1 l min 1000cm 3 60S 3600 l min = 2.9 x10 cm = 2 2 cm nmole 60 cm nmole 1l x50 5 x10 6 50 x10 3 3 3 S l S l 10 cm

=
5 x10 6

Example of Industrial Enzyme Biocatalysis Process

High fructose corn syrup Semisynthetic penicillin Optical resolution of racemic amino acids Conversion of porcine insulin to human insulin

Production of semi-synthetic -lactam antibiotics

Media preparation
Inculum (Penicillium chrysogenum)

Filtration, Extraction

MIBK

Solid Waste

Thermosterilizer

Solvent Recovery

Penicillin G Recovery

Fresh solvent Crystallizer Crude Penicillin Penicillin G

Side Chain molecule

G-acylpenicillanic acid

Enzymatic Reactor

Enzymatic Reactor
Semi-synthetic penicillin purification
formation filling Drug product

KOAc: potassium acetate MIBK: methyl isobutyl ketone

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Semisynthetic Penicillin
H H CH2CONH O N S CH3 CH3 COOK

Enzymatic conversion of porcine into human insulin

Penicillin acylase

H H2 N O N

CH3 CH3 COOH

+ phenyl acetic acid

Penicillin G

High pH deacylation

G-amino penicillanic acid (G-APA)

R C OO

H2 N O N

CH3 CH3 COOH

Penicillin acylase
R CNH O O

H N

CH3 CH3 COOH

G-APA

High pH deacylation

Semisynthetic penicillin

CH NH2

Ampicillin

HO

CH NH2

Amoxicillin

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

Enzymatic Synthesis of Aspartame

Prof. Wei-Shou Hu