Bahir Dar University

Bahir Dar Institute of Technology

Department of Chemical Engineering
Introduction to Biochemical Engineering
For 4th Year Chemical Engineering Students
By Mengistu F.
2013 E.C
Bahir Dar, Ethiopia
 Chapter Three -- Immobilized Enzyme
The term “immobilized enzymes” refers to “enzymes physically
confined or localized in a certain defined region of space with retention
of their catalytic activities, and which can be used repeatedly and
continuously.
It also allows enzymes to be held in place throughout the reaction,
following which they are easily separated from the products and may
be used again.
 Cont.
Since most enzymes are globular protein, they are soluble in
water.
Therefore, it is very difficult or impractical to separate the
enzyme for reuse in a batch process.
Enzymes can be immobilized on the surface of or inside of
an insoluble matrix either by chemical or physical methods.
A main advantage of immobilized enzyme is that it can be
reused since it can be easily separated from the reaction
solution and can be easily retained in a continuous-flow
reactor.
 Advantage of immobilized enzyme

Enzyme is not lost after process and might therefore be used for
greater duration.
Higher enzyme concentrations are attainable without sacrificing these
advantages.
immobilized enzyme may show selectively altered chemical or
physical properties
Some enzymes are more stable if they are immobilized.
 Disadvantage of immobilized enzyme

An additional process (and cost) is associated with enzyme

immobilization.
Enzymes may leak from immobilized state.
Diffusional limitations. Substrate(s) and product(s) must transfer
across a boundary to get to/from active site.
Can be difficult to control environment immediately affecting enzyme
and its activity.

Some enzymes are less stable if they are immobilized.(Structural

change)
 Immobilization Techniques
Enzymes can be immobilized on the surface or inside of an insoluble
matrix either by chemical or physical methods.
Immobilization techniques can be classified by two methods:

1. Chemical Method :-Based on covalent bond formation

 Covalent Attachment
 Cross-linking

2. Physical Method:-Based on non-covalent bond formation

 Adsorption
 Entrapment
 Microencapsulation
 Chemical method
Covalent attachment

This immobilization techniques is the formation of covalent bonds

between the enzyme and the support matrix.
The covalent attachment of enzyme molecules using nonessential
amino acid residues to water insoluble, functionalized supports.
Functional groups of the nonessential amino acid residues that are
suitable for the immobilization process are free α-,ß-, or γ carboxyl
groups, α - or ß-amino groups, and phenyl, hydroxyl, sulfhydryl, or
imidazole groups.
 Cont.
Commonly employed water-insoluble supports for the covalent
attachment of enzymes include:
synthetic supports such as

acryl amide based polymers

anhydride-based polymers
methacrylic acid-based polymers

styrene-based polymers
natural supports such as
agarose , cellulose, dextran

 glass, starch and, polypeptides,

 Cont.

Another variation of immobilization by covalent attachment

is the copolymerization of the enzyme with a reactive
monomer (M) such as
 Cont.

Already active polymers such as maleic anhydride copolymers will

be simply mixed with enzymes to produce immobilized enzymes.

Natural or synthetic polymers need to be activated by treating them

with reagents before adding the enzyme.

The activation involves the chemical conversion of a functional

group of the polymer.

The enzyme's active site should not be involved in the attachment,

in which case the enzyme would lose its activity upon
immobilization.
 Cont.

Water-insoluble enzymes can be prepared by using multifunctional

agents that are all bifunctional in nature and have low molecular
weight, such as glutaraldehyde.

Enzymes can be reacted with multifunctional reagent alone so that

they are cross-linked intermolecularly by the reagent to form a water-
insoluble derivative.

Another method is to adsorb enzymes on a water-insoluble, surface-

active support followed by intermolecular cross-linking with
multifunctional reagents to strengthen the attachment.
 Physical Method

Adsorption:
simplest way to immobilize enzymes.

It is process of adsorbing enzymes physically on a surface-active

adsorbent by contacting an aqueous solution of enzyme with an adsorbent.

Commonly employed adsorbents are:

alumina, anion-exchange resins, calcium carbonate, carbon,

cation-exchange resins, celluloses, clays, collagen,

colloid-ion, conditioned metal, glass plates, diatomaceous earth, and

hydroxyapatite.
 The advantages of adsorption techniques

The procedure of immobilization is simple.

It is possible to separate and purify the enzymes while being
immobilized.
The enzymes are not usually deactivated by adsorption.

The adsorption is a reversible process.

However, adsorption techniques also have several disadvantages:

The bonding strength is weak.
Very sensitive to solution pH, ionic strength, and temperature.

Amount of enzymes loaded on a unit amount of support is usually low.

 Physical Method

Entrapment
Enzymes can be entrapped within cross-linked polymers by forming a
highly cross-linked network of polymer in the presence of an enzyme.
The entrapment method of immobilization is based on the localization
of an enzyme within the lattice of a polymer matrix or membrane
 Cont.

This method has a major advantage

 there is no chemical modification of the enzyme
 the intrinsic properties of an enzyme are not altered.

However this method has disadvantage of

 enzyme may be deactivated during the gel formation
 Enzyme leakage

The most commonly employed cross-linked polymer is

the polyacrylamide gel system.
 Microencapsulation

Enzymes can be immobilized within semipermeable

membrane microcapsules.

This can be done by the interfacial polymerization

technique. Organic solvent containing one component of
copolymer with surfactant is agitated in a vessel and aqueous
enzyme solution is introduced.

The polymer membrane is formed at the liquid-liquid

interface while the aqueous phase is dispersed as small
droplets.
  Criteria for selecting support for immobilization of enzymes

Surface properties of the support.

Interactions between support and enzyme.

Interactions between support and reaction mixture.
Physical and Mechanical properties of the support.

 Surface properties of the support

 The surface should have the capacity to adsorb the enzyme or have
functional groups essential for attachment of enzymes.
 Effect of Mass-Transfer Resistance

 The immobilization of enzymes may introduce a new

problem which is absent in free soluble enzymes.
 It is the mass-transfer resistance due to the large particle
size of immobilized enzyme or due to the inclusion of
enzymes in polymeric matrix.
Cont.
 Cont.

If we follow the hypothetical path of a substrate from the liquid to the
reaction site in an immobilized enzyme, it can be divided into several
steps :

(1) transfer from the bulk liquid to a relatively unmixed liquid layer
surrounding the immobilized enzyme;

(2) diffusion through the relatively unmixed liquid layer; and

 (3) diffusion from the surface of the particle to the active site of the
enzyme in an inert support. Steps 1 and 2 are the external mass-transfer
resistance.

Step 3 is the intraparticle mass transfer resistance.

 External Mass-Transfer Resistance

 If an enzyme is immobilized on the surface of an insoluble particle,

the path is only composed of the first and second steps, external mass-

transfer resistance.

The rate of mass transfer is proportional to the driving force, the

concentration difference, as

Where
 CSb and CS are substrate concentration in the bulk of the solution and
at the immobilized enzyme surface, respectively
 Cont..
The term kS is the mass-transfer coefficient (length/time)
and A is the surface area of one immobilized enzyme
particle.
During the enzymatic reaction of an immobilized enzyme,
the rate of substrate transfer is equal to that of substrate
consumption
Therefore, if the enzyme reaction can be described by the
Michaelis-Menten equation,
 Cont..
where

a is the total surface area per unit volume of reaction solution.

Dimensionless way expression of the equation

Where
 Cont..
NDa is known as Damkohler number, which is the ratio of the
maximum reaction rate over the maximum mass-transfer rate.

1. If NDa<<1, the mass-transfer rate is much greater than the reaction

rate and the overall reaction is controlled by the enzyme reaction,

2. If Nda>>1, the reaction rate is much greater than the mass-transfer

rate and the overall rate of reaction is controlled by the rate of mass
transfer that is a first-order reaction,
 Cont..
To measure the extent which the reaction rate is lowered
because of resistance to mass transfer, we can define
the effectiveness factor of an immobilized enzyme, η as
η = actual reaction rate
rate if not slowed by diffusion
Where: actual reaction rate is:

Rate if not slowed by diffusion is:

but Cs is replaced by Csb
 Cont..
Therefore, the effectiveness factor is:

where the effectiveness factor is a function of xS and β. If

xS is equal to 1, the concentration at the surface CS is equal
to the bulk concentration CSb. Substituting 1 for xS in the
preceding equation yields η = 1, which indicates that there is
no mass-transfer limitation.
 Internal Mass Transfer Effects
A. General Derivation
Enzymes are immobilized to a porous support having large
internal surface areas
If enzymes are immobilized by copolymerization or
microencapsulation, the intraparticle mass-transfer resistance
can affect the rate of enzyme reaction
Assumptions:
Immobilized enzyme is distributed uniformly; reaction involves
no change in pH and electrostatic effects are negligible
Cont.

The reaction occurs at every position within the immobilized enzyme,

and the kinetics of the reaction are of the same form as observed for
free enzyme.
Mass transfer through the immobilized enzyme occurs via molecular
diffusion.
There is no mass-transfer limitation at the outside surface of the
immobilized enzyme.
The immobilized enzyme is spherical. The model developed by these
assumptions is known as the distributed model.
Reading assignment (develop equations using these assumptions for
zero order, first order and M-M kinetics.)
Chapter Four -- Industrial Applications of Enzymes
The applications of enzymes can be classified into three major categories:
industrial enzymes, analytical enzymes, and medical enzymes.
Carbohydrates
 Carbohydrates constitute a major class of naturally occurring organic
compounds, including sugars, starches, and celluloses.
 They are essential to the maintenance of plant and animal life.
 Carbohydrates are classified into three major groups:
 Monosaccharides,

 Oligosaccharides, and
 Polysaccharides.
 Cont.
 Monosaccharides
 Monosaccharides are the simplest carbohydrate units

 They are basic carbohydrate molecules including:-

 polyhydroxy aldehyde,
 polyhydroxy ketone, and their derivatives

 All simple monosaccharides have the general empirical formula,

(CH2O)n, where n is the whole number ranging 3 to 8.

 All monosaccharides can be grouped into two general classes as:

 Aldoses: contain a functional aldehyde grouping (-CHO), or Ketoses

 Cont.
 Disaccharides
 Two sugars can link to each other by losing water from OHs to
form disaccharides
 The four important disaccharides are:-
 Sucrose,
 Lactose,
 Maltose, and

 Cellobiose, which all have the same molecular formulas, C12H22O11

 Sucrose and lactose are the most abundant and most important
disaccharides of natural origin
 Cont.
 Maltose and cellobiose are repeating units of polymeric starch and
cellulose, respectively
 Disaccharides may hydrolyze to form two monosaccharide molecules

 Important disaccharides
 Cont.
 Polysaccharides

 Polysaccharides consist of many simple sugar units linked together

 One of the most important polysaccharides is starch, which is produced by
plants for food storage
 Starch comprises a large percentage of cereals, potatoes, corn, and rice

 Complete hydrolysis of starch yields glucose, but partial hydrolysis gives

maltose as well
 Starch can be separated into two main fractions by treatment with hot water.

 The insoluble component (10 % to 25 %) is amylose (long linear),

 The soluble component (75 % to 90 %) is amylopectin (branched)

 Amylose and amylopectin are degraded by α- and β-amylase, which are

found in the pancreatic juice and saliva of animals
 Cont…

 Enzymatic hydrolysis of starch by sequential treatment with α- amylase and

gluco-amylase will produce glucose as the main final product.
 Dextrins, products of the partial hydrolysis of starch, are polysaccharides of
lower molecular weight than starch
 They are used in infant food because they are easier to digest than starches
 Dextrins are sticky when wet and are used as mucilage on postage stamps
and envelopes
 Cellulose is one of the three major structural components of all plant cell
walls with two other components, hemicellulose and lignin
 Cellulose is the most abundant organic compound of natural origin on the
face of the earth
 Cont.
 Complete hydrolysis of cellulose gives glucose
 The cellulose molecule is comprised of long chains of cellobiose
molecules joined together by β-1,4- glucosidic bonds
 Structure of cellulose
 Starch Conversion

 In recent years, the conversion of starch to fructose has become a very

important commercial process
 High-fructose corn syrup (HFCS) is approximately twice as sweet as
sucrose. It is used in soft drinks, canned fruits, lactic acid beverages,
juice, bread, ice cream, frozen candies, and so on
 HFCS can be obtained from a variety of cereals and vegetables, such
as corn, wheat, rice, potatoes, and cassava
 Corn is the most important source of HFCS because of low costs and
excellent utilities of its by-products, corn meal, oil, gluten, germ, and
fiber
 Corn Wet Milling
 The first step of the HFCS process is the corn wet milling

D.E.: dextrose equivalent

 Cellulose Conversion

 Cellulosic wastes have great potential as a feedstock for producing

fuels and chemicals
 Cellulose is a renewable resource that is inexpensive, widely available
and present in ample quantities
 Large amounts of waste cellulose products are generated by
commercial and agricultural processes
 In addition, municipal facilities must treat or dispose of tremendous
quantities of cellulosic solid waste
Lignocellulosic Materials

 Lignocellulosic materials have a common basic structure, but vary

greatly in chemical composition and physical structure
 Cont..
 Lignocellulosic biomass consists mainly of cellulose, hemicellulose,
lignin, small amounts of extractives, and ash
Typically, these materials contain
 30% to 60 % cellulose,
 10 % to 30 %hemicellulose (polyoses), and

 10 % to 20 % lignin
 Cellulose provides strength and flexibility, while
 Lignin supports and protects the cellulose from biological and
chemical attack and
 Hemicellulose bonds lignin to cellulose
 Cellulose Pretreatment and Hydrolysis

 Major obstacles in the hydrolysis of cellulose are the interference of

lignin (which cements cellulosic fibers together) and the highly
ordered crystalline structure of cellulose
 These obstacles necessitate a costly pretreatment step in which
elementary cellulosic fibrils are exposed and separated
 Many pretreatments have been employed to enhance the degradation
of lignocellulosic materials to glucose
 The treatments fall into two general areas

 Physical pretreatment
 Chemical pretreatment
 Physical pretreatment
 Milling, irradiation, heating, and heating with other pretreatment and

 Chemical pretreatment
 Alkali treatments, acid treatments, delignification, and dissolving
and reprecipitating
 The major drawbacks of the milling are cost and the fact that
noncellulosic substances are not removed
 Common chemical pretreatments of alkali and acid contacting
improve hydrolysis by breaking down the lignin, hemicellulose, and
cellulose
Cont.
 Cont.

 Enhanced conversion of cellulose in the attrition bioreactor is due to a

combination of factors, including:-
 A reduction in crystallinity,
 An increase in pore volume and surface area, and
 An increase in the accessibility of glucosidic bond sites to the cellulase
complex
 Many fungi are capable of producing extracellular enzymes that can degrade
cellulose
 For the cost effective production of bioethanol from lignocellulosic
saccharification and fermentation, there is a necessity for the improved activity
of hydrolytic enzymes followed by efficient utilization of sugar by fermentative
microbes
Th a n k
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