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" Modern Industrial Microbiology and Biotechnology
(d) entrapment of the enzyme molecule inside a water-insoluble polymer lattice or
semi-permeable membrane.
The IUPAC groups can be divided into two basic groups, the chemical and the
physical methods as shown in Fig. 22.4.
The IUPAC emphasizes that in dealing with immobilized enzymes, the properties of
the free enzyme, the type of support used and the methods of support activation and
enzyme attachment must be specified.
22.5.2.1 Immobilization by covalent linkage
This is by far the most widely studied method. The covalent linkage is achieved between
a functional group on the enzyme not essential for catalytic activity and a reactive group
on a solid water-insoluble support. The functional groups available on enzymes for
linkage are amino and carboxyl groups, hydroxyl groups, imidazole groups, indole
groups, phenolic groups and sulphydryl groups. The nature of the enzyme’s functional
group through which immobilization is to be effected determines the reaction which will
be used to bind the enzyme to the support. Some of the reactions which have been used
are acylation, amination and arylation and alkylation.
Some supports which have been used for immobilization include agarose, celluose,
dextran, chitin, starch, polygalacturonic acid (pectin), polyacrylamide, polyvivyl
alcohol, polystyrene, polyprpylene, polyamino acids, polyamide, glass and metal aides
and bentonite. Many of these are organic, but recently the use has been advocated of
inorganic support on the grounds of reuse of inorganic materials, non-toxicity, good half-
life of enzymes immobilized on inorganic supports, and the ease with which inorganic
materials can be fashioned to suit any particular enzyme system.
In the above description the reaction has been a direct one between functional groups
on the enzyme and reactive sites on the support. However, in some cases intermolecular
linkage may occur through the mediation of a crosslinking reagent, In such cases the
cross linking agent has a number of different active sites. Some of these react with the
support and others with the enzyme. One of the most commonly used cross-linking agent
is glutaraldehyde which has two cross-linking sites. Several others are available. The
advantage of the covalent bonding method of enzyme immobilization are:
(i) The coupling of the enzyme to the support is easy to conduct and consists of
allowing support and enzyme to interact and therefore facilitates centrifuging and
washing off any enzymes not bound.
Immobilized Enzyme
Bound (Chemical) Entrapped (Physical containment)
Covalently linked Adsorbed Micro-encapsulation Matrix-entrapped
Fig. 22.4 Methods of Enzyme Immobilization
Biocatalysts: Immobilized Enzymes and Immobilized Cells "
(ii) The enzyme-support derivative is easy to manipulate and adapt because of the
great physical and chemical variation in the available support: they can be used in
a variety of reactors including stirred tank, fluidized bed-reactors and can also be
modified into flat sheets fiber.
(iii) Covalent coupling has been widely described and methods for carrying it out are
readily available in the literature.
(iv) The supports themselves are widely available commercially.
The disadvantages are that some preparations are tedious to make; the chemical
bonding may inactivate the enzyme in some cases; and finally covalently-bound water-
insoluble enzyme-substrate derivatives act poorly on high molecular weight substrates.
22.5.2.2 Immobilization by adsorption
This method is both simple and inexpensive and consists of bringing an enzyme solution
in contact with a water-insoluble solvent surface and washing off the unadsorbed
enzyme. The extent of the adsorption depends on a number of factors including the
nature of the support, pH, temperature, time, enzyme concentration. In principle, though
not always in practice, adsorption is reversible. Adsorbents which have been used
include alumina, bentonite, calcium carbonate, calcium phosphate, carbon, cellulose,
charcoal, clay, collagen, diatomaceous earth, glass, ion-exchange resins, sephadex, and
silica gel. Apart from the ease of the operation, the other advantage is that the enzymes are
unlikely to be inactivated because the system is mild. The disadvantage is that in cases of
weak binding the enzyme may be easily washed away.
22.5.2.3 Immobilization by micro-encapsulation
Micro-encapsulation consists in packaging the enzyme in tiny usually spherical
capsules ranging from 5-300 m in diameter in semi-permeable (permanent) or liquid (non-
permanent) membranes. The former are more commonly employed. To prepare micro-
capsules a high aqueous concentration of the enzyme is first prepared. The aqueous
enzymes solution is then emulsified in an organic solvent or solvents with a surfactant
which is soluble in the organic solvents. Two methods are then used to form micro-
capsules from this enzyme-surfactant emulsion.
In one method known as the interfacial polymerization technique, the enzyme
solution contains the enzyme as well as one component of the membrane that will form
round the micro-capsule. The emulsion is stirred vigorously and more of the organic
solvent(s) containing the rest of the capsule-forming reagent is added.
In the second method, the coacervation-dependent method, the added organic
solvents contain all the components of the polymer. In both cases the enzyme droplets are
formed during the vigorous stirring. The semi-permeable membrane is allowed to harden
around the micro-droplets; the micro-capsules are then washed and then transferred.
Semi-permeable membranes have been made of cellulose nitrate, polystyrene, etc.,
with the coacervation method.
Micro-capsule formation by the interfacial method has been produced from nylon and
widely investigated because of its application in medicine e.g. in urease immobilization
in artifical kidneys. The organic solvent usually used for the polyamide Nylon-6,10
" Modern Industrial Microbiology and Biotechnology
semi-permeable membrane from hexamethylenediamine and sabacoyl chloride is a
chloroform-cyclohezane mixture.
OO
||||
H
2
N – (CH
2
)
6
– NH
2
+ Cl – C – (CH
2
) – C – Cl – HCI
Hexamethlenediamine Sabacoyl chloride
OO OO
||||||||
– NH – (CH
2
)
6
– NH – C – (CH
2
)
8
– C (CH
2
)
6
– NH G – (CH
2
)
8
– C – NH–
Non-permanent liquid membranes are prepared by emulsifying the aqueous enzyme
solution in a surfactant to form the liquid membrane-encapsulated enzyme. It is not
commonly used.
The advantages of the permanent (semi-permeable) micro-capsulation method are:
(i) An extremely large surface is provided by the tiny bubbles of enzymes. A micro-
capsule with 20mm diameter would for example have a surface area of
2,500 cm
2
/ml.
(ii) The specificity of the micro-capsule is increased by the possibility of using a
membrane which will favor the diffusion of substrates of certain types.
The disadvantage is that a high concentration of enzyme is needed and only low
molecular weight substances pass through. With the non-permanent liquid membranes,
the same advantages accrue; the disadvantage however is leakage.
22.5.2.4 Immobilization by entrapment
In the entrapment of enzymes, no reaction occurs between support and the enzyme. A
cross-linked polymeric network is formed around the enzyme; alternatively the enzyme
is placed in a polymeric substance and the polymeric chains cross-linked.
Polyacrylamide gels have been widely used for this purpose, although enzymes do leak
through the network in some cases.
The advantages of the entrapping method are: (i) its simplicity, (ii) the small amount of
enzyme used, (iii) the unlikelihood of damage to the enzyme, (iv) applicability to water
insoluble enzymes.
The disadvantages include leakage of enzymes and some chemical and thermal
enzyme damage during gel formation.
22.5.3 Methods for the Immobilization of Cells
Three general methods are available for immobilizing microbial cells.
(i) Ionic binding to water-soluble ion-exchangers: Cells of E. coli and Azotobacter agile
bound to Dowex-l resin have been studied while mold spores have been bound to
ion-exchange cellulose derivatives. In both cases successful demonstration of
succinic acid oxidation and invertase activity were demonstrated. This method is
however not entirely satisfactory as enzymes may leak out following autolysis
during continuous enzyme reaction.
Biocatalysts: Immobilized Enzymes and Immobilized Cells "!
Fig. 22.5 Methods of Immobilizing Enzymes and Cells
(ii) Immobilization in cross-linked chemicals: Microbial cells have been immobilized by
cross-linking each other with bi-functional reagents such as glutaraldehyde. But
non-cross-linking agents are equally effective.
(iii) Entrapment in a polymer matrix: This appears to be the most widely used method of
immobilizing cells. In this method the cells are entrapped in a polymer matrix
where they are physically restrained. The following matrixes have been used:
polyacrylamide, collagen, cellulose triacetate, agar, alginate and polystyrene.
Methods for immobilization of cells and enzymes are given in Fig. 22.5.
22.5.3.1 Advantages of immobilized cells
Immobilized cells have the following advantages over conventional batch fermentation
as well as over immobilized enzymes.
(i) In batch fermentation, a significant proportion of the substrate is ‘wasted’ for the
growth of the microbial population and for producing other substances other than
enzymes required for the conversion at hand. Once the cells are immobilized
however, they need to be offered nutrients for growth.
(ii) When cells are immobilized the reactions are more homogeneous and can be
treated more like catalysts.
"" Modern Industrial Microbiology and Biotechnology
(iii) The lag period which occurs in a conventional batch fermentation is eliminated for
the accumulation of products associated with non-growth phase of the cells.
(iv) It is more feasible to run immobilized cells continuously at high dilution rates
without the risk of washout which would occur in a conventional continuous
culture system.
(v) Higher and faster yield is possible because of the greater density of cells;
furthermore, toxic materials are continuously removed.
(vi) It is possible to recharge or resuscitate the cells by inducing growth and
reproduction among resting cells.
(vii) A high capital cost is involved in installing, and operating a fermentor; in systems
where comparison have been made, immobilized cells are cheaper than the
conventional batch production.
(viii) The use of immobilized cells eliminates the need for enzyme extraction and
purification. Furthermore, systems involving multi-enzyme reactions can occur
more easily in intact cells harboring these enzymes.
(ix) Immobilized cells are more suited to multiple step processes
(x) Cofactor regeneration is not a problem
Immobilized cells are particularly appropriate under the following conditions:
(i) When the enzymes are intracellular: the use of immobilized cells would eliminate
the need for breaking the cells for enzyme isolation.
(ii) When extracted enzymes are unstable during or after immobilization.
(iii) When the micro-organism does not produce enzymes which can cause
undesirable side reactions; or when such side-reaction producing enzymes can be
readily inactivated.
(iv) When the substrates and products are not high molecular weight substances.
22.5.3.2 Disadvantages of immobilized cells
Some of the disadvantages of the conventional system of cultivation of organisms spill
over to the immobilized cell thus:
(i) The cells may produce enzymes other than the one (s) sought.
(ii) Genetic changes, although with reduced likelihood in comparison with conven-
tional fermentation, can also occur during immobilization.
(iii) Immobilization may result in the loss of a specific catalytic activity due to enzyme
inactivation, resulting from the immobilization process or to diffusional barriers
hindering substrate access, or product removal from the organisms.
(iv) Cells located in the center of a cell flow may be deprived of nutrients or be
inactivated by accumulating toxic wastes.
(v) Contamination by other microorganisms can occur.
22.6 BIOREACTORS DESIGNS FOR
USAGE IN BIOCATALYSIS
A variety of bioreactors are available for immobilizing enzymes and cells and these are
shown in Fig. 22.6.
Biocatalysts: Immobilized Enzymes and Immobilized Cells "#
a). batch stirred fermentor
b). continuous stirred tank
c). continuous packed-bed
i) downward flow
ii). upward flow
iii). Upward flow and re-cycle
d). continuous fluidized-bed
e). continuous ultrafiltration
Fig. 22.6 Various Designs of Bioreactors for Use in Biocatalysis.
"$ Modern Industrial Microbiology and Biotechnology
22.7 PRACTICAL APPLICATION OF IMMOBILIZED
BIOLOGICAL CATALYST SYSTEMS
Immobilized enzymes and cells have been intensively studied in the hope that they can
be used industrially. Only some of the expectations have been realized because of
economic reasons. Soluble ‘once only’ enzymes marketed in the form of powder or liquids
are available at low prices. Amylases and glucoamylases used in the starch industry are
so low-priced comparatively that immobilized forms can hardly compete. The only
immobilized enzyme currently used on a large scale is glucose isomerase used to produce
about 2 million tons of high-fructose syrups around the world, but especially in the USA,
Europe, and Japan. High fructose syrup competes successfully with sugar from beet or
cane.
Several immobilized enzymes or whole cell processes are being applied in the
Japanese pharmaceutical industry. These include L-amino acid from racemic acyl – D – L
– amino acids, L-aspartic acid from ammonium fumarate, L-citrulline from L-arginine,
and the production of 6-amino penicillanic acid (6-APA) for semi synthetic penicillin
production. In the United States and Europe 6-APA is produced with immobilized
enzymes. In Italy whole milk lactose hydrolysis is carried out by fiber entrapped lactase.
Many other applications are nearing the point in their development where they are
ready for commercialization: saccharification of starch by immobilized glucoamylase;
cheese whey lactose hydrolysis by bound >-galactosidase; beer chill-proofing, steroid
transformations, protein-hydrolysis to improve digestibility. Industrial processes do not
receive publicity rapidly and it is not unlikely that some of these may well have been
commercialized already.
Immobilized cells have been used industrially in Japan for the transformations
mentioned above except for L-amino acid isolation from racemic mixtures. The most-
widely employed use of immobilized cells however are glucose isomerization and the
hydrolysis of raffinose in beet sugar using mycelial pellets of the fungus Mortierrella sp.
Raffinose (in beet molasses) is hydrolyzed to sucrose and galactose by >–galactosidase
(mellibiase) produced by the fungus.
The potentials of immobilized enzymes and cells are yet far from realized. When
economic conditions permit them to become so, the whole process of fermentation as we
know it today may be revolutionized and fermentors may become largely for the growth
of cells for subsequent use in immobilized enzyme or cell production.
22.8 MANIPULATION OF MICROORGANISMS FOR
HIGHER YIELD OF ENZYMES
Until recently higher yields in enzyme production have been achieved, as has been the
case with most other industrial microbial products, by empirical means using selection
from natural variants, mutants obtained through treatment with various mutagens, and
improvement in the environmental conditions of the fermentor or semi-solid medium.
With these methods the rate of enzyme production has increased from two to five-
hundred times.
In recent times more knowledge has accumulated about various aspects of enzyme
production including those of molecular and other dimensions, and the manipulation of
Biocatalysts: Immobilized Enzymes and Immobilized Cells "%
industrial organisms in general. In this section some of these new developments and
their use or possible use in increasing enzyme yield will be discussed. Some of them
promise the possibility of producing virtually any enzyme extracelluarly and at will.
22.8.1 Some Aspects of the Biology of Extracellular
Enzyme Production
(i) The nature of extra-cellular enzymes secretion: Extracellular enzymes have been
defined as those which are secreted into the medium outside the cell without involving cell
lysis. This distinction is important because most extraccellular enzyme-producing
organisms are Gram-positive organisms. Gram-negative organisms, in general produce
enzyme in the medium only when the cell is lysed. Most Gram-negative organisms do not
therefore, according to this definition, produce ‘true’ extra-cellular enzymes. However, it
has been found in recent times that some Gram-negative organisms do in fact secrete
extracellular enzymes. Furthermore many Gram-negative organisms do in fact produce
and secrete enzymes across the cytoplasmic membrane. Such enzymes are however held
within the periplasmic zone (Fig. 22.7) of the Gram-negative cell wall and hence do not
find their way to the medium. Thirdly mutants of Gram-negative cells defective in the
ability to synthesize cell wall components continue to synthesize and secrete
polypeptides into the environment. On account of these observations extra-cellular
enzymes have been redefined as those which are secreted across the cell membrane. In
terms of industrial microbiology it is an apt definition as methods for deranging the
molecular arrangements of cell walls exist, which when successfully applied to Gram-
negative bacteria secreting into the periplasmic space convert them to extra-cellular
secreters. Such methods include the formation of protoplasts by the prevention of cell
wall formation using suitable antibiotics, limited digestion by trypsin, solubilization of
the cell wall with a combination of a detergent (e.g. laurodeoxycholate and a chelating
agent).
(ii) Some biochemical properties of extra-cellular enzymes: Bacterial extracellular
enzymes vary in molecular weight from 12,000 to 500,000 but in the main they range from
20,000 to 40,000. Secondly, most but not all bacterial exoproteins lack cysteine. It has been
suggested, but not entirely accepted, that this absence of cysteine will confer the property
of malleability on extra-cellular enzymes thus facilitating their export.
(iii) Site of synthesis of extra-cellular proteins: Even in cells actively secreting extra-
cellular proteins, an examination of the cytoplasm shows a complete absence or only a
trace amount of the enzymes being excreted. Early report for instance claiming that a–
amylase of >–amyloliquifaciens is first produced as a high molecular weight precursor
have been shown to be wrong on the basis of radio-isotope (labeling) experiments. Since
no evidence exists for the cytoplasmic synthesis of extracellular proteins it has been
suggested they are synthesized on ribosomes associated with the cells in much the same
way as in eucaryotic cells. In eucaryotic cells, membrance-bound polysomes are engaged
in secreting proteins for export whereas polysomes secrete non-exportable proteins.
According to the currently accepted model synthesis takes places on the cell
membrane and is secreted directly into pores in the cell membrane. Indeed synthesis and
secretion are one process, following the system in eucaryotic cells. Some evidence for this
are as follows. In many bacteria a considerable but variable fraction of the ribosomes is
"& Modern Industrial Microbiology and Biotechnology
associated with the cytoplasmic membrane. In exponential phase of Bacillus licheniformis
for example, 96% of the ribosomes are membrane bound. It is also know that exoenzyme
synthesis is more sensitive to antiobiotic inhibition than general protein synthesis. This
has been interpreted as being so because of the membrane bound ribosomes are more
accessible to the antibiotic.
(iv) Control of extra-cellular enzyme secretion by gene cloning: When the terminal portion
of the b-galactosidase gene of E. coli was replaced with a gene that codes for a protein of the
outer cell wall membrane of the bacteria, the b-galactosidase activity which is normally
intracellular was formed extracellularly depending on the size of the latter that attached
on the b-galactosidase gene. This and other similar experiments show that in due course it
may be possible to produce virtually any enzyme extracellularly by gene cloning.
(v) Some methods for increasing enzyme yields: The increased yields which have been
observed in enzyme production is based on strain selection, improved environmental
factors, regulatory controls and genetic manipulation.
(a) Strain selection: Strain selection from natural variants of the same species or even
entirely new species have resulted in the array of enzymes available in some
industries e.g. the starch hydrolyzing industries.
The natural strains may then be mutagenized for increased variation in the gene
pool. Strains have been selected in the above two manners for a wide variety of
properties including temperature-tolerant enzymes and resistance to feedback
regulation.
Left = Gram-negative wall; Right = Gram-positive wall; Dotted areas = hydrophobic
zones; cc=capsular carbohydrate; cp = capsular polysaccharide; ec = cytoplasmic
membrane enzymes in the cytoplasmic which synthesize cell wall macro-molecules; lp =
lipoprotein; p = structural and encymic proteins of the outer layers of the Gram-negative
wall; s = structural proteins of cytoplasmic membrane; sp = enzymes in the periplasmic
zone; ps = permeases.
Fig. 22.7 Generalized Structure of the Bacterial Cell Wall
Biocatalysts: Immobilized Enzymes and Immobilized Cells "'
(b) Environmental factors: Exoenzymes may be constitutive, but the majority are
inducible or partially so. Inducers are therefore important in increasing the yields
of many extracellular enzymes. Since many of the substrates are insoluble they
cannot enter the cell, and hence their analogues or gratuitious inducers (those that
induce the enzyme but are not substrates or breakdown products) have been used.
Inducers are usually cheap in order to bring down costs. Thus, corn cobs are
hydrolyzed to produce xylose which act as inducers for glucose isomerase.
Most extracellular enzymes are produced in the idiophase and maximum
production is usually in the late log and early stationary phase. This period
coincides with the period when the organism is released from catabolite
repression. Increased yields may therefore be achieved by feeding low levels of the
substrate or feeding them intermittently. Yields may also be increased by
increasing cell-wall permeability. Surfactants may be incorporated into the
medium for this purpose although how they affect the wall permeability is not fully
understood.
(c) Regulatory control: Control of regulation is through induction and catabolite and
feedback regulations. Mutants resistant to all three have been produced with
consequent boost in production. An example of inducer-resistance is in the case of
the glucose isomerase producing antinomycete Streptomyces phaechromogenes, the
wild strain of which will not germinate on L-lyxose, another form of D-xylose. It
will grow on lyxose only if germination is first obtained on xylose. Mutants were
selected which would germinate directly on lyxose, thus eliminating the need for
xylose.
The bypassing of catabolite repression has led to the production of large
amounts of enzymes. This has been achieved by using toxic analogues of the
substrates. Thus, 2-deoxy-glucose is used as a toxic analogue when seeking for
mutants able to over produce glucosidase.
Feedback regulation is not very applicable to enzyme synthesis, although some
examples are known.
(d) Genetic manipulation: As had been indicated earlier the gene specifying the extra-
cellular secretion may be cloned on those controlling the synthesis of particular
enzymes, thus causing the enzyme to be secreted extracellularly.
The number of copies of specific genes may be increased by gene amplification
methods thus increasing enzyme yield several times. For example, plasmids specifying
particular extra-cellular enzymes may continue to replicate while the parent
chromosome is inhibited by, for example, chloramphenicol thereby permitting an
amplification of the genes – sometimes up to 2,000 copies or up to 40% of the cells total
DNA. The result is increased yield of the enzyme.
SUGGESTED READINGS
Anon. 1995. Classification and Chemical Characteristics of Immobilized Enzymes. (Technical
Report), International Union of Pure and Applied Chemistry. Pure and Applied Chemistry,
67, 597-600.
Butterfoss, G.L., Kuhlman, B. 2006. Computer-Based Design of Novel Protein Structures. Annual
Review of Biophysics and Biomolecular Structure, 35, 49–65.