Effects of Immobilization on Enzyme Stability and Use

1
Effects of Immobilization on Enzyme Stability and
Use

• Design of enzymatic processes requires knowledge
  of
• reactant and product selectivity
• thermodynamic equilibria that may limit product
  yield
• reaction rate as a function of process conditions
  (Enzyme, substrate(s), Inhibitors,
  temperature, pH, )
• Two design issues that we have not considered
  are
• enzyme stability
• efficiency losses associated with the use of
  homogeneous (soluble) catalysts
• Immobilization of an enzyme allows
• it to be retained in a continuous reactor,
• but its initial activity and its stability
• directly influence its usefulness
• in industrial applications.

2
Enzyme Stability

• Although enzyme storage stability is important,
  it is the operational stability of an enzyme that
  governs its reactor performance.
• Operation stability is a complex function of
  temperature, pH, substrate and the presence of
  destabilizing agents.
• Generally, the rate of free enzyme deactivation
  is first order with a deactivation constant, kd
• Integrating this expression
• yields the concentration of
• active enzyme as a function
• of time

3
Effect of Thermolysin Instability on APM
Production

• Recall the rate expression developed for APM
  synthesis by thermolysin
• If thermolysin deactivation were adequately
  described as a first order process, the observed
  reaction rate would have an explicit time
  dependence, as shown below
• where ET,o represents the initial enzyme
  concentration and kd is the deactivation rate
  constant.
• The conversion versus time profile for aspartame
  synthesis by a batch process can be developed
  from this expression by integration.

4
Effect of Thermolysin Instability on APM
Production

• The evolution of L-Asp and conversion with time
  for a batch process is shown below.
• Depending on the relative rates of reaction and
  enzyme deactivation, the ultimate conversion can
  be strongly affected

5
Effect of Immobilization on Operational Stability

• Given that activity of enzymes is dictated by
  structure and conformation, the environmental
  change resulting from immobilization affects not
  only maximum activity, but the stability of the
  enzyme preparation.
• The factors that inactivate enzymes are not
  systematically understood, and depend on the
  intrinsic nature of the enzyme, the method of
  immobilization, and the reaction conditions
  employed.
• In general, immobilized enzyme preparations
  demonstrate better stability.
• Note that the immobilized
• preparation is often more
• stable than the soluble
• enzyme and displays a
• period during which no
• enzyme activity appears to
• be lost.

6
Classification of Immobilization Methods for
Enzymes
7
Immobilization by Entrapment

• Gel entrapment places the enzyme within the
  interstitial
• spaces of crosslinked, water-insoluble polymer
  gels.
• Polyacrylamide gels
• Polysaccharides The solubility of alginate and
  k-Carrageenan varies with the cation, allowing
  these soluble polymers to be crosslinked upon the
  addition of CaCl2 and KCl, respectively.
• Variations of pore size result in enzyme leakage,
  even after washing. The effect of initiator used
  in polyacrylamide gels can be problematic.

8
Immobilization by Entrapment

• Microencapsulation encloses enzymes within
  spherical,
• semi-permeable membranes of 1-100 mm diameter.
• Urethane prepolymers, when mixed with an aqueous
• enzyme solution crosslink via urea bonds to
  generate membranes of varying hydrophilicity.
• Alternatively, photo-
• crosslinkable resins
• can be gelled by
• UV-irradiation.
• Advantage of Entrapment
• Enzymes are immobilized without a chemical or
  structural modification. A very general
  technique.
• Disadvantage of Entrapment
• High molecular weight substrates have limited
  diffusivity, and cannot be treated with entrapped
  enzymes.

9
Immobilization by Carrier Binding

• Attachment of an enzyme to an insoluble carrier
  creates an active surface catalyst. Modes of
  surface attachment classify carrier methods into
  physical adsorption, ionic binding and covalent
  binding.
• Physical Adsorption Enzymes can be bound to
  carriers
• by physical interaction such as hydrogen bonding
  and/or
• van der Waals forces.
• the enzyme structure is unmodified
• carriers include chitosan, acrylamide
  polymers and silica-alumina
• binding strength is usually weak and affected by
  temperature and the concentration of reactants.
• Ionic Binding Stronger enzyme-carrier binding is
  obtained with solid supports containing
  ion-exchange residues.
• cellulose, glass-fibre paper, polystyrene
  sulfonate
• pH and ionic strength effects can be significant

10
Immobilization by Carrier Binding

• Covalent attachment of soluble enzymes to an
  insoluble support is the most common
  immobilization technique.
• Amino acid residues not involved in the active
  site can be used fix the enzyme to a solid
  carrier
• Advantages
• 1. Minimal enzyme leaching from the support
  results
• in stable productivity
• 2. Surface placement permits enzyme contact with
  
• large substrates
• Disadvantages
• 1. Partial modification of residues that
  constitute the active site decreases activity
• 2. Immobilization conditions can be difficult to
  optimize (often done
• in the presence of a competitive inhibitor)

11
Most Convenient Residues for Covalent Binding

• Abundance()Reactions
• 7.0 27
• 3.4 31
• 3.4 16
• 2.2 13
• 4.8 4
• 4.8 4
• 3.8 6

Amino acid residues with polar and reactive
functional groups are best for covalent binding,
given that they are most often found on the
surface of the enzyme. Shown are the most
convenient residues in descending order. The
average percent composition of proteins (reactive
residues only) is shown, along with the number of
potential binding reactions in which the amino
acids partake.
12
Covalent Attachment Techniques

• Cyanogen bromide activates supports with vicinal
  hydroxyl groups (polysaccharides, glass beads) to
  yield reactive imidocarbonate derivatives
• Diazonium derivatives of supports having aromatic
  amino groups are activated for enzyme
  immobilization
• Under the action of condensing agents (Woodwards
  reagent K), carboxyl or amino groups of supports
  and amino acid residues can be condensed to yield
  peptide linkages.
• Other methods include diazo coupling, alkylation,
  etc.

13
Immobilization by Crosslinking

• Bi- or multi-functional compounds serve as
  reagents for intermolecular crosslinking of
  enzymes, creating insoluble aggregates that are
  effective heterogeneous catalysts.
• Reagents commonly have two identical functional
  groups
• which react with specific amino acid residues.
• Common reagents include glutaraldehyde,
• and diisocyanates,
• Involvement of the active site in crosslinking
  can lead to great reductions in activity, and the
  gelatinous nature of the product can complicate
  processing.

14
Effects of Enzyme Immobilization on Activity
15
Selecting an Immobilization Technique

• It is well recognized that no one method can be
  regarded as the universal method for all
  applications or all enzymes. Consider,
• widely different chemical characteristics of
  enzymes
• different properties of substrates and products
• range of potential processes employed