Immobilized biocatalysts

Enzymes or whole cells

• physically confined or localised in a certain

defined region of space

• with retention of their catalytic activities

• and which can be used repeatedly and

continuously
 Immobilized biocatalysts

Composite of two essential components

• Carrier, designed to aid separation and reuse of

the catalyst and facilitates control of the process

• Enzyme, designed to convert the substrates of

interest into the desired products
 Methods of immobilization

Figure 3.1

• Binding to a solid support

• Cross-linking
• Entrapment

• Covalent, ionic, hydrophobic binding (Fig. 3.2)

• Influence of enzyme structure and behaviour

 Colloidal enzymatic nanoreactors

Macromol Biosci (2004) 4:13-16

 Spherical polyelectrolytic brush

Macromol Biosci (2004) 4:13-16

 Preparation of apo flavoprotein

Eur J Biochem (2003) 270: 4227-4242

 Support materials

• large surface area

• functional groups
• insolubility in water
• chemical stability

• stability against microbial attack

especially important for industrial biocatalysts
 Size and shape of insoluble carriers

bead fibre capsule film membrane

 Selection criteria for carriers

• Reactor configurations (batch, stirred-tank, column,

plug-flow)

• Type of reaction medium (aqueous, organic solvent,

two-phase system)

• Reaction system (slurry, liquid-to-liquid, liquid-to-

solid, solid-to-solid)

• Process conditions (pH, temp, pressure)

 Aim of chosen parameters

• Easy separation of the immobilised enzymes from the

reaction mixture

• Flexibility of reactor design

• Broad applicability in various reaction media and

reaction systems

• Facilitate down-stream processing and, in particular,

control of the process.
 Other requirements for ideal BIOCAT

• Recyclable

• Cost effective

• Safe for use

• Competitive and innovative enough to protect the

intellectual property right

• Attractive for end-users

Criteria for robust IMBIOCATs
 Optimization of an industrial BIOCAT

Glutaryl acylase

• stabilisation of the enzyme by multipoint covalent

attachment onto a new amino-epoxy Sepabead

• parameters that effect the enzyme-support interaction

• J Biotechnol (2004) 111: 219-227

 Immobilised environment

Environment different from solution

• effects on [S], pH or ionic strength

• restricted diffusion

• influence on enzyme kinetic properties

• influence on enzyme regio- and enantioselectivity

• immobilised enzyme molecules not all identical

 Immobilised enzymes

Conformational and steric effects

• Location of active site

• Conformational changes

• Partial inactivation by covalent attachment

• Reduced number of active molecules

• Reduced flexibility
 Action of immobilized enzymes

Effects on enzyme kinetics

• Conformational and steric effects

• Partitioning effects (charge)

• Micro-environmental effects on intrinsic catalytic

parameters (ionic strength)

• Diffusional limitation and mass-transfer

 -Lactamase

Biotin-derivatized PEG-coated sensor chip

• Study on oriented attachment and surface activity by

enzyme kinetics and in situ optical sensing

• Sequential adsorption of avidin and biotinylated

-Lactamase or immobilisation of preformed complex

• Langmuir (2004) 20: 10464 -10473

 Enzyme kinetics

Chymotrypsin

• Acyl-enzyme intermediate

• E + S  ES  EA + P1  H2O  E + P2

• Hydrolysis (deacylation) rate limiting step

 Chymotrypsin

Ester hydrolysis

• k2 >> k3 kcat ~ k3
• Km = k3 k-1 / k2 k1

Amide hydrolysis

• k3 >> k2 kcat ~ k2
• Km = k-1 / k1
 Chymotrypsin

Partitioning effects

• Charged matrix can change local pH

• pH activity optimum of chymotrypsin (Fig. 3.8)

• Anionic polymer: higher pH optimum
• Cationic polymer: lower pH optimum
• Effects dependent on ionic strength (Fig. 3.9)
 Enzyme kinetics

Partitioning effects

• Attraction or repulsion of charged substrates

• Change in local concentration S (PS)

• Same charge: Km(app) higher

• Opposite charge: Km(app) lower (Fig. 3.10)
 Chymotrypsin

Micro-environmental effects

• Polyanionic EMA-chymotrypsin: higher kcat

• Polycationic PO-chymotrypsin: lower kcat
(Fig. 3.11)

• Perturbation of kcat greater with amides than

with esters: acylation step more strongly affected
(Table 3.1)
 Immobilized chymotrypsin

Micro-environmental effects

Chymotrypsin copolymers

• Amide substrates: similar Km(app) (Table 3.1)

• Ester substrates: perturbed Km(app)

• Diffusion limitations (effective [S] )

• Change in ratio k3 / k2 and not in k-1 / k1
 Immobilised enzymes

Micro environmental effects

Increase ionic strength

• Increase kcat native enzyme

• Increase kcat PO-chymotrypsin
• No change kcat EMA-chymotrypsin

• Change in charge-charge interaction in active site

before the acylation step
 Immobilised enzymes

Mass transfer effects

• Rate of substrate diffusion lower than rate of catalysis

•  , effectiveness factor: vimm / vsolution

dependent on [S], LB plot not linear

• External and internal diffusion limitation

 Immobilised enzymes

Mass transfer effects

• External diffusion limitation:

Reduced substrate transport from bulk solution to
biocatalyst surface

• Internal diffusion limitation:

Slow diffusion inside porous medium where the
enzyme is immobilised in (substrate size)
 Applications

Dye decolorization

• immobilised laccase enzyme reactor

• on-line spectroscopy

Biotechnol Bioeng (2004) 87: 552-563

 Applications

Full hydrolysis of lactose in milk

• immobilization of lactase from K. lactis

• greatly reduces the inhibition by glucose

Biotechnol Prog (2004) 20: 1259-1262

 Applications

Xanthine oxidase

• binding to heparin-Sepharose 6B
• limits inhibition by clinical relevant inhibitor
oxypurinol

J Biol Chem (2004) 279: 37231-37234

IMBIOCAT and process development
 Carrier-bound enzymes

Savings
• Enzyme re-use
• Downstream processing

Additional costs
• Reaction times (lower activity and productivity)
• Immobilisation process (laborious design)
 Carrier-bound or carrier-free

Cross-linking

• CLE cross-linked dissolved enzyme

• CLEA cross-linked enzyme aggregate
• CLEC cross-linked enzyme crystal
• CLSD cross-linked spray-dried enzyme

Curr Opin Biotechnol (2003) 14: 387-394

Carrier-free immobilised enzymes
 CLECs

Highly active and stable immobilised enzymes of

controllable size

• Selection of right crystal form or size

• Engineering properties crystallization medium
• Activity dependent on size and properties substrates,
reaction medium, reaction type and reaction
conditions

Chemtech (1997) 27: 38-45

 CLEAs
Preparation, optimization and structures

• simplicity of operation
• no need for laboriuos optimization
• no need for pure enzymes
• high-throughput methodologies
• high yield of activity for any enzyme

Biotechnol Bioeng (2004) 86: 273-276

Activity retention and enzyme solubility
 CLEAs: current research topics

Particle size and diffusion constraints

• Broad range of enzymes

• Size control
• New aggregation methods
• New cross-linkers

Biotechnol Bioeng (2004) 86: 273-276