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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