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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

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immobilized biocatalysts
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 biocatalysts2
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
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
Colloidal enzymaticnanoreactors

Macromol Biosci (2004) 4:13-16

slide5

Spherical polyelectrolyticbrush

Macromol Biosci (2004) 4:13-16

slide6

Preparation of apoflavoprotein

Eur J Biochem (2003) 270: 4227-4242

support materials
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
Size and shape of insoluble carriers

bead fibre capsule film membrane

selection criteria for carriers
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
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
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
optimization of an industrial biocat
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
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
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
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
-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
Enzyme kinetics

Chymotrypsin

  • Acyl-enzyme intermediate
  • E + S  ES  EA + P1  H2O  E + P2
  • Hydrolysis (deacylation) rate limiting step
chymotrypsin
Chymotrypsin

Ester hydrolysis

  • k2>> k3kcat~ k3
  • Km = k3k-1 / k2k1

Amide hydrolysis

  • k3>> k2kcat~ k2
  • Km = k-1 / k1
chymotrypsin21
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 kinetics22
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)
chymotrypsin23
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
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 enzymes25
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 enzymes26
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 enzymes27
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
Applications

Dye decolorization

  • immobilised laccase enzyme reactor
  • on-line spectroscopy

Biotechnol Bioeng (2004) 87: 552-563

applications29
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

applications30
Applications

Xanthine oxidase

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

J Biol Chem (2004) 279: 37231-37234

carrier bound enzymes
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
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

clecs
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
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

cleas current research topics
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