Immobilization of Enzymes and Cells

1. 2. 3. 4. 5. 6. 7. 8. 9.

Opening Lecture Introduction to Enzyme Technology Basics of Enzymes as Biocatalysts Kinetics of Enzyme Catalysed Reaction Principles and Application of Enzyme Selectivity Enzymes in Organic Synthesis I. Enzymes in Organic Synthesis II. Enzyme Production and Purification Engineering of Biocatalysts

10. Immobilization of Enzymes and Cells 11. Reaction Engineering and Process Technology 12. Biotechnology Informatics

Immobilization

of

enzymes

definition: Immobilization means that the biocatalysts are limited in moving due to chemically or physically treatment insoluble enzyme - advantages as classical heterogeneous catalyst Reasons reuse of enzyme (reducing cost) easy product separation continous processing
- facilitated process control - low residence time (high volumetric activity) - optimisation of product yield

Limitations cost of carriers and immobilisation changes in properties (selectivity) mass transfer limitations
- problems with cofactor and regeneration - problems with multienzymes systems

stabilization by immobilisation

activity loss during immobilisation

Immobilization

of

enzymes

transformation of enzyme to insoluble form or inclusion to definite space method for reuse and stabilisation of enzyme one-step reactions - domain of immobilized enzymes

Parameters

of

enzyme

immobilization

effective, easy, cheap, acceptable (non-toxic in food and medical applications) rate and yield dependent on the parametrs involved (e.g., type of carrier, concentrations, pH, temperature, method, reaction time) empirical optimization binding to porous carrier - standard method for both, laboratory and industry external protein surface properties (e.g., hydrophobicity, ionic groups, functional groups for covalent binding) protein surface engineering
introduction of functional groups spacers increases binding interactions, stability (e.g., nanoparticles, protecting molecules) and activity (e.g., cofactors) change of pI

Crosslinking
crosslinking is the first option of enzyme immobilisation soluble enzyme (e.g., glutar(di)aldehyde with lysine residues) crystals (enzyme crystallisation and crosslinking - CLEC) aggregates (salt precipitation and crosslinking - CLEA) imprinted molecules (derivatisation of protein and polymerisation, CLIP) whole microorganisms (dead cells) containing enzyme

Inclusion

into

polymeric

network

one of the most convenient method for whole cell immobilization problems with enzyme diametr and leak out of the particle
combination with crosslinking binding to nanoparticles of beads

Binding

to

porous

carrier

standard method for enzyme immobilisation capacity of carrier - adsorption isoterm (often Langmuir type) carrier characteristis
chemical (e.g., hydrophobicity, chemical and microbial stability) morphological (e.g., particle diametr, pore size, (inner) surface for adsorption) mechanical (e.g., resistance to pressure, compressibility, elasticity) general (e.g., food or pharma grade, cost)

carriers with different functional groups accessible carriers classification (e.g., according basic material, origin of source or structure)
inorganic organic from natural sources organic synthetic materials

Inorganic

carriers

high pressure stability may undergo abrasion in stired vessels SiO2 based carriers functionalized by introduction of amino groups (e.g., treating with aminopropyl triethoxysilane) covalent binding carried out using glutaraldehyde commercialy SiO2 available materials
porous glass (Corning, Waters, Schuller) silica (Grace, Solvay, Degussa)

mineral materials (clays)

celite - adsorption and stabilisation of enzyme in organic media bentonite - excellent adsorption capacity (up to 1.5 g protein per g bentonit) used for enzyme isolation by adsorption/desorption crosslinking prevents desorption and the carrier can be entrapped in alginate

Organic

carriers

from

natural

sources

favorable compatibility with proteins range of polysacharides and derivatives used for immobilization
wide network structure hydrophilic properties - weak interactions with proteins Cellulose

cellulose derivatives
DEAE-cellulose (diethylaminoethyl-) CM-cellulose (carboxymethyl-)

dextran
widely used for enzyme immobilization protein chromatography activated by cyanogen bromide mechanical stability limited Sephadex

other polysacharides
agarose, starch, pectine and chitosan

proteins (gelatine)

Organic

synthetic

carriers

high chemical and mechanical stability wide range of carriers with good capacity and simple manipulation (ion-exchange) resins
copolymerization with functional groups (e.g., nitration, sulfonation, carboxylation, epoxydation) resines with XAD adsorbent - hydrophobic characteristics polystyrene polyvinylacetate acrylic polymers polystyrene Eupergit

polyacrylate

Amberlite

Binding

methods

adsorption
binding onto silica, clay or ion-exchange materials by weak interactions (e.g., ionic, electrostatic, hydrophobic) dependent on process conditions (e.g., pH, temperature, ionic strength, hydrophobicity) simple and cost-effective, reversible (stabilized by crosslinking), may cause unfolding

covalent binding
better stabilization of enzyme on carrier introduction of functional group (e.g., amino, epoxy, thiol, cyanide) principle (1. derivatization, 2. activation, 3. binding of enzyme)

Examples:

industrial

enzyme

immobilizations

Immobilization

of

microorganisms

and

cells

first example in 1823, Acetobacter adsorbed to wood chips (acetic acid production) production of secondary metabolites interest in cofactor regeneration and multienzyme systems (e.g., alcohol production) applicable if enzyme(s) difficult to isolate or show low stability/activity outside cell (e.g., nitrile hydratase in acrylamide production)

continous processing with (re)synthesis of enzyme in immobilized living cells mostly resting cells - limited in growth by controlling C-, N- or P- sources industrial applications of immobilized viable cells:
1. 2. 3. beer maturation with yeast cells anchorage-dependent mammalian cell (production of vaccines) environmental technologies using mixed cultures

Immobilization
Advantages

of

microorganisms

and

cells

Limitations insufficient stability, low resistance mass transfer limitation side reactions, degradation of product byproducts from lysis of cell or toxic metabolites (pharma grade) low productivity

no enzyme isolation and purification multienzyme complex reactions cofactor regeneration in native system application of achorage-dependent cells (e.g., mammalian cells)

synthrophic mixed cultures

Fundamental

aspects

modifications in microorganism microenvironment mass transfer affects substrate and product gradient and metabolism effective diffusion coefficients range from 50 to 100% of that in free solution (e.g., in ionotropic gels - diffusion close to that in water) mass transfer reduced with growth and increase of biofilm thickness (>100m) immobilized cell physiology changes:
growth and reproduction (e.g., immobilized yeast cell growth reduced by 45%) specific productivities (e.g., alcohol production 40-50% enhanced) difference of intracellular pH higher resistance

Immobilization

of

microorganisms

and

cells

Immobilization

by

adsorption

adsorption and growth of microorganism on surface is natural phenomenon (e.g., stone, clay, plant material, teeth, plastics) vinegar production - adsorption of Acetobacter sp. onto wood chips (since 1823)
even today 60 m3 reactors trickling generators in use (conversion 90%) curled beechwood filling (pack life 20 years)

manufacture of beer
Saccharomyces cerevisiae immobilized by adsorption on: - Siran porous silica particles (maturation and aroma formation) - polystyrene carrier coated with DEAE-cellulose (alcohol free beer production)

antibodies, therapeutic proteins and vaccines

mammalian cells on microcarriers (surface electrostatically and chemicaly treated) - gelatine and collagen - crosslinked dextran and cellulose - glass - polystyrene

Immobilization

by

adhesion

adhesion - follows adsorption, microorganisms produce secondary matrix of polymers

environmental technology applications

waste water treatment nitrogen elimination exhauste gas purification

Immobilization

by

entrapment

entrapment in polymeric networks recently dominated research and development complete retention of cells with almost unlimited transport of substrate and product gentle conditions combined with variety of low-cost materials (e.g., ionotropic polysacharides)
gelation - simple and gentle, phase transitions in polymer-solvent mixture (e.g., agar or agarose) polyvinylalcohol polymerization - polymer solution cooled to room temperature and mixed with cell suspension, droplets deposited on surface (LentiKats) - Saccharomyces cerevisiae and Leuconostoc oenos (alcohol production) - Escherichia coli (production of tryptophane) - Nitrosomonas and Nitrobacter (oxidation of ammonia to nitrate in water) polyurethanes (under development not applied) polyelectrolytes

Immobilization

by

entrapment

entrapment in ionotropic gels polysacharides:

carboxy- or sulfonyl groups (e.g., alginate, pectin, carrageenan) amino groups (e.g., chitosan - deacetylated chitin)

low cost, non-toxic water soluble polyelectrolytes forming solid polymeric networks (gels) by crosslinking through (poly)cations (e.g., Ca2+, K+) or (poly)anions (e.g., polyphosphates)

Immobilization

by

entrapment

Example: entrapment in alginate gel

water soluble sodium alginate (commercially available) polymerize with Ca2+ destabilisation by chelating agents (e.g., phosphate, citrate, EDTA) or other cations rapid, nontoxic, inexpensive, versatile used for more then 65 years in food and pharma industry

Alginate beads

Examples:

industrial

whole

cell

immobilizations