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Immobilization of Enzymes and Cells
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
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)
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)
Immobilization
by
adhesion
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
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
Alginate beads
Examples:
industrial
whole
cell
immobilizations