Cell and Enzyme Immobilization
Cells and enzymes as biocatalysts
enzyme
S
P
cells
cell based versus enzymatic
processes
glucose
glucose/fructose
glucose isomerase
glucose
ethanol
multi-enzymes acting sequentially
• whole cells preferred when multi-step
• enzymes preferred for 1 or 2 step transformations
• competing side reactions with whole cells
• sterility problems
• cell lysis
• other physiological requirements (nutrients, O2)
Advantages to immobilizing
enzymes and cells
• increased stability,weeks or months
• stable to heat, pH extremes, storage,
reaction
• facilitates recovery for repeated or
continuous use (essential for soluble
enzymes)
• cellular activity is enzymatic activity
(biotransformations)
Immobilization Techniques
bound
entrapped
matrix
encapsulation
adsorbed
microencapsulation
covalently
attached
support
enzyme or
cell
Matrix or lattice entrapment in
polymeric gels
• monomer, crosslinker,
polymerization catalyst, cells or
enzyme
• forms lattice structure, entrapping
cells/enzyme
• eg. polyacrylamide cross-linked
with N,N'-methylenebisacrylamide
(covalent gel)
Alginate and carrageenan non-covalent gels
• Naturally derived polymers extracted from
seaweed
• Used in food industry as a thickener
– ice cream, pudding, frozen drink
concentrates, jam, yoghurt, bakery
products, confectionery
• Dental molds
• Immobilization technology as an
encapsulating matrix
• Natural polymers are highly variable in
composition and their chemistry is generally
not known
• Composition affects properties
Alginate
a-L-mannuronic acid
b-D-guluronic acid
Alginate polymer
Alginate block
structures
(Mikkelsen and Elgsaeter, 1995; Smidsrod and Skjak-Braek, 1990)
Alginate Matrix
Binding of Ca2+ to G
Eggbox model for
Ca2+ binding
Structure of the
Alginate-Ca2+ Matrix
Cell entrapment protocol
- external gelation
M
Ca++
Dropwise addition of
alginate/cells into CaCl2
gelation bath
DNA entrapment protocol
- emulsification/internal gelation
Ca++
M
alginate droplet
containing DNA,
microcrystalline CaCO3
alginate in oil
emulsion
6.5
7.5
CaCO3
M
M
oil recycle
canola oil: 40oC
KCl
M
carrageenan: 40oC
5oC
static mixer
M
separator
settler
yeast
40oC
static mixer
carrageenan beads
to bioreactor
Immobilized yeast
technology
Kenics static mixer to
encapsulate brewing
yeast
Continuous brewing
gas out
beer out
bead disengagement
section
draft tube
temperature
control jacket
medium in
sparger - air in
Labatt continuous
airlift reactor
Tannase from Aspergillus oryzae to
hydrolyze tea tannins
• tannins represent 25% of extractables
in tea leaves
• cause creaming (turbidity) on cooling
• desire tea to be clear and bright
• tannase controlled hydrolysis of tannins,
retaining flavour
• encapsulated tannase remained stable
for 1 month
• 3 successive batch cycles during
48 h processing
Membrane coating
polyanion core
(alginate)
polycation
membrane
• chitosan
• poly-L-lysine
• co-guanidine
DNA microspheres following GI transit
Damon/Connaught process to
encapsulate pancreatic islets
coated with
poly-L-lysine
islets in
alginate bead
liquify alginate
core with citrate
or EDTA
Microencapsulation
• spherical ultrathin semipermeable membrane
enclosing cell/enzyme
suspension/solution
• interfacial polymerization
reaction (nylon)
NH2(CH2)6NH2 + ClCO(CH2)8COCl
NH2(CH2)6NH-CO(CH2)8CONH(CH2)6NH-CO(CH2)8COnylon 6-10 polyamide
+
HCl
Microencapsulation protocol
- interfacial polymerization
oil soluble
cross-linker
M
chitosan
cells/chitosan
in oil emulsion
Encapsulation of lobster carotenoids as
natural food pigment
Adsorption
• simple adsorption of
cell/enzyme onto support
(carrier) with adsorptive
properties
– anion exchange resins (DEAE
cellulose, Sephadex)
– cation exchange resins
(carboxymethylcellulose)
Covalent binding to support
• common technique
• carriers
– natural materials (cellulose, active
carbon)
– inorganic materials (glass, stainless
steel, ceramics (porous), silica (sand)
– enzymes/cells have reactive groups
(NH2, OH, SH, COOH)
– carriers are usually unreactive so
activation step required
Corning glass process (glucose isomerase and lactase)
1. support activation
ceramic + (C2H5O)3Si(CH2)3NH2
ceramic-Si-(C2H5O)2(CH2)3NH2
(3-aminopropyltriethoxysilane)
(activated support)
2. cross-linking of cells/enzyme
cells-NH2
+
OHC-(CH2)3-CHO
(glutaraldehyde)
cell-N=CH(CH2)3CH=N-carrier + H2O
Cross-linking intramolecular or cell to cell
• enzyme or cell cross-linked to
– another enzyme molecule
– another protein (BSA)
– insoluble carrier molecule
• glutaraldehyde cross-links NH2 groups
• hexamethylene diamine links COOH groups
Comparison of immobilization techniques
• adsorption and gel entrapment
– simple, gentle and efficient
– enzyme/cells often released (leaky); solved by cross-linking
– gas buildup may be problem
• microencapsulation
–
–
–
size exclusion (eg. antibodies)
only small substrates can be used
may lead to inactivation
• covalent attachment and cross-linking
– strong attachment
– laborious and expensive
– often leads to significant inactivation
Reaction kinetics or mass transfer control
• diffusional resistances
minimized by
– decreasing particle
size (increase surface
area/volume ratio)
– increasing [R]bulk
– improved mixing,
agitation
– increasing porosity
– optimizing distribution
of enzyme/cells
boundary film
Rbulk
Vmax [R]
v
K m  [R]
CH3CHOHCOOH + O2
CH3COOH + CO2 + H2O
L-lactate-oxygen 2-oxidoreductase