Cell and Enzyme Immobilization
description
Transcript of Cell and Enzyme Immobilization
Cell and Enzyme Immobilization
Cells and enzymes as biocatalysts
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enzyme
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
entrapped bound
matrixencapsulation
microencapsulation
adsorbed covalentlyattached
support enzyme orcell
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
Alginate polymer
Alginate block structures
-D-guluronic acid-L-mannuronic acid
(Mikkelsen and Elgsaeter, 1995; Smidsrod and Skjak-Braek, 1990)
Alginate Matrix
Binding of Ca2+ to G
Eggbox model for Ca2+ binding
Structure of theAlginate-Ca2+ Matrix
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Ca++
Dropwise addition of alginate/cells into CaCl2
gelation bath
Cell entrapment protocol- external gelation
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alginate in oilemulsion
alginate dropletcontaining DNA,
microcrystalline CaCO3
7.56.5Ca++
DNA entrapment protocol- emulsification/internal gelation
CaCO3
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canola oil: 40oC
carrageenan: 40oC
yeast40oC
static mixer
static mixer
5oC
KCl
oil recycle
carrageenan beadsto bioreactor
separatorsettler
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Kenics static mixer toencapsulate brewingyeast
Continuous brewing
Immobilized yeast technology
gas out
beer out
sparger - air in
medium in
draft tube
temperaturecontrol jacket
bead disengagementsection
Labatt continuousairlift reactor
Tannase from Aspergillus oryzae to hydrolyze tea tannins
• tannins represent 25% of extractablesin 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)
polycationmembrane• chitosan• poly-L-lysine• co-guanidine
DNA microspheres following GI transit
Damon/Connaught process to encapsulate pancreatic islets
islets inalginate bead
coated with poly-L-lysine
liquify alginatecore with citrateor EDTA
Microencapsulation
• spherical ultrathin semi-permeable 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)8CO- + HCl
nylon 6-10 polyamide
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Microencapsulation protocol- interfacial polymerization
chitosan
oil solublecross-linker
cells/chitosanin oil emulsion
Encapsulation of lobster carotenoids asnatural 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
Rbulk
boundary film
[R]K
[R]Vv
m
max
CH3CHOHCOOH + O2 CH3COOH + CO2 + H2O
L-lactate-oxygen 2-oxidoreductase