cell and enzyme immobilization. cells and enzymes as biocatalysts s p enzyme cells

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Cell and Enzyme Immobilization

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Page 1: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Cell and Enzyme Immobilization

Page 2: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Cells and enzymes as biocatalysts

S P

enzyme

cells

Page 3: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P 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)

Page 4: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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)

Page 5: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Immobilization Techniques

entrapped bound

matrixencapsulation

microencapsulation

adsorbed covalentlyattached

support enzyme orcell

Page 6: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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)

Page 7: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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

Page 8: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Alginate

Alginate polymer

Alginate block structures

-D-guluronic acid-L-mannuronic acid

(Mikkelsen and Elgsaeter, 1995; Smidsrod and Skjak-Braek, 1990)

Page 9: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Alginate Matrix

Binding of Ca2+ to G

Eggbox model for Ca2+ binding

Structure of theAlginate-Ca2+ Matrix

Page 10: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

M

Ca++

Dropwise addition of alginate/cells into CaCl2

gelation bath

Cell entrapment protocol- external gelation

Page 11: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

M

alginate in oilemulsion

alginate dropletcontaining DNA,

microcrystalline CaCO3

7.56.5Ca++

DNA entrapment protocol- emulsification/internal gelation

CaCO3

Page 12: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

M

M

M

canola oil: 40oC

carrageenan: 40oC

yeast40oC

static mixer

static mixer

5oC

KCl

oil recycle

carrageenan beadsto bioreactor

separatorsettler

M

Page 13: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Kenics static mixer toencapsulate brewingyeast

Continuous brewing

Immobilized yeast technology

Page 14: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

gas out

beer out

sparger - air in

medium in

draft tube

temperaturecontrol jacket

bead disengagementsection

Labatt continuousairlift reactor

Page 15: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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

Page 16: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Membrane coating

polyanion core(alginate)

polycationmembrane• chitosan• poly-L-lysine• co-guanidine

Page 17: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

DNA microspheres following GI transit

Page 18: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Damon/Connaught process to encapsulate pancreatic islets

islets inalginate bead

coated with poly-L-lysine

liquify alginatecore with citrateor EDTA

Page 19: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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

Page 20: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

M

Microencapsulation protocol- interfacial polymerization

chitosan

oil solublecross-linker

cells/chitosanin oil emulsion

Page 21: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Encapsulation of lobster carotenoids asnatural food pigment

Page 22: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

Adsorption

• simple adsorption of cell/enzyme onto support (carrier) with adsorptive properties– anion exchange resins (DEAE

cellulose, Sephadex)– cation exchange resins

(carboxymethylcellulose)

Page 23: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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

Page 24: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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

Page 25: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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

Page 26: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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

Page 27: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

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

Page 28: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells
Page 29: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells

CH3CHOHCOOH + O2 CH3COOH + CO2 + H2O

L-lactate-oxygen 2-oxidoreductase

Page 30: Cell and Enzyme Immobilization. Cells and enzymes as biocatalysts S P enzyme cells