BTK 4102Advanced Enzyme Technology

Assoc. Prof. Dr. Lai Oi Ming

Topic 7:

Gel Entrapment

Learning Outcomes

At the end of the topic, you should know :1. About gel entrapment technique2. Advantages and disadvantages of gel

entrapment3. Factors affecting the gel entrapment of

enzymes and cells

Introduction

• Entrapment is a physicaltechnique.

• Does not involve formation of bonds to the enzyme/cells.

• Enzymes/cells are entrapped in the interstitial spaces of cross-linked polymers.

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• Entrapment is very easy to perform. • Biocatalysts dissolved in polymer’s precursors

solution and polymerisation is initiated. • The biocatalysts will be trapped in the spaces

between the cross-linked polymers.• 2 types of polymer with widest application are:a) polyacrylamide type gels b) naturally derived gel materials such as

cellulose triacetate, agar, gelatin, carrageenanor alginate.

Gel Entrapment Technique

• Major advantage of technique is its simplicity, mild conditions used and the usefulness when immobilising cells.

• However, because average pore size of the gel should be as high as possible to prevent excessive diffusion limitation and the variability of the pore size in such gels, there may be great leakage of the biocatalyst from the gel, particularly low molecular weight enzymes.

• Non-viable cells are usually well retained, but viable, dividing cells may burstfrom the gel material.

• The MW of substrates used must not be too big to allow movement of the substrates into the gel structure.

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• None of the entrapment techniques commonly used are perfect.

• Polyacrylamide gels suffer the drawback that the monomers used and the free radicals generated during polymerisation are toxic, therefore do not use this method when using viable cells or sensitive enzymes.

• Agar and carrageenan have large pore sizes, thus allowing quite sizable (< 10µ) cells to escape quite easily; in addition they are also depolymerised by mild heat.

• Calcium alginate gels are the method of choice for viable cell immobilisation.

• It is disrupted by calcium chelating agents such as citrate or phosphate which may often be necessarily included in the reaction medium.

Types of Supports/Gel Material

a) Polyacrylamide –• This is the most commonly used synthetic gel

matrix for entrapment of enzymes. • Since it is uncharged, the pH profile of the

immobilised enzymes are much the same as the free enzymes.

• Gel is formed by cross linking acrylamide with N,N'-methylene bis-acrylamide (cross linking agent) in the presence of EDTA and Tris-HClbuffer.

• Enzyme is later dissolved in this solution and the sample purged with nitrogen to remove any traces of oxygen.

polymerized

b) K-carrageenan• Carrageenan is the general name for the

galactans extracted from red algae, which consists of galactose units which are partly in the anhydro form and partly esterified with sulfuric acid.

• �-carrageenan(kappa-carrageenan)

• �-carrageenan(iota-carrageenan)

• �-carrageenan(lambda-carrageenan)

• Carrageenan has very similar properties to alginate and agarose.

• Of the 3 known types of carrageenan –gamma, iota and kappa – only kappa and iota are suitable as supports for immobilisation, with the kappa being the most excellent.

• Potassium and calcium salts form gels of carrageenan at ambient temperatures as a result of the formation of double helixes.

• These gels are unstable in the presence of sodium ions and at low pH because of the weak (1→4)-glycosidic bond connecting the galactose residues.

• But above pH 4.5, the gels are very stable even under heat sterilisation conditions.

c) Agar and agarose• Agars and agarose are D-galactans isolated

from certain red seaweeds that form rigid gels when their solutions are cooled to temperatures below 45°C.

• Chemically, agarose is a purified linear-galactan hydrocolloid isolated from agar with a repeating unit, agarobiose, possessing an alternating (1→3)-linked β-D-galactopyranoseand (1→4)-linked, 3,6-anhydro-α-L-galactopyranose structure.

• (see Figure 5)

Structure of agarose

-(1 3)-�-D-galactopyranose-(1 4)-3,6-anhydro-�-L-galactopyranose

• Agarose gels are mechanically more stable and have greater pore size than other gels.

• They are resistant to microbial degradation, as agar degrading enzymes (agarases) have been found in only certain microorganisms living on seaweeds.

• There are certain disadvantages: a) agarose gels cannot be heat sterilisedb) they disintegrate in alkaline solutions and

organic solvents. Even at neutral pH, there is possibility of solubilisation.

c) They must be stored in wet form as they shrink irreversibly on drying.

• Improvement of mechanical properties and chemical resistance can be achieved by cross linking with bifunctional reagents (eg: epichlorohydrin or divinylsulfone) which eliminates most of these deficiencies.

• Cross linked agarose is only used for analytical purposes as it is a very expensive matrix and unsuitable economically for industrialisation.

• Comparatively inexpensive crude agar, which contains negatively-charged sulfate and carboxyl groups that may effect its applicability, can be treated with alkali to remove these groups, and thus make it suitable as immobilisation matrix .

d) Alginate• Alginic acid or alginate is a glycuronan

extracted from brown algae and chemically is an unbranched co-polymer consisting of residues of D-manuronicacid and L-guluronic acid.

• Three structural elements are conventionally considered to occur:

a) (1→4)-α-L-guluronan (G) blocksb) (1→4)-β-D-mannuron (M) blocksc) polyuronid consisting of alternating L-guluronic

and D-mannuronic acid residues arranged in a blockwise manner.

Figure 6: Structural elements in alginate

• Alginates are chemically very stablebetween pH 5 and 10.

• High acid concentrations and high temperatures cause decarboxylation of alginates.

• They are degraded by direct-oxidationagents such as halogens or periodate and by redox systems such as polyphenolsand thiols.

• Alginate gels are mechanically stable except when high concentration of K+, Mg2+, phosphate or chelating agents are present.

• Immobilisation in alginate gel is safe, fast, mild, cheap, simple and versatile. For plant/animal cells, entrapment with calcium alginate or agar is preferred.

e) Collagen• Collagen is the most abundant protein

constituent of higher vertebrates. • It is easily isolated from many biological

sources and can be reconstituted into various forms without losing its native structure.

• Collagens fibrous structure and high swellability in aqueous solutions contributes its use as a support matrix.

• Due to its biological origin, collagen may be very useful in biomedical applications.

f) Gelatin• Gelatin is easily obtained in solution from

collagen by boiling with water.• It does not have the strength of collagen and to

use it as an water insoluble carrier, must cross link gel with multifunctional reagents such as glutaraldehyde.

• Due to its very hydrophilic nature, a homogenous distribution of enzymes and gelatin can be obtained at relatively low temperatures (35-40°C)

• Advantages of Gel Entrapment• Individual enzyme molecules are homogenously

dispersed throughout the gel, thus, giving greater enzyme accessibility to the substrate & faster reaction rates.

• For this method, usually only aqueous solvents are used. Most enzymes denature when placed in non-aqueous solvent. Thus, this method avoids placing enzymes in a destructive environ.

Factors Affecting Gel Entrapment

1. Gel pore size• Gel pore size of 35 angstrom will retain most of

the enzymes. Gel pore size that is formed should be sufficiently big enough to prevent diffusion limitations, but because of these, sometimes the cells and enzymes leak out of the gel.

• Viable and dividing cells may also burst out from the gel material.

• 2. Oxygen• Oxygen inhibits co-polymerisation , thus it

is preferable to deoxygenate the mixture by purging with an inert gas (nitrogen) before the addition of the enzyme solution.

• 3. Photopolymerisation• Certain polymerisation reaction are initiated by

catalysing the solution with either chemicals(polymerisation agents) or light.

• For photocatalysis which is done using a photo floodlamp, the affect of heat may denature the enzyme. Thus, the reaction container should be placed in an ice bath during polymerisation or other cooling means.

Enzyme + polymer solution

ice

Flood lamp

• 4. Gas exchange• Gas exchange between polymer

microenvironment and bulk phase may be limited particularly where the cells have a high metabolic rate.

• Thus, oxygen and carbon dioxide levels may be substantially different in the cells microenvironment and this may directly affect their metabolism and metabolic output.

• 5. Photosyntetic microorganisms• For photosynthetic organism, light intensity

may be reduced, not so much by the polymer but by self shading of the cells deeper in the polymer by those on the outside.

• 6. Microenvironment of the immobilised cells/enzymes

• Metabolising cells will often produce acid or alkali, thus changing the pH or the microenvironment of the cell/enzymes compared to the bulk phase.

• This may affect the metabolic rate and metabolite productivity.

• The accumulation of secondary metabolites due to diffusion limitations around the microenvironment of the cell may switch off its own synthesis.

• This may result in the immobilised cells being less productive than free cells.

• Limitation of growth because of nutrient limitation may also switch the cells into stationary phase where secondary metaboliteproduction may be enhanced.

• The potential limitation of nutrients or light, and the accumulation of toxic or inhibitory products in a cell’s microenvironment as a result of immobilisation, causes most of the cells being concentrated at the periphery. This is probably due to cell death in the centre of the particle coupled to cell growth at the periphery rather than cell migration.

nutrients Cell proliferation at periphery

Cell death

• 7. Mechanical rigidity of gel• Gel with suitable mechanical rigidity should be

employed.• Very soft gel sediments slowly and is unsuitable

for use in flowing system application.• High gel concentration (more rigid) tends to

reduce the activity of the gel and diffusion limitation will become a problem in “hard” gels.

• 8. Absence of rigid cell wall• Unlike plant cells, animal cells do not have

a rigid cell wall, thus making the cells more sensitive to osmotic changes that occur in the gel.

• 9. Shape of gel• Alginate solution are viscous and tend to form

rather large beads. For cell entrapment, cells in the middle of the bead tend to die after a few days and near the surface of the beads, cells tend to proliferate.

• This is believed to be due to poor mass transferof key nutrients within larger beads. This problem can be overcome by forming thin gel sheets/membrane of a alginate reinforced with cotton cloth.

nutrients

Cell

Thin membrane