Přednáška 1

Imobilizované biologické systémy

Immobilized Immobilized BiocatalystsBiocatalysts

Doc. Ing. Jiří SAJDOK,CSc.

1.Introduction1.Introduction

Working Party on Applied Working Party on Applied Biocatalysts within the European Biocatalysts within the European Federation of Biotechnology: Federation of Biotechnology: “Immobilized biocatalysts are “Immobilized biocatalysts are enzymes, cells, or organelles (or enzymes, cells, or organelles (or combinations of them) which are in a combinations of them) which are in a state that permits their reuse” state that permits their reuse”

P. B. Poulsen, Enzyme Microb. Technol. P. B. Poulsen, Enzyme Microb. Technol. 55 (1983) 304 – 307 (1983) 304 – 307

Historical Background:Historical Background:

( 1823 vinegar production, sludge, attachment to ( 1823 vinegar production, sludge, attachment to equipment )equipment )

50s – 60s : immobili50s – 60s : immobilizzation of enzymesation of enzymes( 1916 Nelson – Griffin: invertase ads.on charcoal ( 1916 Nelson – Griffin: invertase ads.on charcoal 1948 Sunmer: jack bean urease)1948 Sunmer: jack bean urease)1950 – 1970: intensive investigations on immobilized 1950 – 1970: intensive investigations on immobilized

enzymes and other proteinsenzymes and other proteins ( e.g.antigens -( e.g.antigens -> affinity chromatography > affinity chromatography ))1969 – first industrial appt.of immobilized enzyme1969 – first industrial appt.of immobilized enzymeOptical resolution of DL aminoacids with immobilized Optical resolution of DL aminoacids with immobilized

amino acylase ( Chibata et al. )amino acylase ( Chibata et al. )Since 1960 investigations on immobilized cellsSince 1960 investigations on immobilized cellsIndustrial applications of immobilized microbial cells:Industrial applications of immobilized microbial cells:19731973 L – aspartic acid -Escherichia coli (aspartase )L – aspartic acid -Escherichia coli (aspartase )1974 L - malic acid – Brevibacterium ammoniagenes 1974 L - malic acid – Brevibacterium ammoniagenes ( fumarase)( fumarase)19821982 L – alanin – Pseudomonas dacunhae ( L-aspartate L – alanin – Pseudomonas dacunhae ( L-aspartate

-decarboxylase ) -decarboxylase )

IntroductionIntroduction

Biocatalysts dissolved in aqueous buffer Biocatalysts dissolved in aqueous buffer solutions solutions

soluble or nativesoluble or native enzymes, cells, cell parts, enzymes, cells, cell parts, or organelles.or organelles.

IImmobilized, fixed, or insolubilizedmmobilized, fixed, or insolubilized enzymes, cells, etc., denote biocatalysts enzymes, cells, etc., denote biocatalysts that are bound to a support. that are bound to a support.

carrier, supportcarrier, support, or , or matrixmatrix cross-linking agent, bifunctional agentcross-linking agent, bifunctional agent, or , or

carrier activatorcarrier activator. .

MembrMembránové bílkovinyánové bílkoviny

2.2. Methods of Enzyme 2.2. Methods of Enzyme

ImmobilizationImmobilization 1.1. modified into a water-insoluble form,modified into a water-insoluble form,

2.2. retained by an ultrafiltration membrane inside retained by an ultrafiltration membrane inside a reactor, ora reactor, or

3.3. bound to another macromolecule to restrict bound to another macromolecule to restrict their mobility.their mobility.

Imobilization TechniquesImobilization Techniques

Figure 1.Figure 1. Classification of enzyme immobilization Classification of enzyme immobilization methodsmethods

Encapsulation of EnzymeEncapsulation of Enzyme

2.2.1. Carriers for Enzyme Immobilization2.2.1. Carriers for Enzyme Immobilization

1. 1. Large surface area and high permeability Large surface area and high permeability2. Sufficient functional groups for enzyme 2. Sufficient functional groups for enzyme attachment under attachment under nondenaturing conditionsnondenaturing conditions3. Hydrophilic character3. Hydrophilic character4. Water insolubility4. Water insolubility5. Chemical and thermal stability5. Chemical and thermal stability6. Mechanical strength6. Mechanical strength7. High rigidity and suitable particle form7. High rigidity and suitable particle form8. Resistance to microbial attack8. Resistance to microbial attack9. Regenerability9. Regenerability10. Toxicological safety10. Toxicological safety11. Low or justifiable price11. Low or justifiable price

Table 2. Chemical classification of matrixes Table 2. Chemical classification of matrixes used for enzyme immobilizationused for enzyme immobilization

Synthetic polymers

Synthetic materials

Polystyrene Nonporous glass

Polyacrylates and poly-

Controlled pore glass

methacrylates Controlled pore metal

Polyacrylamide oxides

Hydroxyalkyl methacrylates Metals

Glycidyl methacrylates  

Maleic anhydride polymers  

Vinyl and allyl polymers  

Polyamides  

Natural polymers Minerals

Polysaccharides Attapulgite clays

Cellulose Bentonite

Starch Kieselghur

Dextran Pumice stone

Agar and agarose  

Alginate  

Carrageenan  

Chitin and chitosan  

Proteins  

Collagen  

Gelatin  

Albumin  

Silk  

Carbon materials  

(activated carbon)  

AgarAgarAgar, also called agar-agar, kanten, or Agar, also called agar-agar, kanten, or gelose, is the oldest known gel-forming gelose, is the oldest known gel-forming

polysaccharidepolysaccharide

Discovered in the 17th century in Japan Discovered in the 17th century in Japan and consumed for 200 years, agar is and consumed for 200 years, agar is

extracted from certain marine red algae extracted from certain marine red algae of the class Rhodophyceae mainly from of the class Rhodophyceae mainly from

GelidiumGelidium and and GracilariaGracilaria species, species, growing essentially along the coasts of growing essentially along the coasts of Morocco, Spain, Portugal, Chile, Japan Morocco, Spain, Portugal, Chile, Japan

and Koreaand Korea

OOrigin of seaweed rigin of seaweed extracts — general extracts — general

classificationclassification

OOrigin of seaweed rigin of seaweed extracts —general extracts —general classificationclassification

a Species of economic a Species of economic significancesignificance

b Contains only component b Contains only component mentionedmentioned

c Contains predominantly c Contains predominantly underlined componentunderlined component

AgarAgarKoch and Petri in 1882 Koch and Petri in 1882 - - medium in which to grow bacteria medium in which to grow bacteria no better solidifying agent in microbiological media has been foundno better solidifying agent in microbiological media has been found

microbiological, biotechnological, and public health laboratories, and microbiological, biotechnological, and public health laboratories, and anan important colloid in other industriesimportant colloid in other industries

permitted gelling, stabilizing, and thickening agent for food permitted gelling, stabilizing, and thickening agent for food applications, authorized in all countries without limitations of daily applications, authorized in all countries without limitations of daily intake intake

(confectionery, bakery, pastry, beverage, sauces, wines, spreads, (confectionery, bakery, pastry, beverage, sauces, wines, spreads, spices and condiments, meats and fishes, dairy, jams, etc.)spices and condiments, meats and fishes, dairy, jams, etc.)

Apart from its ability to gelify aqueous solutions and produce gel Apart from its ability to gelify aqueous solutions and produce gel without the support of other agents, agar can also be used as a without the support of other agents, agar can also be used as a safe source of dietary fiber since it is not digestible by the human safe source of dietary fiber since it is not digestible by the human body.body.

AgarAgar

   

Flow sheet of Flow sheet of traditional agar traditional agar extractionextraction

ExtractionExtraction

PurificationPurification

DehydratationDehydratation

Structure of agarStructure of agaragaroseagarose

1,4-linked 3,6-anhydro-1,4-linked 3,6-anhydro- aa-l-galactose alternating -l-galactose alternating with 1,3-linked with 1,3-linked bb-d--d-galactose galactose

AgaropectinAgaropectin repeating unit as repeating unit as agarose, some of the agarose, some of the 3,6-anhydro-l-galactose 3,6-anhydro-l-galactose residues can be residues can be replaced with l-replaced with l-galactose sulfate galactose sulfate residues and the d-residues and the d-galactose residues are galactose residues are partially replaced with partially replaced with the pyruvic acid acetal the pyruvic acid acetal 4,6-4,6-OO-(1--(1-carboxyethylidene)-d-carboxyethylidene)-d-galactosegalactose

AgarosaAgarosa

Processing to remove SOProcessing to remove SO3 3 NaBHNaBH4/4/-OH-OH

Macropourus, hydrophylicMacropourus, hydrophylic

Comercialy availabilityComercialy availability

Chemically stableChemically stable

Low non-specific bindingLow non-specific binding

Resistent to MOResistent to MO

AgarosaAgarosa

AgarosaAgarosa

Zlepšení mechanických vlastnostíZlepšení mechanických vlastností

Prokřížení např. epichlorhydrinemProkřížení např. epichlorhydrinem

Gelling mechanism Gelling mechanism

Hysteresis of 1.5 % agar gelsHysteresis of 1.5 % agar gels

Three equatorial hydrogen atoms of the 3,6-Three equatorial hydrogen atoms of the 3,6-anhydro-anhydro- a a-l-galactose residue are responsible -l-galactose residue are responsible for constraining the molecule so as to form a for constraining the molecule so as to form a helix with a threefold screw axishelix with a threefold screw axis

Gel formation mechanism in aqueous agar solutions

Quick Soluble AgarQuick Soluble Agar

Comparison of production processes of traditional agar and QSA

Patent manufacturing process without any chemical or genetic modifications