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Imobilizované biologické systémy

Immobilized
Biocatalysts








Doc. Ing. Ji
ří SAJDOK,CSc.

1.Introduction


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



P. B. Poulsen, Enzyme Microb. Technol.
5

(1983) 304



307

Historical Background:


( 1823 vinegar production, sludge, attachment to equipment )

50s


60s : immobili
z
ation of enzymes

( 1916 Nelson


Griffin: invertase ads.on charcoal

1948 Sunmer: jack bean urease)

1950


1970: intensive investigations on immobilized enzymes and
other proteins


( e.g.antigens
-
> affinity chromatography
)

1969


first industrial appt.of immobilized enzyme

Optical resolution of DL aminoacids with immobilized amino acylase
( Chibata et al. )

Since 1960 investigations on immobilized cells

Industrial applications of immobilized microbial cells:

1973

L


aspartic acid
-
Escherichia coli (aspartase )

1974 L
-

malic acid


Brevibacterium ammoniagenes

( fumarase)

1982

L


alanin


Pseudomonas dacunhae ( L
-
aspartate


-
decarboxylase )

Introduction


Biocatalysts dissolved in aqueous buffer
solutions


soluble or native

enzymes, cells, cell parts, or
organelles.


I
mmobilized, fixed, or insolubilized

enzymes,
cells, etc., denote biocatalysts that are bound to
a support.


carrier, support
, or
matrix


cross
-
linking agent, bifunctional agent
, or
carrier
activator
.

Membr
ánové bílkoviny

2.2.

Methods of Enzyme Immobilization


1.
modified into a water
-
insoluble form,


2.

retained by an ultrafiltration membrane inside a
reactor, or


3.
bound to another macromolecule to restrict their
mobility.

Imobilization Techniques


Figure 1.

Classification of enzyme immobilization
methods




Encapsulation of Enzyme

2.2.1.

Carriers for Enzyme Immobilization



1.

Large surface area and high permeability

2. Sufficient functional groups for enzyme attachment
under

nondenaturing conditions

3. Hydrophilic character

4. Water insolubility

5. Chemical and thermal stability

6. Mechanical strength

7. High rigidity and suitable particle form

8. Resistance to microbial attack

9. Regenerability

10. Toxicological safety

11. Low or justifiable price



Table

2. Chemical classification of matrixes used 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)



Agar

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


Discovered in the 17th century in Japan and
consumed for 200 years, agar is extracted
from certain marine red algae of the class
Rhodophyceae mainly from
Gelidium

and
Gracilaria

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

O
rigin of seaweed extracts



general classification

O
rigin of seaweed extracts


general classification


a

Species of economic
significance


b

Contains only component
mentioned

c

Contains predominantly
underlined component


Agar

Koch and Petri in 1882
-

medium in which to grow bacteria

no better solidifying agent in microbiological media has been found


microbiological, biotechnological, and public health laboratories, and an

important colloid in other industries


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

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


Apart from its ability to gelify aqueous solutions and produce gel 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 body.


Agar




Flow sheet of traditional
agar extraction


Extraction

Purification

Dehydratation




Structure of agar


agarose




1,4
-
linked 3,6
-
anhydro
-

a
-
l
-
galactose alternating
with 1,3
-
linked
b
-
d
-
galactose



Agaropectin



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


Agarosa

Processing to remove SO
3
NaBH
4/
-
OH


Macropourus, hydrophylic

Comercialy availability

Chemically stable

Low non
-
specific binding

Resistent to MO

Agarosa

Agarosa

Zlepšení mechanických vlastností


Prokřížení např. epichlorhydrinem

Gelling mechanism


Hysteresis of 1.5

% agar gels



Three equatorial hydrogen
atoms of the 3,6
-
anhydro
-

a
-
l
-
galactose residue are
responsible for constraining
the molecule so as to form a
helix with a threefold screw
axis

Gel formation mechanism in
aqueous agar solutions



Quick Soluble Agar

Comparison of
production
processes of
traditional agar and
QSA

Patent manufacturing process without any chemical or genetic
modifications