194
László 1988). Because no significant enhancement in specific activity and catalytic
properties was observed after purification, the extra pure enzyme was not produced
for commerce.
Immobilization of the Enzyme
As a final step of developments, the cyclodextrin glucosyltransferase enzyme was
immobilized on an appropriate carrier to reduce the enzyme consumption and simplify the downstream procedure. Using immobilized enzyme, the same enzyme
could be utilized in several conversion cycles either in batch-wise or continuously
operating technologies. To implement above development concept, experiments
started in 1980 using a commercially available carrier, Eupergit-C. Common coupling studies were performed at Reanal Fine Chemicals Co. (Budapest, Hungary)
by researchers with high experience in enzyme immobilization. The cyclodextrin
glucosyltransferase enzyme was reacted with carbodiimide-activated polysaccharidebased carrier. Another immobilizing procedure was invented using carboxyl functional group-containing acrylic polymer backbone (Boross et al. 1986; patent
application was submitted in 1982).
In contrast to activity of enzyme immobilized on cellulose, which was only
26.5 Kitahata units/g dry weight, the enzyme immobilized on polyacrylamide
reached the 230–450 Kitahata units/g. Immobilization shifted the pH optimum from
5.9 to 5.5. The temperature optimum range was rather broad, between 40 and 60 °C,
whereas the soluble enzyme had a relatively sharp activity peak around 60 °C. Halflife was improved considerably at all pH values and temperatures. For example, the
half-life of the soluble enzyme at 70 °C (optimum pH 5.9) is only 1.0 min, while
that of the immobilized enzyme was 24.3 min (optimum pH 5.5) (Ivony et al. 1983a,
b). Degree of immobilization was strongly dependent on the structure of disubstituted carbodiimides (Szajáni et al. 1991).
The continuous mass transfer kinetic of semi-permeable membranes made from
immobilized cyclodextrin glucosyltransferase enzyme-containing acrylic polymers
was thoroughly studied by Professors E. Nagy and Cs. Sisak, researchers at
Technical Chemistry Research Institute, Veszprém (MÜKKI, Hungary). Compared
to dissolved enzyme, enhanced stability and reusability were an important advantage, permitting a possibility for development of continuous cyclodextrin manufacturing procedure, instead of batch-wise processes (Sisak et al. 1996). A stirred tank
reactor system, operated with immobilized cyclodextrin glucosyltransferase enzyme
beads, was developed. This reaction vessel was coupled to a hollow fiber ultrafiltration membrane to separate cyclodextrin from the low molecular reaction products.
This laboratory system worked in recycling mode developed for months. By means
of such arrangement, starch substrate inhibition and the competitive blocking effect
of glucose and short-chain oligosaccharides on the cyclization reaction diminished.
Moreover, the time-dependent decomposition of parent cyclodextrins could be
decreased in this system. Starch degradation and formation of glycosylic cyclodextrin coproducts were also repressed. Due to the change of regime in
É. Fenyvesi et al.
László 1988). Because no significant enhancement in specific activity and catalytic
properties was observed after purification, the extra pure enzyme was not produced
for commerce.
Immobilization of the Enzyme
As a final step of developments, the cyclodextrin glucosyltransferase enzyme was
immobilized on an appropriate carrier to reduce the enzyme consumption and simplify the downstream procedure. Using immobilized enzyme, the same enzyme
could be utilized in several conversion cycles either in batch-wise or continuously
operating technologies. To implement above development concept, experiments
started in 1980 using a commercially available carrier, Eupergit-C. Common coupling studies were performed at Reanal Fine Chemicals Co. (Budapest, Hungary)
by researchers with high experience in enzyme immobilization. The cyclodextrin
glucosyltransferase enzyme was reacted with carbodiimide-activated polysaccharidebased carrier. Another immobilizing procedure was invented using carboxyl functional group-containing acrylic polymer backbone (Boross et al. 1986; patent
application was submitted in 1982).
In contrast to activity of enzyme immobilized on cellulose, which was only
26.5 Kitahata units/g dry weight, the enzyme immobilized on polyacrylamide
reached the 230–450 Kitahata units/g. Immobilization shifted the pH optimum from
5.9 to 5.5. The temperature optimum range was rather broad, between 40 and 60 °C,
whereas the soluble enzyme had a relatively sharp activity peak around 60 °C. Halflife was improved considerably at all pH values and temperatures. For example, the
half-life of the soluble enzyme at 70 °C (optimum pH 5.9) is only 1.0 min, while
that of the immobilized enzyme was 24.3 min (optimum pH 5.5) (Ivony et al. 1983a,
b). Degree of immobilization was strongly dependent on the structure of disubstituted carbodiimides (Szajáni et al. 1991).
The continuous mass transfer kinetic of semi-permeable membranes made from
immobilized cyclodextrin glucosyltransferase enzyme-containing acrylic polymers
was thoroughly studied by Professors E. Nagy and Cs. Sisak, researchers at
Technical Chemistry Research Institute, Veszprém (MÜKKI, Hungary). Compared
to dissolved enzyme, enhanced stability and reusability were an important advantage, permitting a possibility for development of continuous cyclodextrin manufacturing procedure, instead of batch-wise processes (Sisak et al. 1996). A stirred tank
reactor system, operated with immobilized cyclodextrin glucosyltransferase enzyme
beads, was developed. This reaction vessel was coupled to a hollow fiber ultrafiltration membrane to separate cyclodextrin from the low molecular reaction products.
This laboratory system worked in recycling mode developed for months. By means
of such arrangement, starch substrate inhibition and the competitive blocking effect
of glucose and short-chain oligosaccharides on the cyclization reaction diminished.
Moreover, the time-dependent decomposition of parent cyclodextrins could be
decreased in this system. Starch degradation and formation of glycosylic cyclodextrin coproducts were also repressed. Due to the change of regime in
É. Fenyvesi et al.