192
phase; the unreacted proteins, pigments, and further organic and inorganic ingredients could be removed by washing the column with water and diluted sodium
chloride solution thereafter; the column effluent contained unreacted hydrolytic
enzyme constituents, except for cyclodextrin glucosyltransferase active
ingredient.
Step 3: Elution and freeze-drying of purified product – the cyclodextrin glucosyltransferase enzyme bound to the column was eluted by 1% alpha-cyclodextrincontaining buffer; the resulting column effluent was a concentrated solution
having high (2 Kitahata units/100 mg protein) cyclodextrin glucosyltransferase
activity. This alpha-cyclodextrin-containing enzyme solution was diluted with
buffer to 10
4
Kitahata units/mL, filtered through a 0.2 μm pore size membrane
filter, and dispensed into sterile glass lyophilization vials to obtain a uniform filling quantity of 10
4
Kitahata units/vial via freeze-drying; finally, the vials were
closed under vacuum and labeled.
At the time of these technology developments, Hungary belonged to the Soviet
block, and any sophisticated equipment, for example, automatic chromatography
instrument (Pharmacia) or any HPLC analytical system, was embargoed. The spare
parts of the chromatographs needed for automation of affinity chromatography and
protein purification were purchased in the Western countries by the researchers as
tourists and personally imported, violating the embargo. Finally, a chromatograph
was constructed by Chinoin’s researchers, who developed also an elution control
program to obtain the equipment useful for attention-free, semi-automatic enzyme
production.
For pilot plant scale-up, affinity chromatography parameters were optimized:
elution rate, temperature of binding, selection of buffer (acetate or phosphate), pH,
volume of buffer, volume of column packing, and enzyme load. Based on these
experiments, the optimized operation parameters were established, and the documentation of instructions for laboratory-scale production submitted in 1985. The
column packing had the capacity to bind as much as 500 mg enzyme related to 1 mL
gel bed volume from 100 to 1000 mL culture broth in one cycle (Szejtli et al. 1980a).
The industrial cyclodextrin glucosyltransferase manufacturing started at Chinoin
Fermentation Plant in 1981, with the selected best mutant strain of B. macerans.
Both inoculation and fermentation culture media consisted of oat flake and corn
steep liquor as appropriate carbon sources. The essential nitrogen was applied in
ammonium sulfate form. To ensure an optimal enzyme-stabilizing environment, pH
of the oat-flake-containing fermentation broth was maintained to near neutral, with
addition of potassium bisphosphate and calcium carbonate. The main fermentation
was carried out at 37 °C, for 2 days. The fermentation liquor was filtered on a rotary
vacuum drum filter and concentrated to its 1/10 volume in vacuum evaporators. The
macromolecular enzyme fraction of resulting concentrate was salted out with
ammonium sulfate, filtered, and dried. The industrial process technology, suitable
for enzyme production in 10–100 mega Kitahata unit range in 3 m
3
batch volumes,
was realized in 1982. An amount of 150 mega Kitahata unit crude enzyme powder
É. Fenyvesi et al.
phase; the unreacted proteins, pigments, and further organic and inorganic ingredients could be removed by washing the column with water and diluted sodium
chloride solution thereafter; the column effluent contained unreacted hydrolytic
enzyme constituents, except for cyclodextrin glucosyltransferase active
ingredient.
Step 3: Elution and freeze-drying of purified product – the cyclodextrin glucosyltransferase enzyme bound to the column was eluted by 1% alpha-cyclodextrincontaining buffer; the resulting column effluent was a concentrated solution
having high (2 Kitahata units/100 mg protein) cyclodextrin glucosyltransferase
activity. This alpha-cyclodextrin-containing enzyme solution was diluted with
buffer to 10
4
Kitahata units/mL, filtered through a 0.2 μm pore size membrane
filter, and dispensed into sterile glass lyophilization vials to obtain a uniform filling quantity of 10
4
Kitahata units/vial via freeze-drying; finally, the vials were
closed under vacuum and labeled.
At the time of these technology developments, Hungary belonged to the Soviet
block, and any sophisticated equipment, for example, automatic chromatography
instrument (Pharmacia) or any HPLC analytical system, was embargoed. The spare
parts of the chromatographs needed for automation of affinity chromatography and
protein purification were purchased in the Western countries by the researchers as
tourists and personally imported, violating the embargo. Finally, a chromatograph
was constructed by Chinoin’s researchers, who developed also an elution control
program to obtain the equipment useful for attention-free, semi-automatic enzyme
production.
For pilot plant scale-up, affinity chromatography parameters were optimized:
elution rate, temperature of binding, selection of buffer (acetate or phosphate), pH,
volume of buffer, volume of column packing, and enzyme load. Based on these
experiments, the optimized operation parameters were established, and the documentation of instructions for laboratory-scale production submitted in 1985. The
column packing had the capacity to bind as much as 500 mg enzyme related to 1 mL
gel bed volume from 100 to 1000 mL culture broth in one cycle (Szejtli et al. 1980a).
The industrial cyclodextrin glucosyltransferase manufacturing started at Chinoin
Fermentation Plant in 1981, with the selected best mutant strain of B. macerans.
Both inoculation and fermentation culture media consisted of oat flake and corn
steep liquor as appropriate carbon sources. The essential nitrogen was applied in
ammonium sulfate form. To ensure an optimal enzyme-stabilizing environment, pH
of the oat-flake-containing fermentation broth was maintained to near neutral, with
addition of potassium bisphosphate and calcium carbonate. The main fermentation
was carried out at 37 °C, for 2 days. The fermentation liquor was filtered on a rotary
vacuum drum filter and concentrated to its 1/10 volume in vacuum evaporators. The
macromolecular enzyme fraction of resulting concentrate was salted out with
ammonium sulfate, filtered, and dried. The industrial process technology, suitable
for enzyme production in 10–100 mega Kitahata unit range in 3 m
3
batch volumes,
was realized in 1982. An amount of 150 mega Kitahata unit crude enzyme powder
É. Fenyvesi et al.