190
a fractional salting out step into the downstream enzyme processing. This fractional
precipitation was carried out using ammonium sulfate. In this way, the alphaamylase contamination of final product could be reduced. A combined purification
procedure involving gel filtration and isoelectric focusing was applied to get a fine
enzyme preparation and for physicochemical characterization of our new B. macerans mutant enzyme (Seres 1984). As a result of these investigations, the molecular
weight was found 79 kDa, and two isomeric forms were proved (Seres 1986 and
Table 4.2).
In the early 1980s, a new and innovative enzyme purification method was worked
out in cooperation of the Chinoin researchers (Seres’s team) with research members
(Professors E. László, Á. Hoschke, and B. Bánky) of Agrochemical Department of
Budapest University of Technology. This ingenious chromatography procedure was
based on the specific key-lock enzyme substrate affinity property. Alpha-cyclodextrin
was immobilized on an agarose gel matrix at first, to get an immobilized affinity
chromatography stationary phase. When passing the crude cyclodextrin glucosyltransferase concentrate through this alpha-cyclodextrin-agarose gel column, cyclodextrin glucosyltransferase enzyme was selectively affixed to its covalently bound
substrate. After completely washing off the unbound constituents, the enzyme could
be eluted by concentrated solution of alpha-cyclodextrin substrate (Fig. 4.6). The
technology was scaled up to 250-cm
3
volume affinity column, which allowed to
load 1.5–2.0 mega-Kitahata unit crude enzyme concentrate in a single cycle,
Table 4.2 Specification of cyclodextrin glucosyltransferase
Name
Cyclodextrin glucosyltransferase alpha-(1–4)-glucan-4glucosyltransferase E.C.2.4.1.19
Appearance
White powder
Gross activity
10.000 Kitahata units/vial
Ingredients
Protein content minimum 40% of dry material (Lowry method
1951)
The preparation also contains alpha-cyclodextrin and
maltooligosaccharides as stabilizers and sodium phosphate in order
to maintain the pH of the dissolved material at 7.0
Stability
At least 1 year when stored at 4 °C
The solid preparation can lose <5% of its activity within 1 month
when stored at 25 °C
Solubility
The vial content is fully soluble in 1.0–3.0 mL distilled water
Molecular weight
79 kDa
Michaelis-Menten
constant for soluble
amylose as substrate
5.7 mg/mL (Stavn and Granum 1979)
3.9 mg/mL (De Pinto and Campbell 1968)
Isoelectric point (pI)
Two bands by agarose gel isoelectric focusing: pI = 4.45 and
pI = 4.65
pH optimum
5.9
Operating pH interval
5.0 < pH < 8.0
Operating temperature
optimum
60 °C
É. Fenyvesi et al.
a fractional salting out step into the downstream enzyme processing. This fractional
precipitation was carried out using ammonium sulfate. In this way, the alphaamylase contamination of final product could be reduced. A combined purification
procedure involving gel filtration and isoelectric focusing was applied to get a fine
enzyme preparation and for physicochemical characterization of our new B. macerans mutant enzyme (Seres 1984). As a result of these investigations, the molecular
weight was found 79 kDa, and two isomeric forms were proved (Seres 1986 and
Table 4.2).
In the early 1980s, a new and innovative enzyme purification method was worked
out in cooperation of the Chinoin researchers (Seres’s team) with research members
(Professors E. László, Á. Hoschke, and B. Bánky) of Agrochemical Department of
Budapest University of Technology. This ingenious chromatography procedure was
based on the specific key-lock enzyme substrate affinity property. Alpha-cyclodextrin
was immobilized on an agarose gel matrix at first, to get an immobilized affinity
chromatography stationary phase. When passing the crude cyclodextrin glucosyltransferase concentrate through this alpha-cyclodextrin-agarose gel column, cyclodextrin glucosyltransferase enzyme was selectively affixed to its covalently bound
substrate. After completely washing off the unbound constituents, the enzyme could
be eluted by concentrated solution of alpha-cyclodextrin substrate (Fig. 4.6). The
technology was scaled up to 250-cm
3
volume affinity column, which allowed to
load 1.5–2.0 mega-Kitahata unit crude enzyme concentrate in a single cycle,
Table 4.2 Specification of cyclodextrin glucosyltransferase
Name
Cyclodextrin glucosyltransferase alpha-(1–4)-glucan-4glucosyltransferase E.C.2.4.1.19
Appearance
White powder
Gross activity
10.000 Kitahata units/vial
Ingredients
Protein content minimum 40% of dry material (Lowry method
1951)
The preparation also contains alpha-cyclodextrin and
maltooligosaccharides as stabilizers and sodium phosphate in order
to maintain the pH of the dissolved material at 7.0
Stability
At least 1 year when stored at 4 °C
The solid preparation can lose <5% of its activity within 1 month
when stored at 25 °C
Solubility
The vial content is fully soluble in 1.0–3.0 mL distilled water
Molecular weight
79 kDa
Michaelis-Menten
constant for soluble
amylose as substrate
5.7 mg/mL (Stavn and Granum 1979)
3.9 mg/mL (De Pinto and Campbell 1968)
Isoelectric point (pI)
Two bands by agarose gel isoelectric focusing: pI = 4.45 and
pI = 4.65
pH optimum
5.9
Operating pH interval
5.0 < pH < 8.0
Operating temperature
optimum
60 °C
É. Fenyvesi et al.