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beta-cyclodextrin producing ability of the enzyme. Additionally, other carbohydrase
enzymes can disturb the measurement.
In the later conversion stage, cyclodextrin glucosyltransferase promotes decylization and transferring glucosyl moieties to the initially produced alphacyclodextrin, resulting in the formation of higher degree of polymerization beta- and
gamma-cyclodextrins in the conversion mixture. The methods of Fuwa (1954) and
Kitahata et al. (1974) for determination and screening of cyclodextrin glucosyltransferase activities characterized this second step. The reaction mixture containing soluble starch and the enzyme in a pH 5.5 buffer was incubated at 40 °C for
10 min. Thereafter, aliquot of this mixture was added to iodine in potassium iodide
solution and diluted with distilled water. As zero control, aliquot of this reaction
mixture was also added to the iodine solution before the incubation. Absorbance
was measured at 660 nm. According to Fuwa (1954), the enzyme activity is defined
as the amount of enzyme that produced a difference in absorbance of 1.0 unit per
min under the described conditions. According to the later published and generally
accepted Kitahata method, one Kitahata unit corresponds to 1% decrease in light
absorption in 1 min (Kitahata et al. 1974). Because this assay refers to time-lapsed
secondary stage of enzyme starch conversion, it fulfills the development requirements for not only beta-cyclodextrin but gamma-cyclodextrin formation, too. To
match the early-published data, it was postulated that one Tilden-Hudson unit activity corresponds to approximately 3 Kitahata units.
To detect parallel both recycling and glucosyl coupling enzyme activity, a method
utilized in presence of alpha-methyl glucoside acceptor was also developed. The
reaction product of hydrolysis by hog pancreatic alpha-amylase (maltose) can easily
be measured by any conventional reducing sugar determination method. The main
advantage of this method is that the cyclodextrin glucosyltransferase enzyme action
is not disturbed by the presence of common hydrolytic enzymes, because these
enzymes are unable to split alpha-cyclodextrin substrate (Thoma and Stewart 1965).
A similar rapid method for determination of cyclodextrin glucosyltransferase
activity was elaborated in the Chinoin Biotechnology Development Laboratory by
further development of Thoma’s activity assay (Péterfi and Seres 1982). This
method was based also on general glucosyltransferase mechanism: the forming
enzyme splits alpha-cyclodextrin substrate, and –in the presence of hog pancreatic
amylase – the resulting linear fragment is coupled to the C-1 terminal of pnitrophenyl- beta-D-glucoside acceptor. The resulting mixture of saccharides can
easily be detected as reducing sugars by any conventional method measuring reducing ability, by dinitrosalicylic acid assay in this case.
A reliable cyclodextrin glucosyltransferase activity measuring method, which is
based on direct determination of nascent cyclodextrins by means of highperformance liquid chromatography, applies a polymer-based reverse phase column
equipped with a refractive index detector. The advantage of this assay is that enzyme
activity is expressed in SI system in units of micromoles cyclodextrin/g substrate, in
accordance with later regulations of Enzyme Commission (Sato 1985).
Because of the simplicity of Kitahata method, and due to its wide representation
in overall process technology documentation, the proposed more specific and
É. Fenyvesi et al.
beta-cyclodextrin producing ability of the enzyme. Additionally, other carbohydrase
enzymes can disturb the measurement.
In the later conversion stage, cyclodextrin glucosyltransferase promotes decylization and transferring glucosyl moieties to the initially produced alphacyclodextrin, resulting in the formation of higher degree of polymerization beta- and
gamma-cyclodextrins in the conversion mixture. The methods of Fuwa (1954) and
Kitahata et al. (1974) for determination and screening of cyclodextrin glucosyltransferase activities characterized this second step. The reaction mixture containing soluble starch and the enzyme in a pH 5.5 buffer was incubated at 40 °C for
10 min. Thereafter, aliquot of this mixture was added to iodine in potassium iodide
solution and diluted with distilled water. As zero control, aliquot of this reaction
mixture was also added to the iodine solution before the incubation. Absorbance
was measured at 660 nm. According to Fuwa (1954), the enzyme activity is defined
as the amount of enzyme that produced a difference in absorbance of 1.0 unit per
min under the described conditions. According to the later published and generally
accepted Kitahata method, one Kitahata unit corresponds to 1% decrease in light
absorption in 1 min (Kitahata et al. 1974). Because this assay refers to time-lapsed
secondary stage of enzyme starch conversion, it fulfills the development requirements for not only beta-cyclodextrin but gamma-cyclodextrin formation, too. To
match the early-published data, it was postulated that one Tilden-Hudson unit activity corresponds to approximately 3 Kitahata units.
To detect parallel both recycling and glucosyl coupling enzyme activity, a method
utilized in presence of alpha-methyl glucoside acceptor was also developed. The
reaction product of hydrolysis by hog pancreatic alpha-amylase (maltose) can easily
be measured by any conventional reducing sugar determination method. The main
advantage of this method is that the cyclodextrin glucosyltransferase enzyme action
is not disturbed by the presence of common hydrolytic enzymes, because these
enzymes are unable to split alpha-cyclodextrin substrate (Thoma and Stewart 1965).
A similar rapid method for determination of cyclodextrin glucosyltransferase
activity was elaborated in the Chinoin Biotechnology Development Laboratory by
further development of Thoma’s activity assay (Péterfi and Seres 1982). This
method was based also on general glucosyltransferase mechanism: the forming
enzyme splits alpha-cyclodextrin substrate, and –in the presence of hog pancreatic
amylase – the resulting linear fragment is coupled to the C-1 terminal of pnitrophenyl- beta-D-glucoside acceptor. The resulting mixture of saccharides can
easily be detected as reducing sugars by any conventional method measuring reducing ability, by dinitrosalicylic acid assay in this case.
A reliable cyclodextrin glucosyltransferase activity measuring method, which is
based on direct determination of nascent cyclodextrins by means of highperformance liquid chromatography, applies a polymer-based reverse phase column
equipped with a refractive index detector. The advantage of this assay is that enzyme
activity is expressed in SI system in units of micromoles cyclodextrin/g substrate, in
accordance with later regulations of Enzyme Commission (Sato 1985).
Because of the simplicity of Kitahata method, and due to its wide representation
in overall process technology documentation, the proposed more specific and
É. Fenyvesi et al.