183
not conceivable. There were only a few, including Professor Szejtli, who did not
believe in these statements. He was sure in that times that oligosaccharides, like
cyclodextrins, cannot be toxic and their production should be economical in large
scale. It was clarified later that the toxicity was concluded based on erroneous
experiments using unpurified cyclodextrins.
In the time 1975–1976, cyclodextrins were available in small quantities, mainly
as fine chemicals produced on laboratory scale. According to common procedure, a
cyclodextrin glucosyltransferase enzyme was added to a dilute solution of gelatinized starch, and the enzymatic degradation was allowed to proceed for a sufficient
time. The main drawback of this procedure was that acceptable conversion was
achieved only at low substrate concentration owing to high viscosity of >5% potato
starch solution which limits the enzyme reaction. The viscosity was reduced by
means of hydrolyzing starch to small fragments resulting an average dextrose equivalent (the amount of reducing sugars present in a sugar product, expressed as a
percentage on a dry basis relative to dextrose) of <20. This preliminary procedure
allowed to use 30% substrate concentration resulting in cyclodextrin yields high
enough for scaling up the process to industrial level (Armbruster and Kool 1969).
The pre-hydrolysis performed either by acidic hydrolysis (Armbruster and Mukhtar
1968) or by α-amylase resulted in water-soluble starch fractions with reduced tendency for retrogradation. After the partial hydrolysis, the starch was converted to
cyclodextrins by cyclodextrin glucosyltransferase. Utilizing the outstanding inclusion complex-forming ability of cyclodextrins and the low solubility of resulting
complexes, various apolar precipitating agents, such as toluene, trichloroethylene,
and bromobenzene, were recommended for selective beta-cyclodextrin formation
(Armbruster and Kool 1969; Armbruster 1970).
It was already known that the cyclodextrin yield could be highly enhanced by
applying these complexing agents in the cyclodextrin glucosyltransferase-catalyzed
starch conversions. Cyclodextrin reaction products could be collected from the
enzyme-catalyzed reaction mixture by precipitation, inclusion complex formation,
and crystallization (Table 4.1) (Armbruster and Kool 1969). Figure 4.1 refers to the
general principle of cyclodextrin production. It was also recognized that using an
appropriate complexing agent, the conversion equilibria could be shifted toward the
production of desired cyclodextrin. For instance, long alkyl chain alcohols were
found useful for production of alpha-cyclodextrin as dominant cyclodextrin component (Armbruster and Jacaway 1969) (Table 4.1).
Optimizing the reaction conditions, 100% pure alpha-cyclodextrin or betacyclodextrin was obtained by applying a selective precipitant, 1-decanol or toluene,
respectively (Armbruster and Jacaway 1969). Figure 4.2 shows the Schardinger
β-dextrin (beta-cyclodextrin) marketed as cyclohexane complex and purchased
from Sigma, at the end of the 1970s.
Because of the growing interest for cyclodextrin production, the focus was
directed to isolation and purification of cyclodextrin-producing enzymes. Before
the 1970s, only few bacteria, i.e., Bacillus macerans, Bacillus circulans, and
Bacillus polymyxa, were described as appropriate cyclodextrin glucosyltransferase
enzyme sources. According to the patent of Armbruster and Kool (1969), maximum
4 History of Cyclodextrin Production in Hungary
not conceivable. There were only a few, including Professor Szejtli, who did not
believe in these statements. He was sure in that times that oligosaccharides, like
cyclodextrins, cannot be toxic and their production should be economical in large
scale. It was clarified later that the toxicity was concluded based on erroneous
experiments using unpurified cyclodextrins.
In the time 1975–1976, cyclodextrins were available in small quantities, mainly
as fine chemicals produced on laboratory scale. According to common procedure, a
cyclodextrin glucosyltransferase enzyme was added to a dilute solution of gelatinized starch, and the enzymatic degradation was allowed to proceed for a sufficient
time. The main drawback of this procedure was that acceptable conversion was
achieved only at low substrate concentration owing to high viscosity of >5% potato
starch solution which limits the enzyme reaction. The viscosity was reduced by
means of hydrolyzing starch to small fragments resulting an average dextrose equivalent (the amount of reducing sugars present in a sugar product, expressed as a
percentage on a dry basis relative to dextrose) of <20. This preliminary procedure
allowed to use 30% substrate concentration resulting in cyclodextrin yields high
enough for scaling up the process to industrial level (Armbruster and Kool 1969).
The pre-hydrolysis performed either by acidic hydrolysis (Armbruster and Mukhtar
1968) or by α-amylase resulted in water-soluble starch fractions with reduced tendency for retrogradation. After the partial hydrolysis, the starch was converted to
cyclodextrins by cyclodextrin glucosyltransferase. Utilizing the outstanding inclusion complex-forming ability of cyclodextrins and the low solubility of resulting
complexes, various apolar precipitating agents, such as toluene, trichloroethylene,
and bromobenzene, were recommended for selective beta-cyclodextrin formation
(Armbruster and Kool 1969; Armbruster 1970).
It was already known that the cyclodextrin yield could be highly enhanced by
applying these complexing agents in the cyclodextrin glucosyltransferase-catalyzed
starch conversions. Cyclodextrin reaction products could be collected from the
enzyme-catalyzed reaction mixture by precipitation, inclusion complex formation,
and crystallization (Table 4.1) (Armbruster and Kool 1969). Figure 4.1 refers to the
general principle of cyclodextrin production. It was also recognized that using an
appropriate complexing agent, the conversion equilibria could be shifted toward the
production of desired cyclodextrin. For instance, long alkyl chain alcohols were
found useful for production of alpha-cyclodextrin as dominant cyclodextrin component (Armbruster and Jacaway 1969) (Table 4.1).
Optimizing the reaction conditions, 100% pure alpha-cyclodextrin or betacyclodextrin was obtained by applying a selective precipitant, 1-decanol or toluene,
respectively (Armbruster and Jacaway 1969). Figure 4.2 shows the Schardinger
β-dextrin (beta-cyclodextrin) marketed as cyclohexane complex and purchased
from Sigma, at the end of the 1970s.
Because of the growing interest for cyclodextrin production, the focus was
directed to isolation and purification of cyclodextrin-producing enzymes. Before
the 1970s, only few bacteria, i.e., Bacillus macerans, Bacillus circulans, and
Bacillus polymyxa, were described as appropriate cyclodextrin glucosyltransferase
enzyme sources. According to the patent of Armbruster and Kool (1969), maximum
4 History of Cyclodextrin Production in Hungary