Cyclodextrin Glycosyltransferase Research Articles

Purification and Some Properties of Cyclodextrin Glycosyltransferase from a Strain ofBacillusSpecies

An ultracentrifugally homogeneous preparation of the cyclodextrin glycosyltransferase was obtained from the culture broth of a strain of Bacillus species (No. 5 strain) by a purification methods including adsorption on corn starch, ammonium sulfate precipitation, chromatography on DEAE-cellulose column, and gel-filtration on Sephadex G–100. This enzyme preparation (No, 5 enzyme) was separated into two fractions in disc-electrophoresis and Ampholine-electrophoresis (Fr. 1: pI 6.07 and Fr. 2: pI 6.80) which had the ability to form cyclodextrin. These two fractions were similar to each other in the enzymic properties except the isoelectric point. These enzymes differ from the amylase from Bacillus macerans (BMA) in the enzymic properties. Furthermore, No. 5 enzymes differ from BMA in the action on starch. The authors consider that No. 5 enzymes mainly produce the cycloheptaamylose from starch and on the contrary, BMA mainly produces the cyclohexaamylose from starch.

Studies on cyclodextrin glycosyltransferase. II. Action of cyclodextrin glycosyltransferase from Bacillus megaterium strain No. 5 on starch.

The amounts of the cyclodextrins G6, G7 and G8 produced by the action of the enzyme from Bacillus megaterium (No. 5 enzyme) and Bacillus macerans amylase (BMA) on starch-14C (U) were determined by the calculation of radioactivity. Both fractions of No. 5 enzyme produced the cyclodextrin G6, G7 and G8 in the proportion of 1: 2.4: 1. On the other hand, BMA produced the cyclodextrin G6, G7 and G8 in the proportion of 2.7: 1:1. The cyclodextrin G6 and G8 which are smaller parts of the reaction products by both fractions of No. 5 enzyme were found to be produced directly from starch, not from the redecomposition of cyclodextrin G7. The ratio of the cyclodextrin G6, G7 and G8 were almost constant, regardless of the pH range of the reaction system.By using the maltooligosaccharides terminated at the reducing end by radioactive glucose, the action of both fractions of No. 5 enzyme and BMA on the maltooligosaccharides were compared with each other. The results showed that both fractions of No. 5 enzyme acted on ol...

Purification and Some Properties of Cyclodextrin Glycosyltransferase from a Strain of Bacillus Species

An ultracentrifugally homogeneous preparation of the cyclodextrin glycosyltransferase was obtained from the culture broth of a strain of Bacillus species (No. 5 strain) by a purification methods including adsorption on corn starch, ammonium sulfate precipitation, chromatography on DEAE-cellulose column, and gel-filtration on Sephadex G–100. This enzyme preparation (No, 5 enzyme) was separated into two fractions in disc-electrophoresis and Ampholine-electrophoresis (Fr. 1: pI 6.07 and Fr. 2: pI 6.80) which had the ability to form cyclodextrin. These two fractions were similar to each other in the enzymic properties except the isoelectric point. These enzymes differ from the amylase from Bacillus macerans (BMA) in the enzymic properties. Furthermore, No. 5 enzymes differ from BMA in the action on starch. The authors consider that No. 5 enzymes mainly produce the cycloheptaamylose from starch and on the contrary, BMA mainly produces the cyclohexaamylose from starch.

Production of cyclodextrin glycosyltransferase by batch and chemostat culture of Bacillus macerans in chemically defined medium

AbstractA chemically defined medium for cultivation of Bacillus macerans is reported. Growth rates and cyclodextrin glycosyltransferase (CGT) titres obtained were similar to those obtained using media containing potato extract. The proteins in supernatant fluids from a batch culture were separated by disc electrophoresis and results obtained showed that CGT was produced after growth ceased, in agreement with results of activity measurements. The maximum growth rate in the chemostat considerably exceeded that in batch culture; this anomalous effect is unexplained. High CGT titres were produced at low dilution rates (0.03 to 0.05 h−1) but residual starch was present at higher dilution rates and CGT synthesis was repressed. Enzyme titres obtained in chemostat cultures at D = 0.03 h−1 using defined medium containing 13 g starch/1 were 2.75 times greater than the maximum obtainable by batch cultivation and about 20 times greater than those reported by other workers using medium containing diced potato and CaCO3. A two‐stage chemostat cultivation was performed using dilution rates of 0.1 h−1 and 0.033 h−1 in the first and second stages, respectively. The CGT activity in the second stage increased by 57 per cent when a maintenance feed of starch was supplied at 0.08 g g−1 dry biomass h−1. Only negligible CGT titres were obtained when a dilution rate of 0.5 h−1 was used in the first stage. For reasons not understood, DM medium would not support biomass yields greater than 5 g 1−1. This limitation was not due to production of an inhibitor, or to deficiency of N, Fe, Zn, Mn, thiamine or biotin.

Production of cyclodextrin glycosyltransferase by Bacillus macerans in batch cultures

AbstractSatisfactory growth of Bacillus macerans and high activities of cyclodextrin glycosyltransferase (CGT) were obtained in media containing potato extract. The appearance of CGT in culture supernatant fluids during the stationary phase of growth was not due to autolysis, since intracellular levels of the enzyme were very low throughout the cultivation. When cell growth was limited by the starch concentration in the medium, CGT activity remained low during the logarithmic phase of growth but high activities appeared suddenly in the supernatant fluid when growth ceased. Negligible activities were produced by cultures growing in a nitrogen‐deficient medium in which starch was present in excess. CGT therefore appears to be a true extracellular enzyme, produced in response to exhaustion of the starch from the medium. A simple fractionaltion procedure was developed for concentrating the CGT from culture supernatant fluids. This enzyme preparation was used to convert 20% (w/v) starch solutions to cyclodextrins in 24 h and the cyclodextrins recovered were equivalent to 55% of the original starch.