Fundamental Study and Application of Cyclodextrins. (1993)

1. Cyclodextrin-producing enzyme produced by Bacillus macerans (CGTase, macerans enzyme) Crude macerans enzyme preparation was purified and the enzyme was crystallized. Elucidation of the CDs' forming path : By use of various kinds of substrates, well-known phenomenon of p-CD accumulation at final stage of the reaction was explained from time course data of CD formation and the rate of coupling. Elucidation of cyclizing reaction : Cyclizing reaction proceeds from non-reducing ends of the substrates cyclizing 6 glucose units to form α-CD and the remaining fragments. The rate of cyclization was high on substrates having more than 8 glucose units, and accelerated by the addition of helices forming reagents ; moreover, it was found that α-and β-CDs were selectively formed by the addition of 65 and 76 helices forming reagents. Branched CDs were produced by forming helical structure with surface active reagents from substrates having branches, and in the same manner, 1 or 2 hydroxyethyl substituted CDs were produced by the action of macerans enzyme on the helix formed hydroxyethyl starch.Elucidation of coupling action : Glucosyl-α-CD and 14C-labeled glucose were reacted with macerans enzyme, and the structure of the formed branched oligosaccharides was analyzed. The smallest non-radioactive branched oligosaccharide was B4(63-α-glucosylmaltotriose), and at the initial stage of the reaction, radioactive(*) Bs(mainly 66-α-glucosylmaltoheptaose)was formed to be gradually degraded to B7* (mainly 65-α-glucosylmaltohexaose), B6 (mainly 64-α-glucosylmaltopentaose) and B5 (64-α-glucosylmaltotetraose) From the results described above, the author proposed an enzyme model of active site.2. Production of CDs Methods for the production of α-CD, α-CD containing starch syrup, γ-CD were developed, and a large ringed CDs preparation method, by which δ-θ-CDs were produced, was also developed. δ-θ-CDs preparation contained a fair amount of intra branched CDs which have α-1, 6 linkage in the CDs themselves. 3. Production and the properties of branched CDs A method for the production of branched CDs which have branch(es) longer than glucosyl unit was developed, and maltosyl-CDs (G2-CDs) were effectively produced. The number of branches attached to CD ring depends on the size of CD, and 2 and 3 branches were easily attached to a-CD and p-CD, respectively. Panose was also attached to CDs to form panosyl-CDs. Branching moiety of the CDs were degraded by the action of glucoamylase to glucosyl-CDs (G1-CDs), and maltose was again attached to G1-CDs to form doubly branched G1-, G2-CDs by the action of pullulanase. Properties of branched CDs : Solubility was higher than that of original CDs, and degree of the solubility was different depending on the variety of CD, the variety of branch and the number of branches. As for the action of starch-degrading enzymes on the branches, it was found that the action of glucoamylase was considerably different depending on the variety of branched CD. Amylo-1, 6-glucosidase acted on AD and AC type of diglucosyl-α-CD((G1)2-α-CD), but not on AB type. Aspergillus oryzae α-amylase degraded G1-CD, but the rate of degradation was depressed to less than 1/10 in comparison with that of original CD, and multiply branched glucosyl-CDs were scarcely degradable. Pullulanase (branching enzyme) scarcely degrades maltosyl branches of AB type of branched CDs, but acted on the branches of AC and AD types. AD type of (G1)2-α-CD was completely resistant to the action of macerans enzyme ; AB and AC types were reacted in the presence of acceptors such as glucose and maltose. By the combination of the results described above, systems for the separation and produc-tion of AB, AC and AD types of (G1)2-α-CD were established.