C.Edwin Weill

The use of 6-O-methylamylose as a substrate for amylases

Abstract A type of chemically modified amylose, in which a fraction of the primary hydroxyl groups is specifically methylated, has been prepared by the following sequence of reactions: amylose → 6- O -tritylamylose → 2,3-di- O -benzoyl-6- O -tritylamylose → 2,3-di- O -benzoylamylose → 2,3-di- O -benzoyl-6- O -methylamylose → 6- O -methylamylose. Two such preparations of 6- O -methylamylose, having D.S. 0.2 and D.S. 0.4, were examined as a model substrates for a number of starch-degrading enzymes. Neither was detectably attacked by sweet-potato β-amylase or potato phosphorylase. Some hydrolysis (much greater for the 6- O -methylamylose of D.S. 0.2 than for that of D.S. 0.4) did occur with crystalline α-amylase preparations from hog pancreas and Bacillus subtilis . Relatively extensive hydrolysis was effected by an Aspergillus niger enzyme preparation containing both α-amylase and amyloglucosidase. Products of the action of the pancreatic and Bacillus subtilis amylases included maltose and higher saccharides, but neither d -glucose nor 6- O -methyl- d -glucose was detected. Products obtained with the A. niger enzyme preparation included d -glucose and a saccharide tentatively identified as 4- O -(6- O -methyl-α- d -glucopyranosyl)- d -glucose; 6- O -methyl- d -glucose was not detected. The failure of the exo-enzymes to effect any hydrolysis, and the extent of hydrolysis and the nature of the products formed by the two crystalline α-amylases, demonstrate that hydrolysis is blocked in the vicinity of the methylated primary hydroxyl groups. The results suggest that those limitations on amylase action normally attributed to the bulky (1→6)- d -glucose branch are also imposed by a small group, namely, the methyl group.

Unusual carbohydrate transfer reactions of cyclodextrin transglucosylase

Abstract The cyclodextrin transglucosylase from Bacillus macerans has previously been employed to prepare a series of α-methyl malto-oligosaccharides from Schardinger α-dextrin, with α-methyl glueopyranoside as the acceptor. The same enzyme system has now been employed to effect the transfer of glucose units to the methyl glycosides of d -xylose, 6- o -methyl- d -glucose, and 2-deoxy d -glucose. The methyl glycoside of 3- o -methyl- d -glucose does not act as an acceptor

The inhibition of β-amylase by ascorbic acid. II

Abstract Further evidence is presented to demonstrate that the inhibitory effect of ascorbic acid on β-amylase is due to the formation of an inactive cuprous enzyme. Cuprous ion has been shown to give approximately the same amount of inactivation as cupric ion plus ascorbic acid. Chelating agents which specifically bind copper or cuprous ion have been shown to prevent the inactivation of the enzyme. Determination of sulfhydryl content of the enzyme has indicated that these groups are bound in the inactive enzyme and that the degree of sulfhydryl binding is related to enzyme activity.

Random substitution of amylose

Abstract The production of modified amyloses often involves the p-toluenesulfonylation of the primary hydroxyl group of the d -glucopyranosyl residues, but it was not known whether this esterification is random when ∼20% of the d -glucopyranosyl residues have been substituted. The reaction of p-toluenesulfonyl chloride with amylose in pyridine, in C-methylpyridines, in aqueous 2 m KOH, and in dimethyl sulfoxide-pyridine yielded unsatisfactory products. However, p-toluenesulfonylation of 2,3-di-O-acetylamylose in pyridine yielded products that were converted into 2,3-di-O-acetyl-6-deoxy-6-iodoamylose, and thence into 6-deoxyamylose, a suitable substrate for the amylase to be used in the determination of random substitution. The hydrolysis products formed by the action of the crystalline, liquefying amylase of Bacillus subtilis were separated and analyzed. When a summation of the minimum number of separated, modified D -glucopyranosyl residues was compared to a computer-based calculation of the clustering expected, the results showed random esterification.

Thermal behavior of some reducing disaccharides

Abstract Thermogravimetry was used to monitor the loss of weight of a series of reducing disaccharides during a heating process. The resulting heating-curves provide a kinetic fingerprint for the individual compounds. It was shown that the configuration of the disaccharides and the nature of the glycosidic bond affect ( a ) the shape of the heating curve, and ( b ) the temperature at which a given disaccharide loses a fixed percentage of its weight. There is also a positive correlation between the thermogravimetric data and the acid-catalyzed hydrolysis of the disaccharides in solution; the disaccharides that lose water at the lowest temperature are those having the largest rate-constants of acid hydrolysis. Examination of the products of heating indicate polymerization and an intramolecular loss of water, followed by cleavage to form 1,6-anhydro-aldohexopyranoses.