P.J. Somers

The interaction of areneboronic acids with monosaccharides

Abstract The interaction of benzeneboronic acid, 4-methoxybenzeneboronic acid, and 3-nitrobenzeneboronic acid with d -glucose, d -mannose, and d -fructose at various pH values has been investigated by means of optical rotation methods. The effects of ( a ) various molar ratios of sugar and acid and ( b ) overall concentration on the extent of complex formation are reported.

Cross-linked polyacrylamide derivatives (enzacryls) as water-insoluble carriers of amylolytic enzymes

Abstract Methods are described for the water-insolubilisation of alpha- and beta-amylase by chemical coupling to two cross-linked copolymers of acrylamide. For one copolymer, coupling was effected either by diazo or isothiocyanato groups, and for the other, acid azide groups were employed. Active derivatives of alpha-amylase were prepared by diazo, isothiocyanato, and acid azide coupling, whereas active derivatives of beta-amylase could be prepared only by diazo and isothiocyanato coupling. The stabilities of the active, copolyacrylamide-bound preparations of amylase were comparable to those of the corresponding, cellulose-bound enzymes. In the case of alpha-amylase, the copolyacrylamide derivatives were more stable than the free enzyme in solution.

A Spectrophotometric method for the determination of formic acid in the periodate oxidation of carbohydrates

Abstract A spectrophotometric method of assaying formic acid, in the range 5–200μg, has been devised by using the chromophore (λ max 450 mμ) formed on heating formic acid with 2-thiobarbituric acid under acidic conditions. The method may be used to monitor formic acid production from carbohydrates during periodate oxidation, since any interfering aldehydes are reduced with sodium borohydride following termination of the reaction with ethylene glycol.

Preparation and stability of exo-amylolytic enzymes chemically coupled to microcrystalline cellulose

Abstract The preparation of active, water-insoluble derivatives of β- and γ-amylase by chemical coupling with a diazotised 3-( p -aminophenoxy)-2-hydroxypropyl ether of cellulose, and a water-insoluble derivative of β-amylase by coupling with a 2-hydroxy-3-( p -isothiocyanatophenoxy)propyl ether of cellulose is described. The activity retained on insolubilisation is considerably higher than that commonly retained on water-insolubilisation of hydrolytic enzymes active against macromolecular substrates. It is suggested that this is connected with the exo-hydrolytic character of β- and γ-amylase. The insoluble enzymes are relatively more resistant to heat denaturation than the free enzymes in solution.

Preparation and properties of α-amylase chemically coupled to microcrystalline cellulose

Abstract Methods are reported for the preparation of water-insoluble derivatives of α-amylase, in which the enzyme is covalently linked to microcrystalline cellulose. Coupling was effected via diazotised 3-(p-aminophenoxy)-2-hydroxypropyl ethers and 2-hydroxy-3-(p-isothiocyanatophenoxy)propyl ethers of cellulose having different degrees of substitution; low substitution results in higher retention of α-amylase activity. The water-insoluble enzyme shows greater heat stability than the native α-amylase, 20% of the original activity remaining after 7 days at 45°.

The use of poly(4-vinylbenzeneboronic acid) resins in the fractionation and interconversion of carbohydrates

Abstract Poly(4-vinylbenzeneboronic acid) resins have been prepared and used as chromatographic packings in the fractionation of carbohydrates with water as the eluent. The effect of the pH and temperature of the eluent on the fractionations was investigated. These resins have also been used to displace the pseudo-equilibrium established in aqueous alkali between d -glucose, d -fructose, and d -mannose to give high yields of d -fructose, and the use of a model reactor for the preparation of d -fructose is described.

Separations of carbohydrates based on ion exclusion and gel permeation

Abstract A method is described for the fractionation of oligosaccharides by molecular size on columns of cation-exchange resin with water as the sole eluant. The same system allowed separation of inorganic ions and acidic carbohydrates from neutral components. The elution of amino acids and peptides is described, and the application to separation problems in carbohydrate chemistry is discussed.

Linkage analysis of carbohydrates by using saccharinic acid formation

Abstract Methods for assaying isosaccharinic, metasaccharinic, and saccharinic acids on a microgram scale are described; these may be used to monitor the production of such acids during the alkali-mediated, “peeling” reaction of oligosaccharides. These methods of assay, coupled with determination of the formaldehyde produced following periodate oxidation of the equilibrium degradation mixture, are proposed for use in linkage analysis of oligosaccharides containing hexoses or hexuloses.

Recovery and re-use of water-insoluble amylase derivatives

Abstract The activity retained by water-insoluble derivatives of alpha- and beta-amylase on repeated re-use was investigated. Derivatives of both enzymes were prepared by diazo and isothiocyanato coupling with appropriate cellulose or copolyacrylamide (Enzacryl AA) derivatives. In addition, a copolyacrylamide (Enzacryl AH)-based derivative of alpha-amylase, in which the enzyme was attached by acid azide-coupling, was investigated. Although considerable desorption of physically bound enzyme took place on initial re-use, the cellulose-bound derivatives were subsequently re-used repeatedly, with comparatively little loss of activity. The Enzacryl-based derivatives were superior, in that recovery of activity was good on initial as well as repeated re-use.

Arrangement of the l-rhamnose units in Diplococcus pneumoniae Type II polysaccharide

Abstract Pneumococcus Type II polysaccharide, containing l -rhamnose (47.8%), d -glucose (34.7%), and sodium d -glucuronate (16.2%), induced sequentially in cultures of Klebsiella aerogenes an α- l -rhamnosidase, which liberated O -β- l -rhamnopyranosyl-(1→3)- O -β- l -rhamnopyranosyl-(1→3)- l -rhamnose, and then a β- l -rhamnosidase, which degraded this trisaccharide to l -rhamnose. The arrangement of such rhamnose units in the polysaccharide has been determined by using sequential application of degradation by periodate oxidation, sodium borohydride reduction, and mild acid hydrolysis, and repetitions of this sequence.